bims-muscge Biomed News
on Muscle stem cells and gene therapy
Issue of 2023–10–29
thirty papers selected by
Chance Bowman, Dartmouth College



  1. Exp Physiol. 2023 Oct 26.
      
    Keywords:  differentiation; exercise; mechanical loading; myogenesis; satellite cells; wnt signalling
    DOI:  https://doi.org/10.1113/EP091515
  2. Free Radic Biol Med. 2023 Oct 23. pii: S0891-5849(23)01075-4. [Epub ahead of print]
      Satellite cells are bona fide muscle stem cells that are indispensable for successful post-natal muscle growth and regeneration after severe injury. These cells also participate in adult muscle adaptation in several capacities. microRNA (miRNA) are post-transcriptional regulators of mRNA that are implicated in several aspects of stem cell function. There is evidence to suggest that miRNAs affect satellite cell behavior in vivo and myogenic progenitor behavior in vitro, but the role of miRNAs in adult skeletal muscle satellite cells is less studied. In this review, we provide evidence for how miRNAs control satellite cell behavior with emphasis on satellite cells of adult muscle in vivo. We first outline how miRNAs are indispensable for satellite cell viability and control the phases of myogenesis. Next, we discuss the interplay between miRNAs and myogenic cell redox status, senescence, and communication to other muscle-resident cells during muscle adaptation. Results from recent satellite cell miRNA profiling studies are also summarized. In vitro experiments in primary myogenic cells and cell lines have been invaluable for exploring the influence of miRNAs, but we identify a need for novel genetic tools to further interrogate how miRNAs control satellite cell behavior in adult skeletal muscle in vivo.
    Keywords:  Myogenesis; Senescence; Stem cells; Transcriptomics; myomiRs
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.10.403
  3. STAR Protoc. 2023 Oct 22. pii: S2666-1667(23)00623-8. [Epub ahead of print]4(4): 102656
      Muscle stem cells (MuSCs) are the building blocks for regenerating skeletal muscle after trauma. If we intend to maximize the therapeutic potential of MuSCs, we must further study their molecular and functional properties. Here, we present a protocol for the isolation of mouse MuSCs via a two-step enzymatic and mechanical dissociation of skeletal muscle coupled with fluorescence-activated cell sorting (FACS). FACS-isolated MuSCs can be used for various downstream applications including cell culture, cell transduction, immunofluorescence, and gene expression assays. For complete details on the use and execution of this protocol, please refer to Almada et al. (2021).1.
    Keywords:  Antibody; Cell Differentiation; Cell isolation; Flow Cytometry; Gene Expression; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2023.102656
  4. Nano Converg. 2023 Oct 21. 10(1): 48
      Engineered three-dimensional (3D) tissue constructs have emerged as a promising solution for regenerating damaged muscle tissue resulting from traumatic or surgical events. 3D architecture and function of the muscle tissue constructs can be customized by selecting types of biomaterials and cells that can be engineered with desired shapes and sizes through various nano- and micro-fabrication techniques. Despite significant progress in this field, further research is needed to improve, in terms of biomaterials properties and fabrication techniques, the resemblance of function and complex architecture of engineered constructs to native muscle tissues, potentially enhancing muscle tissue regeneration and restoring muscle function. In this review, we discuss the latest trends in using nano-biomaterials and advanced nano-/micro-fabrication techniques for creating 3D muscle tissue constructs and their regeneration ability. Current challenges and potential solutions are highlighted, and we discuss the implications and opportunities of a future perspective in the field, including the possibility for creating personalized and biomanufacturable platforms.
    Keywords:  Biomaterials; Nanomaterials; Skeletal muscle; Stem cells; Tissue engineering; Tissue regeneration
    DOI:  https://doi.org/10.1186/s40580-023-00398-y
  5. Nutrients. 2023 Oct 16. pii: 4377. [Epub ahead of print]15(20):
      Vitamin D deficiency, prevalent worldwide, is linked to muscle weakness, sarcopenia, and falls. Muscle regeneration is a vital process that allows for skeletal muscle tissue maintenance and repair after injury. PubMed and Web of Science were used to search for studies published prior to May 2023. We assessed eligible studies that discussed the relationship between vitamin D, muscle regeneration in this review. Overall, the literature reports strong associations between vitamin D and skeletal myocyte size, and muscle regeneration. In vitro studies in skeletal muscle cells derived from mice and humans showed vitamin D played a role in regulating myoblast growth, size, and gene expression. Animal studies, primarily in mice, demonstrate vitamin D's positive effects on skeletal muscle function, such as improved grip strength and endurance. These studies encompass vitamin D diet research, genetically modified models, and disease-related mouse models. Relatively few studies looked at muscle function after injury, but these also support a role for vitamin D in muscle recovery. The human studies have also reported that vitamin D deficiency decreases muscle grip strength and gait speed, especially in the elderly population. Finally, human studies reported the benefits of vitamin D supplementation and achieving optimal serum vitamin D levels in muscle recovery after eccentric exercise and surgery. However, there were no benefits in rotator cuff injury studies, suggesting that repair mechanisms for muscle/ligament tears may be less reliant on vitamin D. In summary, vitamin D plays a crucial role in skeletal muscle function, structural integrity, and regeneration, potentially offering therapeutic benefits to patients with musculoskeletal diseases and in post-operative recovery.
    Keywords:  muscle degeneration; skeletal muscle; vitamin D; vitamin D receptor
    DOI:  https://doi.org/10.3390/nu15204377
  6. J Cachexia Sarcopenia Muscle. 2023 Oct 22.
      Skeletal muscle wasting is a complicated metabolic syndrome accompanied by multiple diseases ranging from cancer to metabolic disorders and infectious conditions. The loss of muscle mass significantly impairs muscle function, resulting in poor quality of life and high mortality of associated diseases. The fundamental cellular and molecular mechanisms inducing muscle wasting have been well established, and those related pathways can be activated by a variety of extracellular signals, including inflammatory cytokines and catabolic stimuli. As an emerging messenger of cell-to-cell communications, extracellular vesicles (EVs) also get involved in the progression of muscle wasting by transferring bioactive cargoes including various proteins and non-coding RNAs to skeletal muscle. Like a double-edged sword, EVs play either a pro-wasting or anti-wasting role in the progression of muscle wasting, highly dependent on their parental cells as well as the specific type of cargo they encapsulate. This review aims to illustrate the current knowledge about the biological function of EVs cargoes in skeletal muscle wasting. Additionally, the potential therapeutic implications of EVs in the diagnosis and treatment of skeletal muscle wasting are also discussed. Simultaneously, several outstanding questions are included to shed light on future research.
    Keywords:  Exosomes; Extracellular vesicles; Muscle wasting; Therapeutic implications
    DOI:  https://doi.org/10.1002/jcsm.13364
  7. Adv Sci (Weinh). 2023 Oct 23. e2305080
      Skeletal muscle comprises a large, heterogeneous assortment of cell populations that interact to maintain muscle homeostasis, but little is known about the mechanism that controls myogenic development in response to artificial selection. Different pig (Sus scrofa) breeds exhibit distinct muscle phenotypes resulting from domestication and selective breeding. Using unbiased single-cell transcriptomic sequencing analysis (scRNA-seq), the impact of artificial selection on cell profiles is investigated in neonatal skeletal muscle of pigs. This work provides panoramic muscle-resident cell profiles and identifies novel and breed-specific cells, mapping them on pseudotime trajectories. Artificial selection has elicited significant changes in muscle-resident cell profiles, while conserving signs of generational environmental challenges. These results suggest that fibro-adipogenic progenitors serve as a cellular interaction hub and that specific transcription factors identified here may serve as candidate target regulons for the pursuit of a specific muscle phenotype. Furthermore, a cross-species comparison of humans, mice, and pigs illustrates the conservation and divergence of mammalian muscle ontology. The findings of this study reveal shifts in cellular heterogeneity, novel cell subpopulations, and their interactions that may greatly facilitate the understanding of the mechanism underlying divergent muscle phenotypes arising from artificial selection.
    Keywords:  fibro-adipogenic progenitors; muscle characteristics; neonatal myogenesis; pigs, single-cell RNA-sequencing
    DOI:  https://doi.org/10.1002/advs.202305080
  8. Int J Mol Sci. 2023 Oct 13. pii: 15161. [Epub ahead of print]24(20):
      Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.
    Keywords:  RNA modifications; epitranscriptomics; gene expression; m6A; skeletal muscle
    DOI:  https://doi.org/10.3390/ijms242015161
  9. Geroscience. 2023 Oct 24.
      Skeletal muscle adaptation to external stimuli, such as regeneration following injury and hypertrophy in response to resistance exercise, are blunted with advanced age. The accumulation of senescent cells, along with defects in myogenic progenitor cell (MPC) proliferation, have been strongly linked as contributing factors to age-associated impairment in muscle adaptation. p53 plays an integral role in all these processes, as upregulation of p53 causes apoptosis in senescent cells and prevents mitotic catastrophe in MPCs from old mice. The goal of this study was to determine if a novel pharmaceutical agent (BI01), which functions by upregulating p53 through inhibition of binding to MDM2, the primary p53 regulatory protein, improves muscle regeneration and hypertrophy in old mice. BI01 effectively reduced the number of senescent cells in vitro but had no effect on MPC survival or proliferation at a comparable dose. Following repeated oral gavage with 2 mg/kg of BI01 (OS) or vehicle (OV), old mice (24 months) underwent unilateral BaCl2 injury in the tibialis anterior (TA) muscle, with PBS injections serving as controls. After 7 days, satellite cell number was higher in the TA of OS compared to OV mice, as was the expression of genes involved in ATP production. By 35 days, old mice treated with BI01 displayed reduced senescent cell burden, enhanced regeneration (higher muscle mass and fiber cross-sectional area) and restoration of muscle function relative to OV mice. To examine the impact of 2 mg/kg BI01 on muscle hypertrophy, the plantaris muscle was subjected to 28 days of mechanical overload (MOV) in OS and OV mice. In response to MOV, OS mice had larger plantaris muscles and muscle fibers than OV mice, particularly type 2b + x fibers, associated with reduced senescent cells. Together our data show that BI01 is an effective senolytic agent that may also augment muscle metabolism to enhance muscle regeneration and hypertrophy in old mice.
    Keywords:  Hypertrophy; Regeneration; Senescence; Senolytics; Skeletal Muscle
    DOI:  https://doi.org/10.1007/s11357-023-00976-2
  10. Bioengineering (Basel). 2023 Oct 21. pii: 1232. [Epub ahead of print]10(10):
      Skeletal muscle tissue engineering (TE) and adipose tissue engineering have undergone significant progress in recent years. This review focuses on the key findings in these areas, particularly highlighting the integration of 3D bioprinting techniques to overcome challenges and enhance tissue regeneration. In skeletal muscle TE, 3D bioprinting enables the precise replication of muscle architecture. This addresses the need for the parallel alignment of cells and proper innervation. Satellite cells (SCs) and mesenchymal stem cells (MSCs) have been utilized, along with co-cultivation strategies for vascularization and innervation. Therefore, various printing methods and materials, including decellularized extracellular matrix (dECM), have been explored. Similarly, in adipose tissue engineering, 3D bioprinting has been employed to overcome the challenge of vascularization; addressing this challenge is vital for graft survival. Decellularized adipose tissue and biomimetic scaffolds have been used as biological inks, along with adipose-derived stem cells (ADSCs), to enhance graft survival. The integration of dECM and alginate bioinks has demonstrated improved adipocyte maturation and differentiation. These findings highlight the potential of 3D bioprinting techniques in skeletal muscle and adipose tissue engineering. By integrating specific cell types, biomaterials, and printing methods, significant progress has been made in tissue regeneration. However, challenges such as fabricating larger constructs, translating findings to human models, and obtaining regulatory approvals for cellular therapies remain to be addressed. Nonetheless, these advancements underscore the transformative impact of 3D bioprinting in tissue engineering research and its potential for future clinical applications.
    Keywords:  3D bioprinting; adipose tissue; skeletal muscle tissue engineering; tissue engineering
    DOI:  https://doi.org/10.3390/bioengineering10101232
  11. Cell Rep. 2023 Oct 24. pii: S2211-1247(23)01341-4. [Epub ahead of print]42(11): 113329
      Development is regulated by various factors, including protein methylation status. While PRMT5 is well known for its roles in oncogenesis by mediating symmetric di-methylation of arginine, its role in normal development remains elusive. Using Myod1Cre to drive Prmt5 knockout in embryonic myoblasts (Prmt5MKO), we dissected the role of PRMT5 in myogenesis. The Prmt5MKO mice are born normally but exhibit progressive muscle atrophy and premature death. Prmt5MKO inhibits proliferation and promotes premature differentiation of embryonic myoblasts, reducing the number and regenerative function of satellite cells in postnatal mice. Mechanistically, PRMT5 methylates and destabilizes FoxO1. Prmt5MKO increases the total FoxO1 level and promotes its cytoplasmic accumulation, leading to activation of autophagy and depletion of lipid droplets (LDs). Systemic inhibition of autophagy in Prmt5MKO mice restores LDs in myoblasts and moderately improves muscle regeneration. Together, PRMT5 is essential for muscle development and regeneration at least partially through mediating FoxO1 methylation and LD turnover.
    Keywords:  CP: Developmental biology; CP: Molecular biology; PRMT; PTM; SCs; autophagy; myogenesis; posttranslational modification; protein arginine methyltransferase; satellite cells
    DOI:  https://doi.org/10.1016/j.celrep.2023.113329
  12. Int J Mol Sci. 2023 Oct 18. pii: 15318. [Epub ahead of print]24(20):
      Skeletal muscle, a vital and intricate organ, plays a pivotal role in maintaining overall body metabolism, facilitating movement, and supporting normal daily activities. An accumulating body of evidence suggests that microRNA (miRNA) holds a crucial role in orchestrating skeletal muscle growth. Therefore, the primary aim of this study was to investigate the influence of miR-103-3p on myogenesis. In our study, the overexpression of miR-103-3p was found to stimulate proliferation while suppressing differentiation in C2C12 myoblasts. Conversely, the inhibition of miR-103-3p expression yielded contrasting effects. Through bioinformatics analysis, potential binding sites of miR-103-3p with the 3'UTR region of BTG anti-proliferative factor 2 (BTG2) were predicted. Subsequently, dual luciferase assays conclusively demonstrated BTG2 as the direct target gene of miR-103-3p. Further investigation into the role of BTG2 in C2C12 myoblasts unveiled that its overexpression impeded proliferation and encouraged differentiation in these cells. Notably, co-transfection experiments showcased that the overexpression of BTG2 could counteract the effects induced by miR-103-3p. In summary, our findings elucidate that miR-103-3p promotes proliferation while inhibiting differentiation in C2C12 myoblasts by targeting BTG2.
    Keywords:  BTG2; differentiation; miR-103-3p; myoblasts; proliferation
    DOI:  https://doi.org/10.3390/ijms242015318
  13. ArXiv. 2023 Oct 09. pii: arXiv:2310.06191v1. [Epub ahead of print]
      Experimental observations suggest that the force output of the skeletal muscle tissue can be correlated to the intra-muscular pressure generated by the muscle belly. However, pressure often proves difficult to measure through in-vivo tests. Simulations on the other hand, offer a tool to model muscle contractions and analyze the relationship between muscle force generation and deformations as well as pressure outputs, enabling us to gain insight into correlations among experimentally measurable quantities such as principal and volumetric strains, and the force output. In this work, a correlation study is performed using Pearson's and Spearman's correlation coefficients on the force output of the skeletal muscle, the principal and volumetric strains experienced by the muscle and the pressure developed within the muscle belly as the muscle tissue undergoes isometric contractions due to varying activation profiles. The study reveals strong correlations between force output and the strains at all locations of the belly, irrespective of the type of activation profile used. This observation enables estimation on the contribution of various muscle groups to the total force by the experimentally measurable principal and volumetric strains in the muscle belly. It is also observed that pressure does not correlate well with force output due to stress relaxation near the boundary of muscle belly.
  14. Biomedicines. 2023 Sep 30. pii: 2683. [Epub ahead of print]11(10):
      Facioscapulohumeral dystrophy (FSHD) is a muscle disease caused by inappropriate expression of the double homeobox 4 (DUX4) gene in skeletal muscle, and its downstream activation of pro-apoptotic transcriptional programs. Inhibitors of DUX4 expression have the potential to treat FSHD. Apabetalone is a clinical-stage bromodomain and extra-terminal (BET) inhibitor, selective for the second bromodomain on BET proteins. Using primary human skeletal muscle cells from FSHD type 1 patients, we evaluated apabetalone for its ability to counter DUX4's deleterious effects and compared it with the pan-BET inhibitor JQ1, and the p38 MAPK inhibitor-and DUX4 transcriptional repressor-losmapimod. We applied RNA-sequencing and bioinformatic analysis to detect treatment-associated impacts on the transcriptome of these cells. Apabetalone inhibited the expression of DUX4 downstream markers, reversing hallmarks of FSHD gene expression in differentiated muscle cells. JQ1, but not apabetalone, was found to induce apoptosis. While both BET inhibitors modestly impacted differentiation marker expression, they did not affect myotube fusion. Losmapimod also reduced expression of DUX4 target genes but differed in its impact on FSHD-associated pathways. These findings demonstrate that apabetalone inhibits DUX4 target gene expression and reverses transcriptional programs that contribute to FSHD pathology, making this drug a promising candidate therapeutic for FSHD.
    Keywords:  BET inhibitor; DUX4; FSHD; RNA; apabetalone; bromodomain and extra-terminal domain; epigenetics; facioscapulohumeral muscular dystrophy; transcriptome
    DOI:  https://doi.org/10.3390/biomedicines11102683
  15. Cell Rep. 2023 Oct 23. pii: S2211-1247(23)01300-1. [Epub ahead of print]42(11): 113288
      Skeletal muscle function declines in the aging process or disease; however, until now, skeletal muscle has remained one of the organs most undertreated with medication. In this study, naringenin (NAR) was found to build muscle endurance in wild-type mice of different ages by increasing oxidative myofiber numbers and aerobic metabolism, and it ameliorates muscle dysfunction in mdx mice. The transcription factor Sp1 was identified as a direct target of NAR and was shown to mediate the function of NAR on muscle. Moreover, the binding site of NAR on Sp1 was further validated as GLN-110. NAR enhances the binding of Sp1 to the CCCTGCCCTC sequence of the Esrrg promoter by promoting Sp1 phosphorylation, thus upregulating Esrrg expression. The identification of the Sp1-ERRγ transcriptional axis is of great significance in basic muscle research, and this function of NAR has potential implications for the improvement of muscle function and the prevention of muscle atrophy.
    Keywords:  CP: Metabolism; CP: Molecular biology; Duchenne muscular dystrophy; energy metabolism; fiber type; muscle atrophy; naringenin; skeletal muscle
    DOI:  https://doi.org/10.1016/j.celrep.2023.113288
  16. FEBS J. 2023 Oct 24.
      Maintaining cellular homeostasis necessitates precise control of gene expression, a process that molds both the transcriptome and proteome to adapt to internal and external changes effectively. MicroRNAs (miRNAs) are small RNAs (~ 22nucleotides) belonging to a broad family of non-coding RNAs and are important regulators of gene expression. While numerous studies have advanced our understanding of the common processes underlying miRNA biogenesis and function, individual cell types in diverse organisms have evolved distinct mechanisms for regulating them. In this current issue, Satoshi Oikawa and colleagues delve into the molecular dynamics of miRNAs in adult skeletal muscles. Their research introduces intriguing new inquiries for further investigations to uncover alternative mechanisms of miRNA biogenesis in skeletal muscle.
    Keywords:  Dicer knockout; miRNA biogenesis; miRNA turnover; skeletal muscles
    DOI:  https://doi.org/10.1111/febs.16975
  17. Biomolecules. 2023 Oct 17. pii: 1536. [Epub ahead of print]13(10):
      Muscular dystrophies are a heterogeneous group of genetic muscle-wasting disorders that are subdivided based on the region of the body impacted by muscle weakness as well as the functional activity of the underlying genetic mutations. A common feature of the pathophysiology of muscular dystrophies is chronic inflammation associated with the replacement of muscle mass with fibrotic scarring. With the progression of these disorders, many patients suffer cardiomyopathies with fibrosis of the cardiac tissue. Anti-inflammatory glucocorticoids represent the standard of care for Duchenne muscular dystrophy, the most common muscular dystrophy worldwide; however, long-term exposure to glucocorticoids results in highly adverse side effects, limiting their use. Thus, it is important to develop new pharmacotherapeutic approaches to limit inflammation and fibrosis to reduce muscle damage and promote repair. Here, we examine the pathophysiology, genetic background, and emerging therapeutic strategies for muscular dystrophies.
    Keywords:  DUX4; dysferlin; dystroglycan; dystrophin; emerin; fibrosis; inflammation; lamin A; muscular dystrophy; sarcoglycan
    DOI:  https://doi.org/10.3390/biom13101536
  18. Biomolecules. 2023 Sep 22. pii: 1434. [Epub ahead of print]13(10):
      Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a superfamily of RNA-binding proteins consisting of more than 20 members. These proteins play a crucial role in various biological processes by regulating RNA splicing, transcription, and translation through their binding to RNA. In the context of muscle development and regeneration, hnRNPs are involved in a wide range of regulatory mechanisms, including alternative splicing, transcription regulation, miRNA regulation, and mRNA stability regulation. Recent studies have also suggested a potential association between hnRNPs and muscle-related diseases. In this report, we provide an overview of our current understanding of how hnRNPs regulate RNA metabolism and emphasize the significance of the key members of the hnRNP family in muscle development. Furthermore, we explore the relationship between the hnRNP family and muscle-related diseases.
    Keywords:  alternative splicing; hnRNPs; muscle development; muscle disorders
    DOI:  https://doi.org/10.3390/biom13101434
  19. Int J Biol Macromol. 2023 Oct 24. pii: S0141-8130(23)04512-9. [Epub ahead of print] 127614
      An emerging research focus is the role of m6A modifications in mediating the post-transcriptional regulation of mRNA during mammalian development. Recent evidence suggests that m6A methyltransferases and demethylases play critical roles in skeletal muscle development. Ythdf2 is a m6A "reader" protein that mediates mRNA degradation in an m6A-dependent manner. However, the specific function of Ythdf2 in skeletal muscle development and the underlying mechanisms remain unclear. Here, we observed that Ythdf2 expression was significantly upregulated during myogenic differentiation, whereas Ythdf2 knockdown markedly inhibited myoblast proliferation and differentiation. Combined analysis of high-throughput sequencing, Co-IP, and RIP assay revealed that Ythdf2 could bind to m6A sites in STK11 mRNA and form an Ago2 silencing complex to promote its degradation, thereby regulating its expression and consequently, the AMPK/mTOR pathway. Furthermore, STK11 downregulation partially rescued Ythdf2 knockdown-induced impairment of proliferation and myogenic differentiation by inhibiting the AMPK/mTOR pathway. Collectively, our results indicate that Ythdf2 mediates the decay of STK11 mRNA, an AMPK activator, in an Ago2 system-dependent manner, thereby driving skeletal myogenesis by suppressing the AMPK/mTOR pathway. These findings further enhance our understanding of the molecular mechanisms underlying RNA methylation in the regulation of myogenesis and provide valuable insights for conducting in-depth studies on myogenesis.
    Keywords:  Myogenesis; RNA degradation; STK11; Ythdf2; m(6)A modification
    DOI:  https://doi.org/10.1016/j.ijbiomac.2023.127614
  20. J Transl Med. 2023 10 24. 21(1): 748
       INTRODUCTION: The promising potential of adeno-associated virus (AAV) gene delivery strategies to treat genetic disorders continues to grow with an additional three AAV-based therapies recently approved by the Food and Drug Administration and dozens of others currently under evaluation in clinical trials. With these developments, it has become increasingly apparent that the high doses currently needed for efficacy carry risks of toxicity and entail enormous manufacturing costs, especially for clinical grade products. Strategies to increase the therapeutic efficacy of AAV-mediated gene delivery and reduce the minimal effective dose would have a substantial impact on this field. We hypothesized that an exercise-induced redistribution of tissue perfusion in the body to favor specific target organs via acute aerobic exercise prior to systemic intravenous (IV) AAV administration could increase efficacy.
    BACKGROUND: Aerobic exercise triggers an array of downstream physiological effects including increased perfusion of heart and skeletal muscle, which we expected could enhance AAV transduction. Prior preclinical studies have shown promising results for a gene therapy approach to treat Barth syndrome (BTHS), a rare monogenic cardioskeletal myopathy, and clinical studies have shown the benefit of low intensity exercise in these patients, making this a suitable disease in which to test the ability of aerobic exercise to enhance AAV transduction.
    METHODS: Wild-type (WT) and BTHS mice were either systemically administered AAV9 or completed one episode of low intensity treadmill exercise immediately prior to systemic administration of AAV9.
    RESULTS: We demonstrate that a single episode of acute low intensity aerobic exercise immediately prior to IV AAV9 administration improves marker transgene delivery in WT mice as compared to mice injected without the exercise pre-treatment. In BTHS mice, prior exercise improved transgene delivery and additionally increased improvement in mitochondrial gene transcription levels and mitochondrial function in the heart and gastrocnemius muscles as compared to mice treated without exercise.
    CONCLUSIONS: Our findings suggest that one episode of acute low intensity aerobic exercise improves AAV9 transduction of heart and skeletal muscle. This low-risk, cost effective intervention could be implemented in clinical trials of individuals with inherited cardioskeletal disease as a potential means of improving patient safety for human gene therapy.
    Keywords:  AAV; AAV9; Adeno-associated virus; Aerobic exercise; Barth syndrome; Cardioskeletal myopathy; Gene therapy; Systemic gene delivery; TAFAZZIN
    DOI:  https://doi.org/10.1186/s12967-023-04626-1
  21. J Nutr Biochem. 2023 Oct 25. pii: S0955-2863(23)00240-1. [Epub ahead of print] 109507
      In recent years, the impact of bile acids and their representative G protein-coupled bile acid receptor 1 (TGR5) signaling pathway on muscle function and metabolic health has gained considerable interest. Increasing the content of slow muscle fibers has been recognized as an effective strategy to improve metabolic health. Oleanolic acid (OA) is a naturally occurring triterpenoid compound derived from plants, which can activate TGR5. The aim of this study was to investigate the effect of OA and TGR5 on muscle fiber types and further explore the underlying TGR5-dependent mechanisms. In this study, mice were divided into three groups and dietary supplementation with 0, 50, or 100 mg/kg OA. In addition, C2C12 cells were treated with OA at concentrations of 0, 5, 10, and 20 μM. Our studies revealed that OA promoted the conversion of fast to slow muscle fibers. In addition, it was found that OA activated the TGR5-mediated calcineurin (CaN)/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway. Further mechanistic investigations demonstrated that inhibiting TGR5 and CaN abolished the effects of OA on muscle fiber types transformation. In conclusion, this study found that OA promotes the transformation of fast muscle fibers to slow muscle fibers through the TGR5-mediated CaN/NFATc1 signaling pathway.
    Keywords:  NFATc1; Oleanolic acid; TGR5; calcineurin; muscle fiber type transformation
    DOI:  https://doi.org/10.1016/j.jnutbio.2023.109507
  22. Med Sci Sports Exerc. 2023 Oct 26.
       PURPOSE: The purpose of this study was to determine how the intersection of coactivator-associated arginine methyltransferase 1 (CARM1) and biological sex impacts skeletal muscle adaptations to chronic physical activity.
    METHODS: 12-week-old female (F) and male (M) wild-type (WT) and CARM1 skeletal muscle-specific knockout mice (mKO) were randomly assigned to sedentary (SED) or voluntary wheel running (VWR) experimental groups. For 8 weeks, the animals in the VWR cohort had volitional access to running wheels. Subsequently, we performed whole-body functional tests, and 48 hours later muscles were harvested for molecular analysis. Western blotting, enzyme activity assays, as well as confocal and transmission electron microscopy (TEM) were used to examine skeletal muscle biology.
    RESULTS: Our data reveal a sex-dependent reduction in VWR volume caused by muscle-specific ablation of CARM1, as F CARM1 mKO mice performed less chronic, volitional exercise than their WT counterparts. Regardless of VWR output, exercise-induced adaptations in physiological function were similar between experimental groups. A broad panel of protein arginine methyltransferase (PRMT) biology measurements, including markers of arginine methyltransferase expression and activity, were unaffected by VWR, except for CARM1 and PRMT7 protein levels, which decreased and increased with VWR, respectively. Changes in myofiber morphology and mitochondrial protein content showed similar trends among animals. However, a closer examination of TEM images revealed contrasting responses to VWR in CARM1 mKO mice compared to WT littermates, particularly in mitochondrial size and fractional area.
    CONCLUSIONS: The present findings demonstrate that CARM1 mKO reduces daily running volume in F mice, as well as exercise-evoked skeletal muscle mitochondrial plasticity, which indicates that this enzyme plays an essential role in sex-dependent differences in exercise performance and mitochondrial health.
    DOI:  https://doi.org/10.1249/MSS.0000000000003333
  23. J Physiol. 2023 Oct 25.
      Our laboratory previously showed lipid hydroperoxides and oxylipin levels are elevated in response to loss of skeletal muscle innervation and are associated with muscle pathologies. To elucidate the pathological impact of lipid hydroperoxides, we overexpressed glutathione peroxidase 4 (GPx4), an enzyme that targets reduction of lipid hydroperoxides in membranes, in adult CuZn superoxide dismutase knockout (Sod1KO) mice that show accelerated muscle atrophy associated with loss of innervation. The gastrocnemius muscle from Sod1KO mice shows reduced mitochondrial respiration and elevated oxidative stress (F2 -isoprostanes and hydroperoxides) compared to wild-type (WT) mice. Overexpression of GPx4 improved mitochondrial respiration and reduced hydroperoxide generation in Sod1KO mice, but did not attenuate the muscle loss that occurs in Sod1KO mice. In contrast, contractile force generation is reduced in EDL muscle in Sod1KO mice relative to WT mice, and overexpression of GPx4 restored force generation to WT levels in Sod1KO mice. GPx4 overexpression also prevented loss of muscle contractility at the single fibre level in fast-twitch fibres from Sod1KO mice. Muscle fibres from Sod1KO mice were less sensitive to both depolarization and calcium at the single fibre level and exhibited a reduced activation by S-glutathionylation. GPx4 overexpression in Sod1KO mice rescued the deficits in both membrane excitability and calcium sensitivity of fast-twitch muscle fibres. Overexpression of GPx4 also restored the sarco/endoplasmic reticulum Ca2+ -ATPase activity in Sod1KO gastrocnemius muscles. These data suggest that GPx4 plays an important role in preserving excitation-contraction coupling function and Ca2+ homeostasis, and in maintaining muscle and mitochondrial function in oxidative stress-induced sarcopenia. KEY POINTS: Knockout of CuZn superoxide dismutase (Sod1KO) induces elevated oxidative stress with accelerated muscle atrophy and weakness. Glutathione peroxidase 4 (GPx4) plays a fundamental role in the reduction of lipid hydroperoxides in membranes, and overexpression of GPx4 improves mitochondrial respiration and reduces hydroperoxide generation in Sod1KO mice. Muscle contractile function deficits in Sod1KO mice are alleviated by the overexpression of GPx4. GPx4 overexpression in Sod1KO mice rescues the impaired muscle membrane excitability of fast-twitch muscle fibres and improves their calcium sensitivity. Sarco/endoplasmic reticulum Ca2+ -ATPase activity in Sod1KO muscles is decreased, and it is restored by the overexpression of GPx4. Our results confirm that GPx4 plays an important role in preserving excitation-contraction coupling function and Ca2+ homeostasis, and maintaining muscle and mitochondrial function in oxidative stress-induced sarcopenia.
    Keywords:  Ca2+ sensitivity; E-C coupling; SERCA activity; lipid peroxidation; mitochondria; muscle function; oxidative stress
    DOI:  https://doi.org/10.1113/JP285259
  24. Trends Biotechnol. 2023 Oct 19. pii: S0167-7799(23)00284-6. [Epub ahead of print]
      Synthetic transcription factors (synTFs) that control beneficial transgene expression are an important method to increase the safety and efficacy of cell and gene therapy. Reliance on synTF components from non-human sources has slowed progress in the field because of concerns about immunogenicity and inducer drug properties. Recent advances in human-derived DNA-binding domains (DBDs) and transcriptional activation domains (TADs) paired with novel control modules responsive to clinically approved small molecules have poised the synTF field to overcome these hurdles. Advances include controllers inducible by autonomous signaling inputs and more complex, multi-input synTF circuits. Demonstrations of advanced control strategies with human-derived transcription factor components in clinically relevant vectors and in vivo models will facilitate progression into the clinic.
    Keywords:  autonomous control; cell therapy; gene therapy; synthetic biology; transcription factors
    DOI:  https://doi.org/10.1016/j.tibtech.2023.09.010
  25. Hum Mol Genet. 2023 Oct 26. pii: ddad183. [Epub ahead of print]
      Mutations in skeletal muscle α-actin (Acta1) cause myopathies. In a mouse model of congenital myopathy, heterozygous Acta1 (H40Y) knock-in (Acta1+/Ki) mice exhibit features of human nemaline myopathy, including premature lethality, severe muscle weakness, reduced mobility, and the presence of nemaline rods in muscle fibers. In this study, we investigated the impact of Acta1 (H40Y) mutation on the neuromuscular junction (NMJ). We found that the NMJs were markedly fragmented in Acta1+/Ki mice. Electrophysiological analysis revealed a decrease in amplitude but increase in frequency of miniature end-plate potential (mEPP) at the NMJs in Acta1+/Ki mice, compared with those in wild type (Acta1+/+) mice. Evoked end-plate potential (EPP) remained similar at the NMJs in Acta1+/Ki and Acta1+/+ mice, but quantal content was increased at the NMJs in Acta1+/Ki, compared with Acta1+/+ mice, suggesting a homeostatic compensation at the NMJs in Acta1+/Ki mice to maintain normal levels of neurotransmitter release. Furthermore, short-term synaptic plasticity of the NMJs was compromised in Acta1+/Ki mice. Together, these results demonstrate that skeletal Acta1 H40Y mutation, albeit muscle-origin, leads to both morphological and functional defects at the NMJ.
    Keywords:  acetylcholine receptor; nemaline myopathy; neuromuscular junction; skeletal muscle α-actin; synapse
    DOI:  https://doi.org/10.1093/hmg/ddad183
  26. Int J Mol Sci. 2023 Oct 18. pii: 15291. [Epub ahead of print]24(20):
      Over the past 20 years, the use of pluripotent stem cells to mimic the complexities of the human neuromuscular junction has received much attention. Deciphering the key mechanisms underlying the establishment and maturation of this complex synapse has been driven by the dual goals of addressing developmental questions and gaining insight into neuromuscular disorders. This review aims to summarise the evolution and sophistication of in vitro neuromuscular junction models developed from the first differentiation of human embryonic stem cells into motor neurons to recent neuromuscular organoids. We also discuss the potential offered by these models to decipher different neuromuscular diseases characterised by defects in the presynaptic compartment, the neuromuscular junction, and the postsynaptic compartment. Finally, we discuss the emerging field that considers the use of these techniques in drug screening assay and the challenges they will face in the future.
    Keywords:  human neuromuscular junction; in vitro models; microfluidics and organoids; pluripotent stem cells
    DOI:  https://doi.org/10.3390/ijms242015291
  27. Nat Commun. 2023 Oct 26. 14(1): 6827
      Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research.
    DOI:  https://doi.org/10.1038/s41467-023-42252-z
  28. Yi Chuan. 2023 Oct 20. 45(10): 859-873
      Massively parallel reporter assay (MPRA) is a high-throughput analysis method that can simultaneously investigate the activity of thousands of regulatory elements in the genome. MPRA introduces a uniquely identified barcode on a conventional luciferase reporter gene vector, sequences the DNA barcode before transfection and the mRNA barcode after transfection by next-generation sequencing technology, and uses the ratio of mRNA and DNA barcode reads to analyze the activity of cis-regulatory elements. Since MPRA was proposed, it has been widely used in the identification of genomic cis-regulatory elements and functional variants, the effect of post-transcriptional regulation on phenotypes and so on. In this review, we summarize the development history, basic principles, experimental procedures and statistical analysis methods of MPRA, and its applications in post-transcriptional regulation and cis-regulatory elements. It also provides prospects for its development and useful references for researchers in related fields to understand and apply MPRA.
    Keywords:  cis-regulatory elements; gene expression regulation; massively parallel reporter assay; post-transcriptional regulation
    DOI:  https://doi.org/10.16288/j.yczz.23-180
  29. Mol Ther Nucleic Acids. 2023 Dec 12. 34 102044
      Single-cell studies have demonstrated that somatic cell reprogramming is a continuous process of cell fates transition. Only partial reprogramming intermediates can overcome the molecular bottlenecks to acquire pluripotency. To decipher the underlying decisive factors driving cell fate, we identified induced pluripotent stem cells or stromal-like cells (iPSCs/SLCs) and iPSCs or trophoblast-like cells (iPSCs/TLCs) fate bifurcations by reconstructing cellular trajectory. The mesenchymal-epithelial transition and the activation of pluripotency networks are the main molecular series in successful reprogramming. Correspondingly, intermediates diverge into SLCs accompanied by the inhibition of cell cycle genes and the activation of extracellular matrix genes, whereas the TLCs fate is characterized by the up-regulation of placenta development genes. Combining putative gene regulatory networks, seven (Taf7, Ezh2, Klf2, etc.) and three key factors (Cdc5l, Klf4, and Nanog) were individually identified as drivers of the successful reprogramming by triggering downstream pluripotent networks during iPSCs/SLCs and iPSCs/TLCs fate bifurcation. Conversely, 11 factors (Cebpb, Sox4, Junb, etc.) and four factors (Gata2, Jund, Ctnnb1, etc.) drive SLCs fate and TLCs fate, respectively. Our study sheds new light on the understanding of decisive factors driving cell fate, which is helpful for improving reprogramming efficiency through manipulating cell fates to avoid alternative fates.
    Keywords:  MT: Bioinformatics; cellular trajectory; decisive factors; fate bifurcation; gene regulatory network; somatic cell reprogramming
    DOI:  https://doi.org/10.1016/j.omtn.2023.102044