bims-muscge 2023-10-08 papers

bims-muscge

Biomed News

on Muscle stem cells and gene therapy

Issue of 2023–10–08
thirteen papers selected by
Chance Bowman, Dartmouth College

Influence of sexual dimorphism on satellite cell regulation and inflammatory response during skeletal muscle regeneration.
Tenascin-C-enriched regeneration-specific extracellular matrix guarantees superior muscle regeneration in Ambystoma mexicanum.
Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9.
Automated Image Analysis Pipeline Development to Monitor Disease Progression in Muscular Dystrophy Using Cell Profiler.
The role of fibrosis in the pathophysiology of muscular dystrophy.
Nanotopographical Cues Tune the Therapeutic Potential of Extracellular Vesicles for the Treatment of Aged Skeletal Muscle Injuries.
Activation of Lactate Receptor Positively Regulates Skeletal Muscle Mass in Mice.
PAM-flexible genome editing with an engineered chimeric Cas9.
Human Skeletal myopathy myosin mutations disrupt myosin head sequestration.
The Plateau in Muscle Growth with Resistance Training: An Exploration of Possible Mechanisms.
International society of sports nutrition position stand: essential amino acid supplementation on skeletal muscle and Performance.
Innovative Strategies of Reprogramming Immune System Cells by Targeting CRISPR/Cas9-Based Genome-Editing Tools: A New Era of Cancer Management.
An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

Physiol Rep. 2023 Oct;11(19): e15798

Influence of sexual dimorphism on satellite cell regulation and inflammatory response during skeletal muscle regeneration.

Charline Jomard, Julien Gondin.

  After injury, skeletal muscle regenerates thanks to the key role of satellite cells (SC). The regeneration process is supported and coordinated by other cell types among which immune cells. Among the mechanisms involved in skeletal muscle regeneration, a sexual dimorphism, involving sex hormones and more particularly estrogens, has been suggested. However, the role of sexual dimorphism on skeletal muscle regeneration is not fully understood, likely to the use of various experimental settings in both animals and human. This review aims at addressing how sex and estrogens regulate both the SC and the inflammatory response during skeletal muscle regeneration by considering the different experimental designs used in both animal models (i.e., ovarian hormone deficiency, estrogen replacement or supplementation, treatments with estrogen receptors agonists/antagonists and models knockout for estrogen receptors) and human (hormone therapy replacement, pre vs. postmenopausal, menstrual cycle variation…).

Keywords:  estrogens; inflammation; satellite cells; skeletal muscle

DOI:  https://doi.org/10.14814/phy2.15798
Dev Biol. 2023 Sep 29. pii: S0012-1606(23)00166-5. [Epub ahead of print]

Tenascin-C-enriched regeneration-specific extracellular matrix guarantees superior muscle regeneration in Ambystoma mexicanum.

Ayaka Ohashi, Suzuno Terai, Saya Furukawa, Sakiya Yamamoto, Rena Kashimoto, Akira Satoh.

  Severe muscle injury causes distress and difficulty in humans. Studying the high regenerative ability of the axolotls may provide hints for the development of an effective treatment for severe injuries to muscle tissue. Here, we examined the regenerative process in response to a muscle injury in axolotls. We found that axolotls are capable of complete regeneration in response to a partial muscle resection called volumetric muscle loss (VML), which mammals cannot perfectly regenerate. We investigated the mechanisms underlying this high regenerative capacity in response to VML, focusing on the migration of muscle satellite cells and the extracellular matrix (ECM) formed during VML injury. Axolotls form tenascin-C (TN-C)-enriched ECM after VML injury. This TN-C-enriched ECM promotes the satellite cell migration. We confirmed the importance of TN-C in successful axolotl muscle regeneration by creating TN-C mutant animals. Our results suggest that the maintenance of a TN-C-enriched ECM environment after muscle injury promotes the release of muscle satellite cells and supports eventually high muscle regenerative capacity. In the future, better muscle regeneration may be achieved in mammals through the maintenance of TN-C expression.

Keywords:  Axolotl; Muscle; Tenascin-C (TN-C); Volumetric muscle loss (VML)

DOI:  https://doi.org/10.1016/j.ydbio.2023.09.012
Nat Commun. 2023 09 30. 14(1): 6116

Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9.

Marco Thürkauf, Shuo Lin, Filippo Oliveri, Dirk Grimm, Randall J Platt, Markus A Rüegg.

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

DOI: https://doi.org/10.1038/s41467-023-41769-7
Arch Microbiol Immunol. 2023 ;7(3): 178-187

Automated Image Analysis Pipeline Development to Monitor Disease Progression in Muscular Dystrophy Using Cell Profiler.

Alexandra Brown, Brooklyn Morris, John Karanja Kamau, Abdullah A Alshudukhi, Abdulrahman Jama, Hongmei Ren.

  Muscular dystrophies are inherited disorders that are characterized by progressive muscle degeneration. These disorders are caused by mutations in the genes encoding structural elements within the muscle, which leads to increased vulnerability to mechanical stress and sarcolemma damage. Although myofibers have the capacity to regenerate, the newly formed myofibers still harbor genetic mutation, which induces continuous cycles of muscle fiber death and regeneration. This repeated cycling is accompanied by an inflammatory response which eventually provokes excessive fibrotic deposition. The histopathological changes in skeletal muscle tissue are central to the disease pathogenesis. Analysis of muscle histopathology is the gold standard for monitoring muscle health and disease progression. However, manual, or semi-manual quantification methods, are not only immensely tedious but can be subjective. Here, we present four image analysis pipelines built in CellProfiler which enable users without a background in computer vision or programming to quantitatively analyze biological images. These image analysis pipelines are designed to quantify skeletal muscle histopathological staining for membrane damage, the abundance and size distribution of regenerating muscle fibers, inflammation via quantification of CD68+ M1 macrophages, and collagen deposition. Additionally, we discuss methods to address common errors associated with the quantification of microscopy images. These automated tools can not only improve workflow efficiency but can provide a better understanding of the histopathological progression of muscular dystrophy.

Keywords:  Automated Histopathology Analysis; CellProfiler; Evan’s Blue Dye; Fibrosis; Muscle Regeneration; Muscular Dystrophy

DOI:  https://doi.org/10.26502/ami.936500115
Am J Physiol Cell Physiol. 2023 Oct 02.

The role of fibrosis in the pathophysiology of muscular dystrophy.

Farnaz Mogharehabed, Michael Paul Czubryt.

  Muscular dystrophy exerts significant and dramatic impacts on affected patients, including progressive muscle wasting leading to lung and heart failure, and resulting in severely curtailed lifespan. While the focus for many years has been on the dysfunction induced by loss of function of dystrophin or related components of the striated muscle costamere, recent studies have demonstrated that accompanying pathologies, particularly muscle fibrosis, also contribute adversely to patient outcomes. A significant body of research has now shown that therapeutically targeting these accompanying pathologies via their underlying molecular mechanisms may provide novel approaches to patient management that can complement the current standard of care. In this review, we discuss the interplay between muscle fibrosis and muscular dystrophy pathology. A better understanding of these processes will result in improved patient care options, restoration of muscle function, and reduced patient morbidity and mortality.

Keywords:  Becker muscular dystrophy; Duchenne muscular dystrophy; cellular signaling; fibrosis; therapy

DOI:  https://doi.org/10.1152/ajpcell.00196.2023
ACS Nano. 2023 Oct 05.

Nanotopographical Cues Tune the Therapeutic Potential of Extracellular Vesicles for the Treatment of Aged Skeletal Muscle Injuries.

Kai Wang, Nolan Frey, Andres Garcia, Kun Man, Yong Yang, Alice Gualerzi, Zachary J Clemens, Marzia Bedoni, Philip R LeDuc, Fabrisia Ambrosio.

  Skeletal muscle regeneration relies on the tightly temporally regulated lineage progression of muscle stem/progenitor cells (MPCs) from activation to proliferation and, finally, differentiation. However, with aging, MPC lineage progression is disrupted and delayed, ultimately causing impaired muscle regeneration. Extracellular vesicles (EVs) have attracted broad attention as next-generation therapeutics for promoting tissue regeneration. As a next step toward clinical translation, strategies to manipulate EV effects on downstream cellular targets are needed. Here, we developed an engineering strategy to tune the therapeutic potential of EVs using nanotopographical cues. We found that EVs released by young MPCs cultured on flat substrates (fEVs) promoted the proliferation of aged MPCs while EVs released by MPCs cultured on nanogratings (nEVs) promoted myogenic differentiation. We then employed a bioengineered 3D muscle aging model to optimize the administration protocol and test the therapeutic potential of fEVs and nEVs in a high-throughput manner. We found that the sequential administration first of fEVs during the phase of MPC proliferative expansion (i.e., 1 day after injury) followed by nEV administration at the stage of MPC differentiation (i.e., 3 days after injury) enhanced aged muscle regeneration to a significantly greater extent than fEVs and nEVs delivered either in isolation or mixed. The beneficial effects of the sequential EV treatment strategy were further validated in vivo, as evidenced by increased myofiber size and improved functional recovery. Collectively, our study demonstrates the ability of topographical cues to tune EV therapeutic potential and highlights the importance of optimizing the EV administration strategy to accelerate aged skeletal muscle regeneration.

Keywords:  aging; cell-free therapy; exosomes; nanotopography; skeletal muscle repair

DOI:  https://doi.org/10.1021/acsnano.3c02269
Physiol Res. 2023 Aug 31. 72(4): 465-473

Activation of Lactate Receptor Positively Regulates Skeletal Muscle Mass in Mice.

Y Ohno, M Nakatani, T Ito, Y Matsui, K Ando, Y Suda, K Ohashi, S Yokoyama, K Goto.

G protein-coupled receptor 81 (GPR81), a selective receptor for lactate, expresses in skeletal muscle cells, but the physiological role of GPR81 in skeletal muscle has not been fully elucidated. As it has been reported that the lactate administration induces muscle hypertrophy, the stimulation of GPR81 has been suggested to mediate muscle hypertrophy. To clarify the contribution of GPR81 activation in skeletal muscle hypertrophy, in the present study, we investigated the effect of GPR81 agonist administration on skeletal muscle mass in mice. Male C57BL/6J mice were randomly divided into control group and GPR81 agonist-administered group that received oral administration of the specific GPR81 agonist 3-Chloro-5-hydroxybenzoic acid (CHBA). In both fast-twitch plantaris and slow-twitch soleus muscles of mice, the protein expression of GPR81 was observed. Oral administration of CHBA to mice significantly increased absolute muscle weight and muscle weight relative to body weight in the two muscles. Moreover, both absolute and relative muscle protein content in the two muscles were significantly increased by CHBA administration. CHBA administration also significantly upregulated the phosphorylation level of p42/44 extracellular signal-regulated kinase-1/2 (ERK1/2) and p90 ribosomal S6 kinase (p90RSK). These observations suggest that activation of GRP81 stimulates increased the mass of two types of skeletal muscle in mice in vivo. Lactate receptor GPR81 may positively affect skeletal muscle mass through activation of ERK pathway.
Nat Commun. 2023 10 04. 14(1): 6175

PAM-flexible genome editing with an engineered chimeric Cas9.

Lin Zhao, Sabrina R T Koseki, Rachel A Silverstein, Nadia Amrani, Christina Peng, Christian Kramme, Natasha Savic, Martin Pacesa, Tomás C Rodríguez, Teodora Stan, Emma Tysinger, Lauren Hong, Vivian Yudistyra, Manvitha R Ponnapati, Joseph M Jacobson, George M Church, Noah Jakimo, Ray Truant, Martin Jinek, Benjamin P Kleinstiver, Erik J Sontheimer, Pranam Chatterjee.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

DOI: https://doi.org/10.1038/s41467-023-41829-y
JCI Insight. 2023 Oct 03. pii: e172322. [Epub ahead of print]

Human Skeletal myopathy myosin mutations disrupt myosin head sequestration.

Glenn Carrington, Hoi Ting Abbi Hau, Sarah Kosta, Hannah F Dugdale, Francesco Muntoni, Adele D'Amico, Peter Y K Van den Bergh, Norma B Romero, Edoardo Malfatti, Juan J Vilchez, Anders Oldfors, Sander Pajusalu, Katrin Õunap, Marta Giralt-Pujol, Edmar Zanoteli, Kenneth S Campbell, Hiroyuki Iwamoto, Michelle Peckham, Julien Ochala.

  Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle, and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyse the effects of common MYH7 and MYH2 mutations in the light meromyosin region of myosin (LMM). Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in-silico modelling showed that myosin coiled-coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients, and Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with X-ray diffraction measurements to estimate myosin head order we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofibre mechanics experiments to investigate contractile function showed myofibre contractility was not affected. These findings indicate that the structural remodelling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.

Keywords:  Genetic diseases; Muscle Biology; Neuromuscular disease

DOI:  https://doi.org/10.1172/jci.insight.172322
Sports Med. 2023 Oct 03.

The Plateau in Muscle Growth with Resistance Training: An Exploration of Possible Mechanisms.

Ryo Kataoka, William B Hammert, Yujiro Yamada, Jun Seob Song, Aldo Seffrin, Anna Kang, Robert W Spitz, Vickie Wong, Jeremy P Loenneke.

It is hypothesized that there is likely a finite ability for muscular adaptation. While it is difficult to distinguish between a true plateau following a long-term training period and short-term stalling in muscle growth, a plateau in muscle growth has been attributed to reaching a genetic potential, with limited discussion on what might physiologically contribute to this muscle growth plateau. The present paper explores potential physiological factors that may drive the decline in muscle growth after prolonged resistance training. Overall, with chronic training, the anabolic signaling pathways may become more refractory to loading. While measures of anabolic markers may have some predictive capabilities regarding muscle growth adaptation, they do not always demonstrate a clear connection. Catabolic processes may also constrain the ability to achieve further muscle growth, which is influenced by energy balance. Although speculative, muscle cells may also possess cell scaling mechanisms that sense and regulate their own size, along with molecular brakes that hinder growth rate over time. When considering muscle growth over the lifespan, there comes a point when the anabolic response is attenuated by aging, regardless of whether or not individuals approach their muscle growth potential. Our goal is that the current review opens avenues for future experimental studies to further elucidate potential mechanisms to explain why muscle growth may plateau.

DOI: https://doi.org/10.1007/s40279-023-01932-y
J Int Soc Sports Nutr. 2023 Dec;20(1): 2263409

International society of sports nutrition position stand: essential amino acid supplementation on skeletal muscle and Performance.

Arny A Ferrando, Robert R Wolfe, Katie R Hirsch, David D Church, Shiloah A Kviatkovsky, Michael D Roberts, Jeffrey R Stout, Drew E Gonzalez, Ryan J Sowinski, Richard B Kreider, Chad M Kerksick, Nicholas A Burd, Stefan M Pasiakos, Michael J Ormsbee, Shawn M Arent, Paul J Arciero, Bill I Campbell, Trisha A VanDusseldorp, Ralf Jager, Darryn S Willoughby, Douglas S Kalman, Jose Antonio.

  Position Statement: The International Society of Sports Nutrition (ISSN) presents this position based on a critical examination of literature surrounding the effects of essential amino acid (EAA) supplementation on skeletal muscle maintenance and performance. This position stand is intended to provide a scientific foundation to athletes, dietitians, trainers, and other practitioners as to the benefits of supplemental EAA in both healthy and resistant (aging/clinical) populations. EAAs are crucial components of protein intake in humans, as the body cannot synthesize them. The daily recommended intake (DRI) for protein was established to prevent deficiencies due to inadequate EAA consumption. The following conclusions represent the official position of the Society: 1. Initial studies on EAAs' effects on skeletal muscle highlight their primary role in stimulating muscle protein synthesis (MPS) and turnover. Protein turnover is critical for replacing degraded or damaged muscle proteins, laying the metabolic foundation for enhanced functional performance. Consequently, research has shifted to examine the effects of EAA supplementation - with and without the benefits of exercise - on skeletal muscle maintenance and performance. 2. Supplementation with free-form EAAs leads to a quick rise in peripheral EAA concentrations, which in turn stimulates MPS. 3. The safe upper limit of EAA intake (amount), without inborn metabolic disease, can easily accommodate additional supplementation. 4. At rest, stimulation of MPS occurs at relatively small dosages (1.5-3.0 g) and seems to plateau at around 15-18 g. 5. The MPS stimulation by EAAs does not require non-essential amino acids. 6. Free-form EAA ingestion stimulates MPS more than an equivalent amount of intact protein. 7. Repeated EAA-induced MPS stimulation throughout the day does not diminish the anabolic effect of meal intake. 8. Although direct comparisons of various formulas have yet to be investigated, aging requires a greater proportion of leucine to overcome the reduced muscle sensitivity known as "anabolic resistance." 9. Without exercise, EAA supplementation can enhance functional outcomes in anabolic-resistant populations. 10. EAA requirements rise in the face of caloric deficits. During caloric deficit, it's essential to meet whole-body EAA requirements to preserve anabolic sensitivity in skeletal muscle.

Keywords:  Protein; exercise

DOI:  https://doi.org/10.1080/15502783.2023.2263409
Int J Nanomedicine. 2023 ;18 5531-5559

Innovative Strategies of Reprogramming Immune System Cells by Targeting CRISPR/Cas9-Based Genome-Editing Tools: A New Era of Cancer Management.

Khaled S Allemailem, Mohammed A Alsahli, Ahmad Almatroudi, Faris Alrumaihi, Waleed Al Abdulmonem, Amira A Moawad, Wanian M Alwanian, Nahlah Makki Almansour, Arshad Husain Rahmani, Amjad Ali Khan.

  The recent developments in the study of clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system have revolutionized the art of genome-editing and its applications for cellular differentiation and immune response behavior. This technology has further helped in understanding the mysteries of cancer progression and possible designing of novel antitumor immunotherapies. CRISPR/Cas9-based genome-editing is now often used to engineer universal T-cells, equipped with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR). In addition, this technology is used in cytokine stimulation, antibody designing, natural killer (NK) cell transfer, and to overcome immune checkpoints. The innovative potential of CRISPR/Cas9 in preparing the building blocks of adoptive cell transfer (ACT) immunotherapy has opened a new window of antitumor immunotherapy and some of them have gained FDA approval. The manipulation of immunogenetic regulators has opened a new interface for designing, implementation and interpretation of CRISPR/Cas9-based screening in immuno-oncology. Several cancers like lymphoma, melanoma, lung, and liver malignancies have been treated with this strategy, once thought to be impossible. The safe and efficient delivery of CRISPR/Cas9 system within the immune cells for the genome-editing strategy is a challenging task which needs to be sorted out for efficient immunotherapy. Several targeting approaches like virus-mediated, electroporation, microinjection and nanoformulation-based methods have been used, but each procedure offers some limitations. Here, we elaborate the recent updates of cancer management through immunotherapy in partnership with CRISPR/Cas9 technology. Further, some innovative methods of targeting this genome-editing system within the immune system cells for reprogramming them, as a novel strategy of anticancer immunotherapy is elaborated. In addition, future prospects and clinical trials are also discussed.

Keywords:  CRISPR/Cas9; cancer immunotherapy; clinical study; immune response; molecular targeted therapy; nanotechnology; tumor microenvironment

DOI:  https://doi.org/10.2147/IJN.S424872
Cell. 2023 Sep 22. pii: S0092-8674(23)00963-7. [Epub ahead of print]

An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

Tomohiro Hino, Satoshi N Omura, Ryoya Nakagawa, Tomoki Togashi, Satoru N Takeda, Takafumi Hiramoto, Satoshi Tasaka, Hisato Hirano, Takeshi Tokuyama, Hideki Uosaki, Soh Ishiguro, Madina Kagieva, Hiroyuki Yamano, Yuki Ozaki, Daisuke Motooka, Hideto Mori, Yuhei Kirita, Yoshiaki Kise, Yuzuru Itoh, Satoaki Matoba, Hiroyuki Aburatani, Nozomu Yachie, Tautvydas Karvelis, Virginijus Siksnys, Tsukasa Ohmori, Atsushi Hoshino, Osamu Nureki.

  SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.

Keywords:  CRISPR-Cas; animal experiments; cryo-EM; deep mutational scanning; gene therapy; genome editing; iPS cells

DOI:  https://doi.org/10.1016/j.cell.2023.08.031