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BioRxiv : the Preprint Server For... Nov 2023Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Our previous work has shown cofilin () knockdown causes...
Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Our previous work has shown cofilin () knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by knockdown would impact other aspects of muscle development, and, thus, conducted an RNA sequencing analysis which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand our understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology.
PubMed: 38045306
DOI: 10.1101/2023.11.21.568166 -
Molecular Therapy : the Journal of the... Sep 2023Rhabdomyosarcoma is the most common pediatric soft tissue tumor, comprising two major subtypes: the PAX3/7-FOXO1 fusion-negative embryonal and the PAX3/7-FOXO1...
Rhabdomyosarcoma is the most common pediatric soft tissue tumor, comprising two major subtypes: the PAX3/7-FOXO1 fusion-negative embryonal and the PAX3/7-FOXO1 fusion-positive alveolar subtype. Here, we demonstrate that the expression levels of the transcriptional repressor TRPS1 are specifically enhanced in the embryonal subtype, resulting in impaired terminal myogenic differentiation and tumor growth. During normal myogenesis, expression levels of TRPS1 have to decrease to allow myogenic progression, as demonstrated by overexpression of TRPS1 in myoblasts impairing myotube formation. Consequentially, myogenic differentiation in embryonal rhabdomyosarcoma in vitro as well as in vivo can be achieved by reducing TRPS1 levels. Furthermore, we show that TRPS1 levels in RD cells, the bona fide model cell line for embryonal rhabdomyosarcoma, are regulated by miR-1 and that TRPS1 and MYOD1 share common genomic binding sites. The myogenin (MYOG) promoter is one of the critical targets of TRPS1 and MYOD1; we demonstrate that TRPS1 restricts MYOG expression and thereby inhibits terminal myogenic differentiation. Therefore, reduction of TRPS1 levels in embryonal rhabdomyosarcoma might be a therapeutic approach to drive embryonal rhabdomyosarcoma cells into myogenic differentiation, thereby generating postmitotic myotubes.
Topics: Humans; Child; Rhabdomyosarcoma, Embryonal; Myogenin; Cell Differentiation; MicroRNAs; Muscle Development; Cell Line, Tumor; Repressor Proteins
PubMed: 37452493
DOI: 10.1016/j.ymthe.2023.07.003 -
Development (Cambridge, England) Jul 2023The earliest skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are often identified by factors expressed by a diverse number of...
The earliest skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are often identified by factors expressed by a diverse number of progenitors. An early transcriptional checkpoint that defines myogenic commitment could improve hPSC differentiation to skeletal muscle. Analysis of several myogenic factors in human embryos and early hPSC differentiations found SIX1+PAX3+ co-expression was most indictive of myogenesis. Using dCas9-KRAB hPSCs, we demonstrate that early inhibition of SIX1 alone significantly decreased PAX3 expression, reduced PAX7+ SMPCs, and myotubes later in differentiation. Emergence of SIX1+PAX3+ precursors can be improved by manipulating seeding density, monitoring metabolic secretion and altering the concentration of CHIR99021. These modifications resulted in the co-emergence of hPSC-derived sclerotome, cardiac and neural crest that we hypothesized enhanced hPSC myogenic differentiation. Inhibition of non-myogenic lineages modulated PAX3 independent of SIX1. To better understand SIX1 expression, we compared directed differentiations to fetal progenitors and adult satellite cells by RNA-seq. Although SIX1 continued to be expressed across human development, SIX1 co-factor expression was dependent on developmental timing. We provide a resource to enable efficient derivation of skeletal muscle from hPSCs.
Topics: Adult; Humans; PAX3 Transcription Factor; Pluripotent Stem Cells; Cell Differentiation; Muscle, Skeletal; Muscle Development; PAX7 Transcription Factor; Homeodomain Proteins
PubMed: 37366057
DOI: 10.1242/dev.201509 -
Journal of Cachexia, Sarcopenia and... Feb 2024Skeletal muscle atrophy, particularly ageing-related muscular atrophy such as sarcopenia, is a significant health concern. Despite its prevalence, the underlying...
BACKGROUND
Skeletal muscle atrophy, particularly ageing-related muscular atrophy such as sarcopenia, is a significant health concern. Despite its prevalence, the underlying mechanisms remain poorly understood, and specific approved medications are currently unavailable. Deleted in breast cancer 1 (DBC1) is a well-known regulator of senescence, metabolism or apoptosis. Recent reports suggest that DBC1 may also potentially regulate muscle function, as mice lacking DBC1 exhibit weakness and limpness. However, the function of DBC1 in skeletal muscle and its associated molecular mechanisms remain unknown, thus prompting the focus of this study.
METHODS
Tibialis anterior (TA) muscle-specific DBC1 knockdown C57BL/6J male mice were generated through a single injection of 2.00 E + 11 vg of adeno-associated virus 9 delivering single-guide RNA for DBC1. Grip strength and endurance were assessed 2 months later, followed by skeletal muscle harvest. Muscle atrophy model was generated by cast immobilization of the mouse hindlimb for 2 weeks. Molecular markers of atrophy were probed in muscles upon termination. Cardiotoxin (CTX) was injected in TA muscles of DBC1 knockdown mice, and muscle regeneration was assessed by immunohistochemistry, quantitative PCR and western blotting. DBC1 knockdown C2C12 cells and myotubes were investigated using immunofluorescence staining, Seahorse, immunohistology, fluorescence-activated cell sorting and RNA-sequencing analyses.
RESULTS
DBC1 knockdown in skeletal muscle of young mice led to signatures of muscle atrophy, including a 28% reduction in muscle grip force (P = 0.023), a 54.4% reduction in running distance (P = 0.002), a 14.3% reduction in muscle mass (P = 0.007) and significantly smaller myofibre cross-sectional areas (P < 0.0001). DBC1 levels decrease in age-related or limb immobilization-induced atrophic mouse muscles and overexpress DBC1-attenuated atrophic phenotypes in these mice. Muscle regeneration was hampered in mice with CTX-induced muscle injury by DBC1 knockdown, as evidenced by reductions in myofibre cross-sectional areas of regenerating myofibres with centralized nuclei (P < 0.0001), percentages of MyoG nuclei (P < 0.0001) and fusion index (P < 0.0001). DBC1 transcriptionally regulated mouse double minute 2 (MDM2), which mediated ubiquitination and degradation of forkhead box O3 (FOXO3). Increased FOXO3 proteins hampered myogenesis in DBC1 knockdown satellite cells by compromising around 50% of mitochondrial functions (P < 0.001) and exacerbated atrophy in DBC1 knockdown myofibres by activating the ubiquitin-proteasome and autophagy-lysosome pathways.
CONCLUSIONS
DBC1 is essential in maintaining skeletal muscle integrity by protecting against myofibres wasting and enhancing muscle regeneration via FOXO3. This research highlights the significance of DBC1 for healthy skeletal muscle function and its connection to muscular atrophy.
Topics: Animals; Male; Mice; Cachexia; Mice, Inbred C57BL; Muscle Development; Muscle, Skeletal; Muscular Atrophy; RNA, Guide, CRISPR-Cas Systems
PubMed: 38062876
DOI: 10.1002/jcsm.13398 -
Biochemical and Biophysical Research... Nov 2023Skeletal muscle myogenesis represents one of the most intensively and extensively examined systems of cell differentiation, tissue formation, and regeneration. Muscle... (Review)
Review
Skeletal muscle myogenesis represents one of the most intensively and extensively examined systems of cell differentiation, tissue formation, and regeneration. Muscle regeneration provides an in vivo model system of postnatal myogenesis. It comprises multiple steps including muscle stem cell (or satellite cell) quiescence, activation, migration, myogenic determination, myoblast proliferation, myocyte differentiation, myofiber maturation, and hypertrophy. A variety of extracellular signaling and subsequent intracellular signal transduction pathways or networks govern the individual steps of postnatal myogenesis. Among them, MAPK pathways (the ERK, JNK, p38 MAPK, and ERK5 pathways) and PI3K-Akt signaling regulate multiple steps of myogenesis. Ca, cytokine, and Wnt signaling also participate in several myogenesis steps. These signaling pathways often control cell cycle regulatory proteins or the muscle-specific MyoD family and the MEF2 family of transcription factors. This article comprehensively reviews molecular mechanisms of the individual steps of postnatal skeletal muscle myogenesis by focusing on signal transduction pathways or networks. Nevertheless, no or only a partial signaling molecules or pathways have been identified in some responses during myogenesis. The elucidation of these unidentified signaling molecules and pathways leads to an extensive understanding of the molecular mechanisms of myogenesis.
Topics: Cell Differentiation; Mitogen-Activated Protein Kinases; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 37826946
DOI: 10.1016/j.bbrc.2023.09.048 -
IScience Mar 2024Fat infiltration in skeletal muscle (also known as myosteatosis) is now recognized as a distinct disease from sarcopenia and is directly related to declining muscle... (Review)
Review
Fat infiltration in skeletal muscle (also known as myosteatosis) is now recognized as a distinct disease from sarcopenia and is directly related to declining muscle capacity. Hence, understanding the origins and regulatory mechanisms of fat infiltration is vital for maintaining skeletal muscle development and improving human health. In this article, we summarized the triggering factors such as aging, metabolic diseases and metabolic syndromes, nonmetabolic diseases, and muscle injury that all induce fat infiltration in skeletal muscle. We discussed recent advances on the cellular origins of fat infiltration and found several cell types including myogenic cells and non-myogenic cells that contribute to myosteatosis. Furthermore, we reviewed the molecular regulatory mechanism, detection methods, and intervention strategies of fat infiltration in skeletal muscle. Based on the current findings, our review will provide new insight into regulating function and lipid metabolism of skeletal muscle and treating muscle-related diseases.
PubMed: 38433917
DOI: 10.1016/j.isci.2024.109221 -
ELife Jul 2023Ubiquitin-proteasome system (UPS) dysfunction is associated with the pathology of a wide range of human diseases, including myopathies and muscular atrophy. However, the...
Ubiquitin-proteasome system (UPS) dysfunction is associated with the pathology of a wide range of human diseases, including myopathies and muscular atrophy. However, the mechanistic understanding of specific components of the regulation of protein turnover during development and disease progression in skeletal muscle is unclear. Mutations in , an E3 ubiquitin ligase cullin3 (CUL3) substrate-specific adapter protein, result in severe congenital nemaline myopathy, but the events that initiate the pathology and the mechanism through which it becomes pervasive remain poorly understood. To characterize the KLHL40-regulated ubiquitin-modified proteome during skeletal muscle development and disease onset, we used global, quantitative mass spectrometry-based ubiquitylome and global proteome analyses of mutant zebrafish during disease progression. Global proteomics during skeletal muscle development revealed extensive remodeling of functional modules linked with sarcomere formation, energy, biosynthetic metabolic processes, and vesicle trafficking. Combined analysis of mutant muscle proteome and ubiquitylome identified thin filament proteins, metabolic enzymes, and ER-Golgi vesicle trafficking pathway proteins regulated by ubiquitylation during muscle development. Our studies identified a role for KLHL40 as a regulator of ER-Golgi anterograde trafficking through ubiquitin-mediated protein degradation of secretion-associated Ras-related GTPase1a (Sar1a). In KLHL40-deficient muscle, defects in ER exit site vesicle formation and downstream transport of extracellular cargo proteins result in structural and functional abnormalities. Our work reveals that the muscle proteome is dynamically fine-tuned by ubiquitylation to regulate skeletal muscle development and uncovers new disease mechanisms for therapeutic development in patients.
Topics: Animals; Humans; Muscle Proteins; Zebrafish; Proteome; Muscle, Skeletal; Ubiquitination; Sarcomeres; Ubiquitin; Endoplasmic Reticulum; Muscle Development; Disease Progression
PubMed: 37432316
DOI: 10.7554/eLife.81966 -
International Journal of Molecular... Aug 2023Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration....
Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration. The genetic defect results in a reduced number of self-renewing muscle stem cells (MuSCs) and an impairment of their activation and differentiation, which lead to the exhaustion of skeletal muscle regeneration potential and muscle replacement by fibrotic and fatty tissue. In this study, we focused on an unexplored strategy to improve MuSC function and to preserve their niche based on the regenerative properties of mesenchymal stromal cells from the amniotic membrane (hAMSCs), that are multipotent cells recognized to have a role in tissue repair in different disease models. We demonstrate that the hAMSC secretome (CM hAMSC) and extracellular vesicles (EVs) isolated thereof directly stimulate the in vitro proliferation and differentiation of human myoblasts and mouse MuSC from dystrophic muscles. Furthermore, we demonstrate that hAMSC secreted factors modulate the muscle stem cell niche in dystrophic--mice. Interestingly, local injection of EV hAMSC in muscles correlated with an increase in the number of activated Pax7+/Ki67+ MuSCs and in new fiber formation. EV hAMSCs also significantly reduced muscle collagen deposition, thus counteracting fibrosis and MuSCs exhaustion, two hallmarks of DMD. Herein for the first time we demonstrate that CM hAMSC and EVs derived thereof promote muscle regeneration by supporting proliferation and differentiation of resident muscle stem cells. These results pave the way for the development of a novel treatment to counteract DMD progression by reducing fibrosis and enhancing myogenesis in dystrophic muscles.
Topics: Humans; Animals; Mice; Mice, Inbred mdx; Amnion; Muscle, Skeletal; Dystrophin; Muscular Dystrophy, Duchenne; Satellite Cells, Skeletal Muscle; Mesenchymal Stem Cells; Extracellular Vesicles; Disease Models, Animal
PubMed: 37569832
DOI: 10.3390/ijms241512457 -
Frontiers in Neuroendocrinology Oct 2023Androgens' pleiotropic actions in promoting sex differences present not only a challenge to providing a comprehensive account of their function, but also an opportunity... (Review)
Review
Androgens' pleiotropic actions in promoting sex differences present not only a challenge to providing a comprehensive account of their function, but also an opportunity to gain insights by comparing androgenic actions across organ systems. Although often overlooked by neuroscientists, skeletal muscle is another androgen-responsive organ system which shares with the nervous system properties of electrochemical excitability, behavioral relevance, and remarkable capacity for adaptive plasticity. Here we review androgenic regulation of mitogenic plasticity in skeletal muscle with the goal of identifying areas of interest to those researching androgenic mechanisms mediating sexual differentiation of neurogenesis. We use an organizational-activational framework to relate broad areas of similarity and difference between androgen effects on mitogenesis in muscle and brain throughout the lifespan, from early organogenesis, through pubertal organization, adult activation, and aging. The focus of the review is androgenic regulation of muscle-specific stem cells (satellite cells), which share with neural stem cells essential functions in development, plasticity, and repair, albeit with distinct, muscle-specific features. Also considered are areas of paracrine and endocrine interaction between androgen action on muscle and nervous system, including mediation of neural plasticity of innervating and distal neural populations by muscle-produced trophic factors.
Topics: Female; Male; Humans; Androgens; Receptors, Androgen; Longevity; Neurogenesis; Muscle, Skeletal; Muscle Development
PubMed: 37669703
DOI: 10.1016/j.yfrne.2023.101101 -
Seminars in Cell & Developmental Biology Jul 2023Mitochondria play a major role in apoptotic signaling. In addition to its role in eliminating dysfunctional cells, mitochondrial apoptotic signaling is implicated as a... (Review)
Review
Mitochondria play a major role in apoptotic signaling. In addition to its role in eliminating dysfunctional cells, mitochondrial apoptotic signaling is implicated as a key component of myogenic differentiation and skeletal muscle atrophy. For example, the activation of cysteine-aspartic proteases (caspases; CASP's) can aid in the initial remodeling stages of myogenic differentiation by cleaving protein kinases, transcription factors, and cytoskeletal proteins. Precise regulation of these signals is needed to prevent excessive cell disassemble and subsequent cell death. During skeletal muscle atrophy, the activation of CASP's and mitochondrial derived nucleases participate in myonuclear fragmentation, a potential loss of myonuclei, and cleavage of contractile structures within skeletal muscle. The B cell leukemia/lymphoma 2 (BCL2) family of proteins play a significant role in regulating myogenesis and skeletal muscle atrophy by governing the initiating steps of mitochondrial apoptotic signaling. This review discusses the role of mitochondrial apoptotic signaling in skeletal muscle remodeling during myogenic differentiation and skeletal muscle pathological states, including aging, disuse, and muscular dystrophy.
Topics: Humans; Apoptosis; Caspases; Muscle Development; Muscle, Skeletal; Muscular Atrophy; Mitochondria, Muscle
PubMed: 35241367
DOI: 10.1016/j.semcdb.2022.01.011