Skeletal Muscle最新文献

Background: Dystrophin mRNA is produced from a very large genetic locus and transcription of a single mRNA requires approximately 16 h. This prolonged interval between initiation and completion results in unusual transcriptional behaviour: in skeletal muscle, myonuclei express dystrophin continuously and robustly, yet degrade mature transcripts shortly after completion. Consequently, most dystrophin mRNA is nascent, not mature. This implies expression is principally controlled post-transcriptionally, a mechanism that circumvents transcriptional delay, allowing rapid responses to change in demand. Dystrophin protein is however highly stable, with slow turnover: in healthy muscle, despite constant production of dystrophin mRNA, demand is low and the need for responsive expression is minimal. We reasoned this system instead exists to control dystrophin expression during rare periods of elevated but changing demand, such as during muscle development or repair, when newly formed fibres must establish sarcolemmal dystrophin rapidly.

Methods: We assessed dystrophin mRNA (both nascent and mature) and dystrophin protein in regenerating skeletal muscle following injury, using a combination of qPCR, immunofluorescence and in-situ hybridisation to determine timing and location of expression during the repair process.

Results: We reveal a complex program that suggests control at multiple levels: nascent transcription is detectable even prior to overt myoblast fusion, suggesting cells 'pay in advance' to minimise subsequent delay. During myotube differentiation and maturation, when sarcolemmal demands are high, initiation increases only modestly while mature transcript stability increases markedly to generate high numbers of mature dystrophin transcripts, a state that persists until repair is complete, when oversupply and degradation resumes.

Conclusion: Our data demonstrate that dystrophin mRNA is indeed chiefly controlled by turnover, not initiation: degradation consequently represents a potential therapeutic target for maximising efficacy of even modest dystrophin restoration.

Duchenne muscular dystrophy (DMD) is a severe, progressive genetic disorder caused by mutations in the DMD gene, resulting in the absence of dystrophin-a key structural protein at the sarcolemma. As the disease progresses, cardiac involvement becomes a leading cause of morbidity and mortality. By adolescence or early adulthood, many patients develop dilated cardiomyopathy and arrhythmias. Like skeletal muscle, cardiac muscle in DMD patients lacks dystrophin and undergoes similar degenerative changes, ultimately leading to ventricular dilation, systolic dysfunction, and heart failure. Early detection and proactive management of cardiac dysfunction are essential for optimizing outcomes. Despite significant advances and decades of research, a definitive cure for DMD remains elusive. In recognition of World Duchenne Awareness Day, this review highlights current and emerging therapeutic strategies with the potential to transform cardiac care in DMD and improve the lives of those affected.

Background: The development of functional muscles in Drosophila melanogaster relies on precise spatial and temporal transcriptional control, orchestrated by complex gene regulatory networks. Central to this regulation are cis-regulatory modules (CRMs), which integrate inputs from transcription factors to fine-tune gene expression during myogenesis. In this study, we investigate the transcriptional regulation of the LIM-homeodomain transcription factor Tup (Tailup/Islet-1), a key regulator of dorsal muscle development.

Methods: Using a combination of CRISPR-Cas9-mediated deletion and transcriptional analyses, we examined the role of multiple CRMs in regulating tup expression.

Results: We demonstrate that tup expression is controlled by multiple CRMs that function redundantly to maintain robust tup transcription in dorsal muscles. These mesodermal tup CRMs act sequentially and differentially during the development of dorsal muscles and other tissues, including heart cells and alary muscles. We show that activity of the two late-acting CRMs govern late-phase tup expression through positive autoregulation, whereas an early enhancer initiates transcription independently. Deletion of both late-acting CRMs results in muscle identity shifts and defective muscle patterning. Detailed morphological analyses reveal muscle misalignments at intersegmental borders.

Conclusions: Our findings underscore the importance of CRM-mediated autoregulation and redundancy in ensuring robust and precise tup expression during muscle development. These results provide insights into how multiple CRMs coordinate gene regulation to ensure proper muscle identity and function.

Double Homeobox 4 (DUX4) is a potent transcription factor encoded by a retrogene mapped in D4Z4 repeated elements on chromosome 4q35. DUX4 has emerged as pivotal in the pathomechanisms of facioscapulohumeral muscular dystrophy (FSHD), a relatively common hereditary muscle wasting condition, although classified as a rare disease. DUX4 contributes to zygote genome activation before its expression is repressed in most somatic tissues through epigenetic mechanisms, including DNA methylation and chromatin modifications. In FSHD, inappropriate activation of DUX4 expression is driven by a complex interplay of genomic and epigenetic alterations. The ectopic presence of DUX4 in skeletal muscle cells activates genes, viral elements and pathways that are typical of very early embryonic development, disturbing cell function and ultimately contributing to muscle weakness and wasting. This review first traces the history of DUX4, from the FSHD genetic linkage studies in the early 1990s, through to identification and characterization of the DUX4 gene in 1999. We then discuss the seminal studies that showed how and why DUX4 is expressed in FSHD and the effects of this ectopic expression in muscle, notably cellular toxicity. Other pathological roles of DUX4, such as participation in cancer and viral infection, are also highlighted. Maintenance of DUX4 in the genome was explained by discovery of the function of DUX4 in zygotic genome activation to institute the totipotent cells of the embryo. Thus, we encompass the gradual transition of DUX4 over the past 25 years from being considered a pseudogene in "junk DNA" to becoming central to understanding the molecular pathogenesis of FSHD and the primary focus for FSHD therapeutics.

Background: Pathogenic variants in RYR1 cause a spectrum of rare congenital myopathies associated with intracellular calcium dysregulation. Glutathione redox imbalance has been reported in several Ryr1 disease model systems and clinical studies. NAD⁺ and NADP are essential cofactors in cellular metabolism and redox homeostasis. NAD⁺ deficiency has been associated with skeletal muscle bioenergetic deficits in mitochondrial myopathy and sarcopenia.

Methods: Using a new colorimetric assay and large control dataset (n = 299), we assessed redox balance (glutathione, NAD⁺, and NADP) in whole blood from 28 RYR1-RM affected individuals (NCT02362425). Analyses were expanded to human skeletal muscle (n = 4), primary myotube cultures (n = 5), and whole blood and skeletal muscle specimens from Ryr1 Y524S mice. The in vitro effects of nicotinamide riboside (NR) on cellular NAD⁺ content and mitochondrial respirometry were also tested.

Results: At baseline, a majority of affected individuals exhibited systemic NAD⁺ deficiency (19/28 [68%] < 21 µM) and increased NADPH concentrations (22/26 [85%] > 1.6 µM). When compared to controls, decreased NAD⁺/NADH and NADP/NADPH ratios were observed in 9/28 and 23/26 individuals, respectively. In patient-derived myotube cultures (n = 5), NR appeared to increase cellular NAD⁺ concentrations in a dose and time-dependent manner at 72-h only and favorably modified maximal respiration and ATP production. Average whole blood GSH/GSSG ratio was comparable between groups, and redox imbalance was not observed in Ryr1 Y524S specimens.

Conclusions: NAD⁺ and NADP dyshomeostasis was identified in a subset of RYR1-RM affected individuals. Further experiments are warranted to confirm if NAD⁺ repletion could be an attractive therapeutic approach given the favorable outcomes reported in other neuromuscular disorders.

Background: Tongue muscles contain a much greater number of residual adipocytes than other muscles do, which makes them susceptible to obesity-induced muscle fat remodeling. Tongue fat remodeling leads to obesity-induced obstructive sleep apnea (OSA), which is a common sleep disorder characterized by repeated episodes of upper airway collapse during sleep, resulting in fragmented sleep and oxygen deprivation. Although the obstructive role of fat remodeling in tongue muscles for OSA has been confirmed, the cellular and molecular mechanisms regulating fat remodeling in tongue and its impact on tongue muscles have not been well explored.

Methods: To study the impact of obesity on adipocytes and neuromuscular junctions (NMJs) in tongue muscles, we used a high-fat diet (HFD)-induced obese preclinical model.

Results: The results demonstrated hypertrophy of adipocytes and denervation at NMJs in tongue muscles by a HFD. Mechanistically, we revealed that a HFD repressed the expression of growth differentiation factor 10 (GDF10), which is expressed mainly in fibroadipogenic progenitors (FAPs) in skeletal muscles, repressing adipogenesis and maintaining the integrity of neuromuscular connections. We identified sex differences and muscle specificity of Gdf10 mRNA expression in FAPs. To understand how a HFD significantly reduces the level of Gdf10 mRNA expression in FAPs of the tongue, we investigated the epigenetic regulation of Gdf10. We found that a HFD increases miR-144-3p in tongue FAPs, which interferes with Gdf10 mRNA expression and induces adipogenesis. GDF10 overexpression by viral delivery effectively prevented HFD-induced fat remodeling of tongue and limb muscles.

Conclusion: These findings provide important insight into the role of FAP-derived GDF10 in the interplay between fat contents and tongue muscles in response to obesity and suggest potential therapeutic targets for OSA treatment.

Background: The RNA-binding protein hnRNPK is essential for animal growth and development, with a particular emphasis in myogenesis. Despite its importance, the precise mechanisms by which hnRNPK influences skeletal muscle physiology and development remain inadequately characterized.

Methods: To explore its regulatory function, we developed a Myf5-cre-mediated myoblast precursor-specific knockout mouse model (Hnrnpk mKO), an Acta1-CreEsr1-mediated myofiber-specific inducible knockout mouse model (Hnrnpk aKO), and an AAV9-mediated skeletal muscle-specific overexpression mouse model (AAV9-hnRNPK). Morphological alterations in skeletal muscle were assessed using hematoxylin and eosin (HE) staining subsequent to hnRNPK knockout or overexpression. Global gene expression changes in the tibialis anterior (TA) muscle were assessed via RNA sequencing (RNA-seq). Furthermore, reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot analysis, immunofluorescence, immunohistochemistry, co-immunoprecipitation (Co-IP), dual luciferase analysis, and reactive oxygen species (ROS) detection were utilized to elucidate the molecular mechanisms by which hnRNPK contributes to skeletal muscle development.

Results: Our findings indicate that the ablation of hnRNPK in myoblast precursors significantly impairs muscle development, disrupts fetal myogenesis, and results in embryonic lethality. In adult mice, both the loss and gain of hnRNPK function led to reduced muscle mass, decreased fiber size, and compromised skeletal muscle homeostasis. Importantly, the knockout of hnRNPK had a more substantial impact on skeletal muscle development compared to its overexpression, with myofiber-specific knockout leading to mortality within two weeks. Mechanistically, hnRNPK deficiency was associated with increased apoptosis and muscle atrophy, characterized by elevated expression of genes involved in apoptosis, muscle atrophy, and protein catabolism, along with impaired muscle contraction and extracellular matrix (ECM) organization. Conversely, hnRNPK overexpression was correlated with enhanced ferroptosis pathway and improved ECM organization, but was also associated with reduced oxidative phosphorylation and protein synthesis. The overexpression likely promotes ferroptosis via the hnRNPK/P53/Slc7a11/Gpx4 pathway, thereby accelerating muscle aging and reducing muscle mass.

Conclusion: In conclusion, our findings underscore the critical importance of precise hnRNPK expression levels in maintaining skeletal muscle health. Both deficiency and overexpression of hnRNPK disrupt skeletal muscle development, highlighting its pivotal role in muscle physiology.

Clinical trial number: Not applicable.

Background: Small mammals such as mice rely on type IIb myofibers, which express the fast-contracting myosin heavy chain isoform Myh4, to achieve rapid movements. In contrast, larger mammals, including humans, have lost MYH4 expression. Thus, they favor slower-contracting myofiber types. However, the mechanisms underlying this evolutionary shift remain unclear. We recently identified the large Maf transcription factor family (Mafa, Mafb, and Maf) as key regulators of type IIb myofiber specification in mice. In this study, we investigate whether large MAFs play a conserved role in the induction of MYH4 expression and glycolytic metabolism in human and bovine skeletal muscle.

Methods: We performed adenovirus-mediated overexpression of large MAFs in iPSC-derived human myotubes and primary bovine myotubes. We subsequently quantified MYH4 expression using RT-qPCR, RNA sequencing (RNA-seq), and LC-MS/MS analysis. Glycolytic capacity was assessed using a flux analyzer and metabolic gene expression profiling. Additionally, RNA-seq analysis of human muscle biopsy samples was conducted to determine the correlations between large MAFs and the expression of MYH4 and other myosin genes, as well as their association with fast fiber composition and athletic training.

Results: Overexpression of large MAFs in human and bovine myotubes robustly induced MYH4 expression, with mRNA levels increasing by 100- to 1000-fold. LC-MS/MS analysis provided clear evidence of MYH4 protein expression in human myotubes, where it was previously undetectable. RNA-seq and flux analyzer data revealed that large MAFs significantly enhanced glycolytic capacity by upregulating the expression of key genes involved in glucose metabolism. Moreover, RNA-seq analysis of human muscle biopsy samples revealed a positive correlation between MAFA, MAF, and MYH4 expression. Furthermore, MAFA and MAF expression levels were elevated in power-trained individuals, accompanied by increased expression of MYH4 and other fast myosin genes.

Conclusions: Our findings establish large MAF transcription factors as key regulators of MYH4 expression and glycolytic metabolism in human skeletal muscle. This discovery provides novel insights into the evolutionary loss of type IIb myofibers in larger mammals and suggests potential strategies for enhancing muscle performance and mitigating fast-twitch fiber loss associated with aging and muscle degeneration.

Background: The genioglossus (GG) muscle, the largest upper airway dilator muscle, plays a crucial role in maintaining pharyngeal airway patency. It is innervated by hypoglossal motoneurons, and its tone is often reduced in patients with obstructive sleep apnea (OSA), leading to tongue collapse and airway obstruction during sleep. Although the mechanisms underlying this disorder are not fully understood, the neuromuscular junction (NMJ) of the GG muscle, essential for communication between motor neurons and skeletal muscle, has largely been overlooked.

Methods: In this study, we explored whether obesity impacts the NMJ of the GG muscle. Using the leptin-deficient obese mouse model, Lep^ob/ob, which exhibits pharyngeal collapsibility and hypoventilation, we analyzed the GG muscle and its NMJ in both male and female mice. We conducted morphological and histochemical studies of the GG muscle; quantitative fluorescence imaging to assess the density and dynamics of nicotinic acetylcholine receptors (nAChRs) at the NMJ; high-resolution confocal microscopy to evaluate structural changes in the pre- and postsynaptic apparatus; and transmission electron microscopy for ultrastructural analysis. Additionally, we examined the diaphragm (DIA) and sternomastoid (ST) muscles for comparative analysis.

Results: Our results show that the GG muscle and its NMJs exhibit significant alterations in Lep^ob/ob male mice, while the ST and DIA muscles remain unaffected. Lep^ob/ob males displayed altered GG muscle morphology, changes in synapse structure, and reduced postsynaptic AChR density compared to both controls and Lep^ob/ob females. Additionally, AChR turnover and the morphology of the presynaptic apparatus were impaired in Lep^ob/ob male mice. In contrast, Lep^ob/ob females exhibited NMJs similar to those of wild-type mice.

Conclusions: These findings suggest that the GG muscle is particularly susceptible to degeneration in obesity induced by leptin deficiency, with distinct alterations observed in both the muscle and the NMJ. This specificity underscores the complex impact of obesity on NMJ health and highlights the need for further investigation into muscle-specific responses to obesity-related stress. Additionally, the degeneration of the GG muscle appears to reflect a sex-specific impact of obesity on neuromuscular integrity and may contribute to the pathogenesis of OSA.