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Spinal muscle atrophy (SMA) is a genetic disorder produced by mutations in the SMN1 gene leading to degeneration of lower motor neurones and, consequently, denervation of the skeletal muscles. The molecular pathways activated in the denervated muscles in humans are far to be known. We have applied single nuclei RNA sequencing (snRNAseq) to seven muscle samples of patients with genetically confirmed SMA type 3 with at least three copies of SMN2 gene and five age and gender matched controls. Bioinformatic analysis was performed to identify cell types present in muscle samples and changes in their gene expression profile. Metascape was used to identify the molecular pathways dysregulated in the different cell populations. We observed a statistically significant increase in nuclei expressing markers of satellite cells (6.5% vs 2.1%, p<0.01) and in myonuclei expressing markers of fast muscle fibers (66.2% vs 54.8%, p<0.05) in SMA patients versus controls. 195 genes were upregulated at least 0.5 folds in SMA muscle samples, being more than 80% of these expressed by muscle fibers. When analyzing genes dysregulated in myonuclei only, 219 genes were upregulated in SMA myofibes compared to controls, being Col19a1, Rnls, Emc10, Myh8 and Runx the top five genes increased. We identified that the top upregulated molecular pathways in muscles fibers were related with neuromuscular junction remodelling, neuron projection development and axon guidance, renewal of the myofibrillar system and protein digestion and absorption. We validated the expression of genes related with these pathways using immunofluorescence in muscle samples of SMA patients. SMA satellite cells expressed high levels of genes involved in maturation of the myofiber system and Wnt pathway. We did not identify significant changes in the number of fibropadipogenic progenitor cells (FAPs), however when analyzing which genes were upregulated, we observed many of them to be involved in the production of extracellular matrix components but also axonogenesis, neuron projection and axon guidance. Our results show that denervated muscle fibers try to compensate the loss of motor neurons by releasing genes promoting neuronal growth and axon guidance in parallel to remodelling the neuromuscular junction. FAPs seems to collaborate in this function, by releasing molecules involved in axon growth and guidance as well. In parallel, we identified an active process of muscle protein digestion in muscle fibers, which can be responsible of the characteristic presence of atrophic fibers. These results open new avenues for developing therapies trying to compensate the process of muscle degeneration in SMA enhancing the growth of motor axons. [ABSTRACT FROM AUTHOR]

Late-onset Pompe disease (LOPD) is a rare genetic disorder caused by the deficiency of acid alpha-glucosidase leading to progressive cellular dysfunction owing to the accumulation of glycogen in the lysosome. The mechanism of relentless muscle damage (a classic manifestation of the disease) has been studied extensively by analysing the whole-muscle tissue; however, little, if anything, is known about transcriptional heterogeneity among nuclei within the multinucleated skeletal muscle cells. This is the first report of application of single-nucleus RNA sequencing to uncover changes in the gene expression profile in muscle biopsies from eight patients with LOPD and four muscle samples from age- and sex-matched healthy controls. We matched these changes with histological findings using GeoMx spatial transcriptomics to compare the transcriptome of control myofibres from healthy individuals with non-vacuolated (histologically unaffected) and vacuolated (histologically affected) myofibres of LODP patients. We observed an increase in the proportion of slow and regenerative muscle fibres and macrophages in LOPD muscles. The expression of the genes involved in glycolysis was reduced, whereas the expression of the genes involved in the metabolism of lipids and amino acids was increased in non-vacuolated fibres, indicating early metabolic abnormalities. Additionally, we detected upregulation of autophagy genes and downregulation of the genes involved in ribosomal and mitochondrial function leading to defective oxidative phosphorylation. Upregulation of genes associated with inflammation, apoptosis and muscle regeneration was observed only in vacuolated fibres. Notably, enzyme replacement therapy (the only available therapy for the disease) showed a tendency to restore dysregulated metabolism, particularly within slow fibres. A combination of single-nucleus RNA sequencing and spatial transcriptomics revealed the landscape of the normal and diseased muscle and highlighted the early abnormalities associated with disease progression. Thus, the application of these two new cutting-edge technologies provided insight into the molecular pathophysiology of muscle damage in LOPD and identified potential avenues for therapeutic intervention., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Becker muscular dystrophy (BMD) is characterised by fiber loss and expansion of fibrotic and adipose tissue. Several cells interact locally in what is known as the degenerative niche. We analysed muscle biopsies of controls and BMD patients at early, moderate and advanced stages of progression using Hyperion imaging mass cytometry (IMC) by labelling single sections with 17 markers identifying different components of the muscle. We developed a software for analysing IMC images and studied changes in the muscle composition and spatial correlations between markers across disease progression. We found a strong correlation between collagen-I and the area of stroma, collagen-VI, adipose tissue, and M2-macrophages number. There was a negative correlation between the area of collagen-I and the number of satellite cells (SCs), fibres and blood vessels. The comparison between fibrotic and non-fibrotic areas allowed to study the disease process in detail. We found structural differences among non-fibrotic areas from control and patients, being these latter characterized by increase in CTGF and in M2-macrophages and decrease in fibers and blood vessels. IMC enables to study of changes in tissue structure along disease progression, spatio-temporal correlations and opening the door to better understand new potential pathogenic pathways in human samples., (© 2024. The Author(s).)

Duchenne muscular dystrophy is a genetic disease produced by mutations in the dystrophin gene characterized by early onset muscle weakness leading to severe and irreversible disability. The cellular and molecular consequences of the lack of dystrophin in humans are only partially known, which is crucial for the development of new therapies aiming to slow or stop the progression of the disease. Here we have analyzed quadriceps muscle biopsies of seven DMD patients aged 2 to 4 years old and five age and gender matched controls using single nuclei RNA sequencing (snRNAseq) and correlated the results obtained with clinical data. SnRNAseq identified significant differences in the proportion of cell population present in the muscle samples, including an increase in the number of regenerative fibers, satellite cells, and fibro-adipogenic progenitor cells (FAPs) and a decrease in the number of slow fibers and smooth muscle cells. Muscle samples from the younger patients with stable mild weakness were characterized by an increase in regenerative fibers, while older patients with moderate and progressive weakness were characterized by loss of muscle fibers and an increase in FAPs. An analysis of the gene expression profile in muscle fibers identified a strong regenerative signature in DMD samples characterized by the upregulation of genes involved in myogenesis and muscle hypertrophy. In the case of FAPs, we observed upregulation of genes involved in the extracellular matrix regeneration but also several signaling pathways. Indeed, further analysis of the potential intercellular communication profile showed a dysregulation of the communication profile in DMD samples identifying FAPs as a key regulator of cell signaling in DMD muscle samples. In conclusion, our study has identified significant differences at the cellular and molecular levels in the different cell populations present in skeletal muscle samples of patients with DMD compared to controls., (© 2023. The Author(s).)