Decoding the muscle transcriptome of patients with late onset Pompe disease reveals markers of disease progression.

Late-onset Pompe Disease (LOPD) is a rare genetic disorder caused by the deficiency of acid alpha-glucosidase leading to progressive cellular dysfunction due to the accumulation of glycogen in the lysosome. The mechanism of relentless muscle damage - a classic manifestation of the disease - has been extensively studied by analysing the whole muscle tissue; however, little, if any, is known about transcriptional heterogeneity among nuclei within the multinucleated skeletal muscle cells. This is the first report of application of single nuclei 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 gender matched healthy controls. We matched these changes with histology findings using GeoMx Spatial Transcriptomics to compare the transcriptome of control myofibers from healthy individuals with non-vacuolated (histologically unaffected) and vacuolated (histologically affected) myofibers of LODP patients. We observed an increase in the proportion of slow and regenerative muscle fibers 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 fibers, 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. The upregulation of the genes associated with inflammation, apoptosis and muscle regeneration was observed only in vacuolated fibers. Notably, enzyme replacement therapy - the only available therapy for the disease - showed a tendency to restore metabolism dysregulation, particularly within slow fibers. A combination of single nuclei RNA sequencing and spatial transcriptomics revealed the landscape of normal and the diseased muscle, and highlighted the early abnormalities associated with the 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.

Single cell RNA sequencing of human FAPs reveals different functional stages in Duchenne muscular dystrophy.

Background: 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. Muscle degeneration involves a complex interplay between multiple cell lineages spatially located within areas of damage, termed the degenerative niche, including inflammatory cells, satellite cells (SCs) and fibro-adipogenic precursor cells (FAPs). FAPs are mesenchymal stem cell which have a pivotal role in muscle homeostasis as they can either promote muscle regeneration or contribute to muscle degeneration by expanding fibrotic and fatty tissue. Although it has been described that FAPs could have a different behavior in DMD patients than in healthy controls, the molecular pathways regulating their function as well as their gene expression profile are unknown. Methods: We used single-cell RNA sequencing (scRNAseq) with 10X Genomics and Illumina technology to elucidate the differences in the transcriptional profile of isolated FAPs from healthy and DMD patients. Results: Gene signatures in FAPs from both groups revealed transcriptional differences. Seurat analysis categorized cell clusters as proliferative FAPs, regulatory FAPs, inflammatory FAPs, and myofibroblasts. Differentially expressed genes (DEGs) between healthy and DMD FAPs included upregulated genes CHI3L1, EFEMP1, MFAP5, and TGFBR2 in DMD. Functional analysis highlighted distinctions in system development, wound healing, and cytoskeletal organization in control FAPs, while extracellular organization, degradation, and collagen degradation were upregulated in DMD FAPs. Validation of DEGs in additional samples (n = 9) using qPCR reinforced the specific impact of pathological settings on FAP heterogeneity, reflecting their distinct contribution to fibro or fatty degeneration in vivo. Conclusion: Using the single-cell RNA seq from human samples provide new opportunities to study cellular coordination to further understand the regulation of muscle homeostasis and degeneration that occurs in muscular dystrophies.

Imaging mass cytometry analysis of Becker muscular dystrophy muscle samples reveals different stages of muscle degeneration.

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.