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Spatially resolved transcriptomics reveals innervation-responsive functional cluster in skeletal muscle.

ABSTRACT: Striated muscle is a highly organized structure composed by well-defined anatomical domains with integrated but distinct assignments. Indeed, the lack of a direct correlation between tissue architecture and gene expression has thus far limited our understanding of how each unit responds to physio-pathologic contexts. Here, we show how the combined use of spatially resolved transcriptomics and immunofluorescence can bridge this gap by enabling the unbiased identification of such domains and the characterization of their response to external perturbations. Using a spatiotemporal analysis, we followed the changes in the transcriptomics profile of specific domains in muscle in a model of denervation. Furthermore, our approach allowed us to identify the spatial distribution and nerve dependence of atrophic signalling pathway and polyamine metabolism to glycolytic fibres. Indeed, we demonstrate a pronounced alteration of polyamine homeostasis upon denervation.

ORGANISM(S): Mus musculus

PROVIDER: GSE198596 | GEO | 2022/12/19

REPOSITORIES: GEO

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Project description:Loss of muscle proteins and the consequent weakness has important clinical consequences in diseases such as cancer, diabetes, chronic heart failure and in ageing. In fact, excessive proteolysis causes cachexia, accelerates disease progression and worsens life expectancy. Muscle atrophy involves a common pattern of transcriptional changes in a small subset of genes named atrophy-related genes or atrogenes. Whether microRNAs play a role in the atrophy program and muscle loss is debated. To understand the involvement of miRNAs in atrophy we performed miRNA expression profiling of mouse muscles under wasting conditions such as fasting, denervation, diabetes and cancer cachexia. We found that the miRNA signature is peculiar of each catabolic condition. We then focused on denervation and we revealed that changes in transcripts and microRNAs expression did not occur simultaneously but were shifted. Indeed, while the transcriptional control of the atrophy-related genes peaks at 3 days, the changes of miRNA expression maximised at 7 days after denervation. Among the different miRNAs, microRNA-206 and 21 were the most induced in denervated muscles. We characterized their pattern of expression and defined their role in muscle homeostasis. Indeed, in vivo gain and loss of function experiments revealed that miRNA-206 and miRNA-21 were sufficient and required for atrophy program. In silico and in vivo approaches identified the transcription factor YY1 and the translational initiator factor eIF4E3 as downstream targets of these miRNAs. Thus miRNAs are important for the fine-tuning of the atrophy program and their modulation can be a novel potential therapeutic approach to counteract muscle loss and weakness in catabolic conditions. To determine which miRNAs are relevant for the atrophic process, we performed miRNA expression profiles of muscles from different atrophic models (starvation, denervation and streptozotocin-induced diabetes). We checked whether there was a common signature of miRNA expression in different atrophying conditions and we found that every catabolic situation require a peculiar pattern of miRNA. We further focus on the condition of denervation and identified the most up-regulated miRNAs in this condition, miRNA-206 and miRNA-21.

Project description:Background: Skeletal muscle crucially depends on motor innervation, and, when damaged, on the resident muscle stem cells (MuSCs). However, the role and function of MuSCs in the context of denervation remains poorly understood. Methods: Alterations of MuSCs and their myofiber niche after denervation were investigated in a surgery-based mouse model of unilateral sciatic nerve transection. FACS-isolated MuSCs were subjected to RNA-sequencing and mass spectrometry for the analysis of intrinsic changes after denervation and in vivo assays, such as Cardiotoxin-induced muscle injury or MuSC transplantation, were performed to assess MuSC functions after denervation. Bioinformatic and histological analyses were conducted to further examine MuSCs and their myofiber niche after denervation. Results: Muscle cross section analysis revealed a significant increase in Pax7 (p-value= 0.0441), Pax7/Ki67 (p-value= 0.0023), MyoD (p-value= 0.0016) and Myog (p-value= 0.0057) positive cells after denervation, illustrating a break of quiescence and commitment to the myogenic lineage. An Omics approach showed profound intrinsic alterations on the mRNA (2613 differentially expressed genes, p-value <0.05) and protein (1096 differentially abundant proteins, q-value <0.05) level of MuSCs 21 days after denervation. Skeletal muscle injury together with denervation surgery caused deregulated regeneration, indicated by the reduced number of proliferating MuSCs and sustained high levels of developmental myosin heavy chain (Sham: 1 % vs DEN: 40 % of all myofibers), at 21 days post-surgery. In a transplantation assay, MuSCs from a denervated host were still able to engraft and fuse to form new myofibers, irrespective of the innervation status of the recipient muscle. Analysis of myofibers revealed not only massive changes in the expression profile (10492 differentially expressed genes, p-value <0.05) after denervation, but it was also shown that secretion of Opn and Tgfb1 from denervated myofibers was increased 30-fold and 6000-fold, respectively. Bioinformatic analyses indicated strong upregulation of gene expression of the transcription factor Junb in MuSCs from denervated muscles (log2 fold change = 3.27). Of interest, Tgfb1 recombinant protein was able to induce Junb gene expression in vitro, demonstrating that myofiber-secreted ligands can induce gene expression changes in MuSCs, which might result in the phenotypes observed after denervation. Conclusion: Skeletal muscle denervation is altering myofiber secretion, causing MuSC activation and profound intrinsic changes, leading to reduced regenerative capacity. As MuSCs possess a remarkable regenerative potential, they might represent a promising target for novel treatment options for neuromuscular disorders and peripheral nerve injuries.

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Spatially resolved transcriptomics reveals innervation-responsive functional cluster in skeletal muscle.

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