Project description:Background: Skeletal muscle function crucially depends on motor innervation and after injury on the resident muscle stem cells (MuSCs). However, it is poorly understood how innervation affects MuSC properties. Methods: We investigated the alterations of MuSCs and their immediate niche, the myofiber, after denervation in a surgery-based mouse model of unilateral sciatic nerve transection. FACS-isolated MuSCs were subjected to transcriptomics and proteomics analyses to investigate which changes occur after denervation. We performed Cardiotoxin-induced muscle injury, MuSC transplantation and floating myofiber cultures to assess MuSC functionality after denervation in addition to bioinformatics and histological analyses. Results: We observed a significant increase in the number of MuSCs (Pax7 positive; p-value= 0.0441), proliferating MuSCs (Pax7/Ki67 positive; p-value= 0.0023), activated MuSCs (MyoD positive; p-value= 0.0016) and differentiating MuSCs (Myog positive; p-value= 0.0057) after denervation. This aberrant activation and premature commitment of MuSCs to the myogenic lineage was accompanied by profound alterations on the mRNA (2613 differentially expressed genes, adj. p-value <0.05) and protein (1096 differentially abundant proteins, q-value <0.05) level after denervation. MuSCs from denervated hosts still engrafted and fused to form new myofibers irrespective of the innervation status of the recipient, suggesting the MuSC niche is driving alterations in MuSCs after denervation. The myofiber transcriptome after denervation showed massive changes in the general expression profile (10492 DEGs, p-value <0.05) and in several predicted secreted factors. Incubation of myofiber-associated MuSCs with supernatant from denervated myofibers increased cluster formation, reinforcing myofibers as a source of secreted factors driving MuSC alterations after denervation. Opn and Tgfb1 showed an increased secretion by denervated myofibers (30-fold and 6000-fold, respectively), and incubation with Tgfb1 alone induced Junb expression in myogenic cells, one of the genes highly upregulated in MuSCs after denervation (p-value= 1.85e-18, log2fc= 3.27), demonstrating that myofiber-secreted ligands influence MuSC gene expression. A combination of skeletal muscle injury and denervation led to reduced numbers of proliferating MuSCs (Sham: 47 vs DEN: 19.75 cells per cross section 10 days post-injury) and sustained high levels of developmental myosin heavy chain (Sham: 1 % vs DEN: 40 % of all myofibers 21 days post-injury), indicating hampered MuSC functionality due to changes in the microenvironment. Conclusion: Denervation of skeletal muscle causes alterations in myofiber secretion, leading to activation and profound changes of MuSCs, ultimately resulting in a reduced regenerative capacity. As these alterations are partially reversible, MuSCs are a promising target for novel treatment options for neuromuscular disorders and peripheral nerve injuries.

Project description:We report the application of single myofiber ATAC-Seq (smfATAC-Seq) to investigate the chromatin accessibility of a single myofiber without the presence of confounding muscle resident cell types. This method demonstrates that open chromatin regions of myonuclei can be tagmentated and high-quality sequencing ready libraries can be generated from these fragments. To perform comparative analysis as well as to demonstrate the applicability of the smfATAC-Sew to study changes in chromatin of myonuclei within different contexts, smfATAC-Seq was performed both on uninjured myofibers as well as injured myofibers seven days after induced injury. Furthermore, ATAC-Seq on 5000 muscle stem cells (MuSCs) was also performed according to the previously described OMNI ATAC-Seq protocol (Corces, M.R. et al. Nature Methods, 2017) in order to compare the sequencing quality of the smfATAC-Seq as well as to demonstrate the changes in open chromatin that occur from the stem cell state to the fully differentiated myofibers. smfATAC-seq resulted in comparable coverage and sequencing depth to the ATAC-seq performed on MuSCs and allowed for peak calling and differential peak analysis. These analysis revealed that the open chromatin state of uninjured and injured myofibers after seven days are mostly similar although some regions invovled in immune response remain in an open state in the injured myofibers compared to the uninjured myofibers and the regions involved in structural formation of the muscle are more accessible in the case of regeneration. Even though certain differences in the open chromatin are observed, smfATAC-Seq analysis suggest that overall, the open chromatin state of the myonuclei returns back to homeostasis after seven days of regeneration. Furthermore, smfATAC-Seq comparison with the ATAC-Seq from MuSCs show the differences in open chromatin regions between these conditions. Increased accessibility of genes involved in myogenesis and structural components can be observed in the myofibers compared to MuSCs while MuSCs show increased accessibility in regions involved in membrane permeability and signalling pathways. In addition, the regions that are accessible for both conditions include genes involved in mitochondrial transport, regulation of transcription and regulation of metabolites and energy. Overall, this study introduces smfATAC-Seq that succesfully assesses the genome-wide chromatin accessibility of a single myofiber with relatively high sequencing depth. The smfATAC-Seq can be used to perform comparative analysis between different conditions such as injury. However, this method can be readily applied to study differences between young and old myofibers or in the context of muscular dystrophy, cachexia, and exercise.

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Muscle stem cell maintenance and regeneration of skeletal muscle require functional motor innervation

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