Understanding and Rewriting Cellular and Molecular Programs to Restore Skeletal Muscle Homeostasis

Skeletal muscle fibrosis is a common characteristic of different pathologies, including aging, muscular dystrophy, neurodegenerative disease, and severe extremity trauma. The fibrotic supplantation of muscle tissue impacts quality of life through disabilities ranging from reduced function and loss of mobility to osteoarthritis and elected limb amputation. It is the result of a dysregulated immune response that drives excessive extracellular matrix deposition and impairs the tissue resident muscle stem cells (MuSCs), leading to aberrant or muted muscle regeneration, degeneration of neuromuscular synapses, and muscular atrophy. However, the transcriptional networks and epigenetic changes that diminish MuSC function, how MuSC impairments impact innervation, and how exacerbated inflammatory and fibrotic signaling hinder skeletal muscle repair remain poorly understood. This dissertation seeks to elucidate the cellular and molecular mechanisms governing MuSC dysfunction and skeletal muscle fibrosis using high throughput, high dimensional methodologies in combination with various animal models of disease and injury to spatiotemporally profile MuSCs and their microenvironment in regenerative and fibrotic contexts. The first two studies employ a MuSC-centric approach to understand intracellular signaling dysregulation in age and neuromuscular disease and how it functionally impairs MuSC ability to maintain skeletal muscle homeostasis. First, integration of transcriptomics and chromatin accessibility datasets generated from aged and young murine MuSCs across a regenerative injury time course revealed that aging elicits multiple regulatory changes through significant differences in gene expression, metabolic flux, chromatin accessibility, and patterns of transcription factor binding activities. Both in vivo treatment of MuSCs with retinoic acid and silencing the expression of the myogenic co-factor, DNA damage inducible transcript 3, helped restore aged MuSC state and function towards that of young MuSCs. We then showed that following nerve injury, a subset of MuSCs engrafts proximally to the neuromuscular junction (NMJ) in healthy young adult muscles, but that with age or neuromuscular degeneration induced by CuZn superoxide dismutase knockout (SOD1-/-) this localized engraftment behavior is reduced, suggesting that MuSCs lose their ability to support synaptic integrity in these contexts. Single cell transcriptomics of murine MuSCs revealed a subset in aged animals and SOD1-/- animals that shares similarity with synaptic myonuclei, which may reflect a reduced fusion capacity and thus reduced ability to contribute to NMJ regeneration. Moreover, genetically rescuing the motor neurons via over-expressing human SOD1 only in the motor neurons reverted the MuSC response towards that observed in young animals. The final three studies enhance our understanding of the cellular and molecular mechanisms driving the fibrotic degeneration that occurs following severe injury. Using spatial and single cell transcriptomics as well as metabolipidomics and immunofluorescent techniques, we revealed exacerbated and persistent inflammatory and fibrotic signaling mediated by neutrophils, scar-associated macrophages, and fibro-adipogenic progenitors, which is partially antagonized by cytolytic natural killer cells. Elevated transforming growth factor beta (TGF inhibition reduced neutrophil accumulation, partially rescued functional recovery, and reduced collagen accumulation. Finally, restoring the balance of pro-resolving to pro-inflammatory lipid mediators by repleting Maresin 1, a pro-resolving lipid mediator, reduced macrophage and neutrophil accumulation, boosted MuSC proliferation, reduced TGF 1, and improved regeneration. Collectively, this work facilitates a deeper understanding of the regulation that tissue resident stem cells utilize during aging and healing, reveals unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors, and elucidates microenvironmental factors that impede regeneration following traumatic injury.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/176415/1/larouche_2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/176415/2/larouche_1.pd