The metabolic microenvironment regulates myogenic fate decisions through altered histone acetylation
Document TypePhD thesis
Access StatusOpen Access
© 2018 Dr Chi Ly
1. Skeletal muscle has an extensive capacity for regeneration, a property conferred on this tissue by a resident population of skeletal muscle stem cells (MuSCs). Any impairment to this population of stem cells can lead to increased morbidity and mortality. MuSCs normally exist in a quiescent state marked by the paired box transcription factor Pax7. In response to an activating signal, MuSCs rapidly undergo a process of activation and cell-cycle re-entry. At this early stage, MuSCs begin to express the myogenic determination factor MyoD and initiate the process of commitment to the myogenic lineage. Importantly, a sub-population of MuSCs will return to quiescence so as to prevent depletion of the stem cell pool. This decision for MuSCs to either undergo commitment or self-renewal remains ill defined. Studies in cancer, and developmental and stem cell biology has identified cellular metabolism as playing a key role in directing changes associated with stem cell self-renewal, lineage specification and the processes of proliferation and differentiation. Therefore, the aim of this study was to investigate this link between metabolism and the processes of self-renewal and myogenic commitment in MuSCs and to identify how metabolism may regulate these processes. 2. To efficiently isolate a large number of primary MuSCs I utilized Pax7creERT2xROSA26eYFP transgenic mice which allows for the fluorescent labelling of MuSCs and subsequent isolation via fluorescence activated cell sorting. To examine the link between innate cell metabolism and MuSC heterogeneity, single cell RNA sequencing (scRNAseq) was performed on either freshly isolated MuSCs or MuSCs that had been cultured ex vivo for 96 hrs. The scRNAseq results revealed that while freshly isolated MuSCs are largely homogeneous, cultured MuSCs exhibited significant heterogeneity with divergent metabolic signatures. These metabolic signatures marked cells either undergoing myogenic commitment or self-renewal. 3. To examine the role of the metabolic microenvironment in regulating MuSC lineage specification whole skeletal muscle fibres, isolated primary MuSCs or C2C12 cells were cultured in media containing different carbohydrate conditions; high glucose (25 mM glucose, HG), low glucose (5 mM glucose, LG) or galactose (10 mM galactose, GAL). Following culture, the downstream effects on metabolism, including measurements of mitochondrial DNA, mitochondrial abundance, key electron transport chain proteins and cellular bioenergetics was assessed. Myogenic specification was assessed via examination of key myogenic regulatory factors by PCR, western immunoblotting, immunofluorescence, whole transcriptome sequencing and single cell sequencing (scRNAseq). Finally, to link alterations to metabolism to changes in gene transcription, global histone acetylation was examined. Extracellular carbohydrate availability directly regulates both innate cellular metabolism and gene expression via acetyl-CoA availability and histone acetylation. Importantly, use of several pharmacological modulators of metabolism confirm a central role of carbohydrate metabolism in histone acetylation. Combining both whole transcriptome sequencing and scRNAseq, extracellular carbohydrate availability was shown to directly influence lineage fate decisions, with reduced carbohydrate availability linked to a reduction in the proportion of cells undergoing myogenic commitment. The scRNAseq dataset presented provides entirely new information of subpopulations of cells; including true MuSCs, primed MuSCs, early committed muscle progenitors (CMPs) and late CMPs and show that the extracellular metabolic environment directly influences the proportion of cells in each of these subpopulations. Finally, single fibre experiments showed that reduced carbohydrate availability was linked to increased rates of asymmetric division and self-renewal. 4. These results provide the first evidence that the extracellular metabolic microenvironment is able to directly alter MuSC lineage commitment and self-renewal with reduced carbohydrate availability leading to a maintenance of the true MuSC population a result of an increased proportions of asymmetric divisions. Finally, metabolic remodelling can be used to enhance the efficiency of MuSC transplantation.
Keywordsmetabolism; epigenetics; stem cells
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- Physiology - Theses