AbstractDuchenne muscular dystrophy (DMD) is the second most commonly inherited disorder in man, the phenotype of which displays pathological characteristics of altered skeletal muscle function including, amongst others, abnormal Ca2+ homeostasis and cell signaling. This study used the mdx mouse model of DMD to analyse purinergic responses in dystrophic muscle cells in vitro and skeletal muscles in situ. Initial investigations excluded reduction in ATPe hydrolysing potential from explaining previous observations of heightened nucleotide sensitivities in dystrophic myoblasts. Instead, this study demonstrates for the first time that significant P2X7 receptor abnormalities exist in dystrophic myoblasts and skeletal muscles of the adult mdx mouse; significantly elevated levels of P2X7 receptor mRNA and protein expression were found in primary myoblast cultures, myoblast lines and muscles in situ at 4 months of age and this was extended to analysis of changes in individual P2X7 splice variants. These abnormalities were shown to extend to functional responses in cultured myoblast lines, where heightened P2X7 receptor-specific sensitivity was shown to be associated with significantly higher basal and induced levels of, and altered time course of, extracellular-signal regulated kinase (ERK1/2)phosphorylation in dystrophic myoblasts. ERK activation responses were shown to be inducible by ATP and BzATP stimulation, inhibited by P2X7 antagonists, and unresponsive to ivermectin, thus confirming P2X7 receptor involvement. Similar up-regulation of P2X7 receptor expression coinciding with ERK phosphorylation was demonstrated in mdx muscles in vivo. Additionally, NAD was identified as a mediator of P2X7 responses in dystrophic myoblasts. This study has also employed a mass spectrometry based approach to investigate the immediate downstream effects of P2X7 activation in cultured myoblasts; allowing identification of multiple potential signaling relays for future study that are discussed. The potential for a link between P2X7 receptor and dystrophin expression has been suggested here through the demonstration that abnormalities in P2X7 receptor expression and function are corrected by micro-dystrophin expression in dystrophic myoblasts.
In vivo pharmacological P2X7 receptor inhibition using CBBG significantly reduced the number of revertant fibres in dystrophic muscle, indicating a reduction in degenerative/regenerative activity. The data presented in this thesis highlight a novel role for P2X7 receptor signaling in dystrophic myoblasts and muscles in situ; proposing the potential for beneficial therapeutic strategies aimed at manipulating P2X7 signaling responses in vivo, with a view to slowing the progression of what isat present an incurable and invariably fatal disease.
|Date of Award
|Darek Gorecki (Supervisor) & Stephen Arkle (Supervisor)