AbstractDuchenne muscular dystrophy (DMD) is a hereditary, X-linked, muscle wasting disease with no known cure. It is caused by altered mechanical stability of muscle cell membranes combined with altered cell signalling and inflammatory infiltrations due the absence of the cytoskeletal protein dystrophin, the product of the DMD gene. Progressive muscle fibre degeneration combined with chronic inflammation leads to a severe functional impairment, progressive disability, and ultimately to premature death. DMD presents predominantly in skeletal muscles but brain and bone are also affected.
Analyses in the mdx mouse, the most commonly used model of DMD, has led to the identification of an increased expression and function of the P2X7 purinoceptor in dystrophic muscle cells and muscles in situ. This ATP-gated receptor has been implicated in a number of human diseases that, combined with its well-known role in inflammatory cells, suggested that P2X7 upregulation might also be important for the pathogenesis of DMD.
To test the role of the P2X7 purinoceptor in DMD pathogenesis and its potential as a target for treatment, two mdx/P2X7-/- double mutant mouse strains were generated and compared to the mdx mouse with respect to several critical disease parameters during an acute degenerative stage of disease. Histological, molecular, biochemical, and functional analyses revealed reductions in both muscle and non-muscle pathology in mdx/P2X7-/- mice, with significant improvements in key molecular and structural parameters. These included lower serum creatine kinase levels and decreased sarcolemma permeability to blood-born molecules, indicative of less sarcolemma damage. P2X7 ablation also resulted in increased minimum Feret’s diameter, a morphological indicator of muscle regeneration. While the fraction of muscle fibres with centralised nuclei was not significantly different, levels of myogenin, a protein indicator of muscle differentiation, were also higher in mdx/P2X7-/- mice. These changes were concomitant with an overall decreased inflammatory signature in mdx/P2X7-/- mice compared to mdx and an increase in muscle strength, in vitro. Examination of diaphragms (undergoing continuous degeneration/regeneration in the mdx mouse) from 20 month old mice also showed the increase in minimum Feret’s diameter and continued reduced inflammation in the mdx/P2X7-/- group. Aged heart tissue from these mdx/P2X7-/- also presented less inflammatory infiltrate and reduced fibrosis compared to mdx. Moreover, micro-CT analyses showed greatly reduced osteopenia in mdx/P2X7-/- bones when compared to 6 month mdx mice. Amelioration of all symptoms was proportional to the extent of receptor depletion, where single P2X7 isoform loss was less effective than the complete knockout.
These observations support involvement of the P2X7 purinoceptor in the dystrophic pathology in this model of DMD and are consistent with the well-known role of this receptor in other disorders. P2X7 purinoceptors are likely to act via several different molecular pathways, for example increased Ca2+ influx that affects dystrophic cells directly as well as through reduced inflammation. Indeed, this study represents the first known analysis of an effect of P2X7 ablation on a chronic inflammatory phenotype localised to skeletal muscle.
These data from the most widely used model of DMD suggest that the P2X7 receptor can be also involved in human pathology and specific receptor antagonists could be considered for targeted pharmacological intervention to delay progression of this lethal disease.
|Date of Award||Sep 2014|
|Supervisor||Darek Gorecki (Supervisor), Stephen Arkle (Supervisor) & Jerome Swinny (Supervisor)|