Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy.

Mortality of Duchenne Muscular Dystrophy (DMD) is a consequence of progressive wasting of skeletal and cardiac muscle, where dystrophinopathy affects not only muscle fibres but also myogenic cells. Elevated activity of P2X7 receptors and increased store-operated calcium entry have been identified in myoblasts from the mdx mouse model of DMD. Moreover, in immortalized mdx myoblasts, increased metabotropic purinergic receptor response was found. Here, to exclude any potential effects of cell immortalization, we investigated the metabotropic response in primary mdx and wild-type myoblasts. Overall, analyses of receptor transcript and protein levels, antagonist sensitivity, and cellular localization in these primary myoblasts confirmed the previous data from immortalised cells. However, we identified significant differences in the pattern of expression and activity of P2Y receptors and the levels of the "calcium signalling toolkit" proteins between mdx and wild-type myoblasts isolated from different muscles. These results not only extend the earlier findings on the phenotypic effects of dystrophinopathy in undifferentiated muscle but, importantly, also reveal that these changes are muscle type-dependent and endure in isolated cells. This muscle-specific cellular impact of DMD may not be limited to the purinergic abnormality in mice and needs to be taken into consideration in human studies.

Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts.

Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblasts-the effector cells of muscle growth and regeneration-are affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts. In Dmdmdx myoblasts lacking full-length dystrophin, the expression of 170 genes was significantly altered. Myod1 and key genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology analysis indicated enrichment in genes involved in muscle development and function. Functionally, we found increased myoblast proliferation, reduced chemotaxis and accelerated differentiation, which are all essential for myoregeneration. The defects were caused by the loss of expression of full-length dystrophin, as similar and not exacerbated alterations were observed in dystrophin-null Dmdmdx-ßgeo myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and a dystrophic mouse muscle cell line, confirming the cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts showed alterations in the rate of glycolysis/gluconeogenesis, leukotriene metabolism, and mitochondrial beta-oxidation of various fatty acids. These results reveal the disease continuum: DMD defects in satellite cells, the myoblast dysfunction affecting muscle regeneration, which is insufficient to counteract muscle loss due to myofiber instability. Contrary to the established belief, our data demonstrate that DMD abnormalities occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease.

P2X7 Purinoceptor Affects Ectopic Calcification of Dystrophic Muscles.

Ectopic calcification (EC) of myofibers is a pathological feature of muscle damage in Duchenne muscular dystrophy (DMD). Mineralisation of muscle tissue occurs concomitantly with macrophage infiltration, suggesting a link between ectopic mineral deposition and inflammation. One potential link is the P2X7 purinoceptor, a key trigger of inflammation, which is expressed on macrophages but also up-regulated in dystrophic muscle cells. To investigate the role of P2X7 in dystrophic calcification, we utilised the Dmd mdx-ßgeo dystrophin-null mouse model of DMD crossed with a global P2X7 knockout (P2rx7 -/- ) or with our novel P2X7 knockin-knockout mouse (P2x7 KiKo ), which expresses P2X7 in macrophages but not muscle cells. Total loss of P2X7 increased EC, indicating that P2X7 overexpression is a protective mechanism against dystrophic mineralisation. Given that muscle-specific P2X7 ablation did not affect dystrophic EC, this underlined the role of P2X7 receptor expression on the inflammatory cells. Serum phosphate reflected dystrophic calcification, with the highest serum phosphate levels found in genotypes with the most ectopic mineral. To further investigate the underlying mechanisms, we measured phosphate release from cells in vitro, and found that dystrophic myoblasts released less phosphate than non-dystrophic cells. Treatment with P2X7 antagonists increased phosphate release from both dystrophic and control myoblasts indicating that muscle cells are a potential source of secreted phosphate while macrophages protect against ectopic mineralisation. Treatment of cells with high phosphate media engendered mineral deposition, which was decreased in the presence of the P2X7 agonist BzATP, particularly in cultures of dystrophic cells, further supporting a protective role for P2X7 against ectopic mineralisation in dystrophic muscle.

Dystrophic mdx mouse myoblasts exhibit elevated ATP/UTP-evoked metabotropic purinergic responses and alterations in calcium signalling.

Pathophysiology of Duchenne Muscular Dystrophy (DMD) is still elusive. Although progressive wasting of muscle fibres is a cause of muscle deterioration, there is a growing body of evidence that the triggering effects of DMD mutation are present at the earlier stage of muscle development and affect myogenic cells. Among these abnormalities, elevated activity of P2X7 receptors and increased store-operated calcium entry myoblasts have been identified in mdx mouse. Here, the metabotropic extracellular ATP/UTP-evoked response has been investigated. Sensitivity to antagonist, effect of gene silencing and cellular localization studies linked these elevated purinergic responses to the increased expression of P2Y2 but not P2Y4 receptors. These alterations have physiological implications as shown by reduced motility of mdx myoblasts upon treatment with P2Y2 agonist. However, the ultimate increase in intracellular calcium in dystrophic cells reflected complex alterations of calcium homeostasis identified in the RNA seq data and with significant modulation confirmed at the protein level, including a decrease of Gq11 subunit α, plasma membrane calcium ATP-ase, inositol-2,4,5-trisphosphate-receptor proteins and elevation of phospholipase Cß, sarco-endoplamatic reticulum calcium ATP-ase and sodium­calcium exchanger. In conclusion, whereas specificity of dystrophic myoblast excitation by extracellular nucleotides is determined by particular receptor overexpression, the intensity of such altered response depends on relative activities of downstream calcium regulators that are also affected by Dmd mutations. Furthermore, these phenotypic effects of DMD emerge as early as in undifferentiated muscle. Therefore, the pathogenesis of DMD and the relevance of current therapeutic approaches may need re-evaluation.

Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism.

Duchenne muscular dystrophy (DMD) is the most common inherited muscle disorder that causes severe disability and death of young men. This disease is characterized by progressive muscle degeneration aggravated by sterile inflammation and is also associated with cognitive impairment and low bone density. Given that no current treatment can improve the long-term outcome, approaches with a strong translational potential are urgently needed. Duchenne muscular dystrophy (DMD) alters P2RX7 signaling in both muscle and inflammatory cells and inhibition of this receptor resulted in a significant attenuation of muscle and non-muscle symptoms in DMDmdx mouse model. As P2RX7 is an attractive target in a range of human diseases, specific antagonists have been developed. Yet, these will require lengthy safety testing in the pediatric population of Duchenne muscular dystrophy (DMD) patients. In contrast, Nucleoside Reverse Transcriptase Inhibitors (NRTIs) can act as P2RX7 antagonists and are drugs with an established safety record, including in children. We demonstrate here that AZT (Zidovudine) inhibits P2RX7 functions acting via the same allosteric site as other antagonists. Moreover, short-term AZT treatment at the peak of disease in DMDmdx mice attenuated the phenotype without any detectable side effects. Recovery was evident in the key parameters such as reduced sarcolemma permeability confirmed by lower serum creatine kinase levels and IgG influx into myofibres, decreased inflammatory cell numbers and inflammation markers in leg and heart muscles of treated mice. Moreover, this short-term therapy had some positive impact on muscle strength in vivo and no detrimental effect on mitochondria, which is the main side-effect of Nucleoside Reverse Transcriptase Inhibitors (NRTIs). Given these results, we postulate that AZT could be quickly re-purposed for the treatment of this highly debilitating and lethal disease. This approach is not constrained by causative DMD mutations and may be effective in alleviating both muscle and non-muscle abnormalities.

Sustained activation of P2X7 induces MMP-2-evoked cleavage and functional purinoceptor inhibition.

P2X7 purinoceptor promotes survival or cytotoxicity depending on extracellular adenosine triphosphate (ATP) stimulus intensity controlling its ion channel or P2X7-dependent large pore (LP) functions. Mechanisms governing this operational divergence and functional idiosyncrasy are ill-understood. We have discovered a feedback loop where sustained activation of P2X7 triggers release of active matrix metalloproteinase 2 (MMP-2), which halts ion channel and LP responses via the MMP-2-dependent receptor cleavage. This mechanism operates in cells as diverse as macrophages, dystrophic myoblasts, P2X7-transfected HEK293, and human tumour cells. Given that serum-born MMP-2 activity also blocked receptor functions, P2X7 responses in vivo may decrease in organs with permeable capillaries. Therefore, this mechanism represents an important fine-tuning of P2X7 functions, reliant on both cell-autonomous and extraneous factors. Indeed, it allowed evasion from the ATP-induced cytotoxicity in macrophages and human cancer cells with high P2X7 expression levels. Finally, we demonstrate that P2X7 ablation eliminated gelatinase activity in inflamed dystrophic muscles in vivo. Thus, P2X7 antagonists could be used as an alternative to highly toxic MMP inhibitors in treatments of inflammatory diseases and cancers.

Store-operated calcium entry contributes to abnormal Ca²âº signalling in dystrophic mdx mouse myoblasts.

Sarcolemma damage and activation of various calcium channels are implicated in altered Ca(2+) homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca(2+) concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease.

Purinergic receptors in skeletal muscles in health and in muscular dystrophy.

The P2 purinergic (nucleotide) receptor super-family comprises of two families of protein. The P2X, which are channel-forming ionotropic receptors and the P2Y metabotropic receptors activating G protein-mediated signalling pathways. Members of both groups have been identified in skeletal muscle cells at different stages of differentiation. It is well documented that sequential expression and down-regulation of particular P2 receptors on the surface of sarcolemma is closely associated with muscle maturation during embryogenesis and postnatal growth. P2 receptors are also involved in muscle regeneration following injury. Moreover, enhanced expression of specific purinergic receptors together with increased availability of extracellular ATP in dystrophic muscles are important elements of the dys- trophic pathophysiology considerably increasing severity.