A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

<div><p>P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the <i>Dmd^mdx</i> mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of <i>p2rx7</i> also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.</p></div

Store-operated calcium entry contributes to abnormal Ca²⁺ signalling in dystrophic mdx mouse myoblasts

Sarcolemma damage and activation of various calcium channels are implicated in altered Ca2+ 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 Ca2+ 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

P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: Relationship to pathology and potential target for treatment.

Open AccessDuchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca2 homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up-regulated but without alterations of specific splice variant patterns. P2X7 protein was also up-regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca2 and extracellular signal-regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca2 influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin-1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degeneration–regeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease

P2RX7 Purinoceptor: A Therapeutic Target for Ameliorating the Symptoms of Duchenne Muscular Dystrophy

open access articleDuchenne muscular dystrophy (DMD) is the most common inherited muscle disease, leading to severe disability and death in young men. Death is caused by the progressive degeneration of striated muscles aggravated by sterile inflammation. The pleiotropic effects of the mutant gene also include cognitive and behavioral impairments and low bone density. Current interventions in DMD are palliative only as no treatment improves the long-term outcome. Therefore, approaches with a translational potential should be investigated, and key abnormalities downstream from the absence of the DMD product, dystrophin, appear to be strong therapeutic targets. We and others have demonstrated that DMD mutations alter ATP signaling and have identified P2RX7 purinoceptor up-regulation as being responsible for the death of muscles in the mdx mouse model of DMD and human DMD lymphoblasts. Moreover, the ATP–P2RX7 axis, being a crucial activator of innate immune responses, can contribute to DMD pathology by stimulating chronic inflammation. We investigated whether ablation of P2RX7 attenuates the DMD model mouse phenotype to assess receptor suitability as a therapeutic target

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

How should we secure the cystic duct during laparoscopic cholecystectomy? A UK-wide survey of clinical practice and systematic review of the literature with meta-analysis

Introduction: It is currently unknown which method of cystic duct closure is most effective at reducing the risk of bile leak after laparoscopic cholecystectomy. The aims of this work were to determine the most common closure methods used in the UK and review available evidence on which method has the lowest risk of bile leak. Methods: We conducted an online survey through the Association of Upper Gastrointestinal Surgeons (AUGIS). We also undertook a systematic review using PubMed, EMBASE, MEDLINE and the Cochrane Library for studies that compared different methods for cystic duct occlusion and reported postoperative bile leak. Findings: There was significant variation in practice between consultant surgeons. For routine laparoscopic cholecystectomy metal clips were used most (64%) followed by locking polymer clips (33%) and suture ties (3%). In cases of a dilated cystic duct, preferences were locking polymer clips (60%), suture ties (30%) and metal clips (5%). We included six studies in our review with a total of 8,011 patients. Metal clips were associated with an increased odds of bile leak compared with locking polymer clips (OR 5.66, 95% CI 1.13–28.41, p=0.04) or suture ties (OR 4.17, 95% CI 0.72–24.31, p=0.12). Most studies were retrospective, unlikely to be adequately powered, and vulnerable to selection bias. Conclusions: Limited available evidence suggests that metal clips have the highest risk of bile leak, but results are not strong enough to recommend a change in current clinical practice. A trial is now required to determine the best method of cystic duct closure

An investigation of the ionophoric characteristics of destruxin A

Destruxin A, a cyclohexadepsipeptide related to the enniatins and beauvericin, exhibits ionophoric properties. Calcium ion mobilization across liposomal membrane barriers, for example, has been demonstrated using the calcium ion-sensitive dyes Arsenazo III and Fura-2. Initial molecular mechanics/molecular dynamics calculations indicate the potential for destruxin A to form a coordination complex with calcium in which the divalent cation is bound at the center of a sandwich formed by two molecules of destruxin A. This novel calcium ion binding may help explain the diverse biological effects exhibited by the destruxins

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

The file attached to this record is the author's final peer reviewed version.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 den-sity. Given that no current treatment can improve the long-term outcome, approaches with a strong translational potential are urgently needed. DMD alters P2RX7 signaling in both muscle and inflamma-tory 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 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 attenu-ated 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 therapy had some positive impact on muscle strength in vivo and no detrimental effect on mitochondria, which is the main side-effect of 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

Additional file 1: of Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism

Figure S1. Human, giant panda and mouse P2RX7 peptide sequence alignments. (PDF 779 kb