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

Róg, Justyna; Oksiejuk, Aleksandra; Gosselin, Maxime R F; Brutkowski, Wojciech; Dymkowska, Dorota; Nowak, Natalia; Robson, Samuel; Górecki, Dariusz C.; Zabłocki, Krzysztof

doi:10.1016/j.bbadis.2019.01.002

Cited by 17 publications

(24 citation statements)

References 48 publications

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“…These defects are cell autonomous rather than caused by the inflammatory environment of the dystrophic niche because they persist in the dystrophic cell line maintained long-term in culture: it reproduced the key transcriptomic anomalies and has been found before to have functional alterations found in primary cells [67].…”

Section: Discussionmentioning

confidence: 99%

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

Gosselin

Mournetas

Borczyk

et al. 2021

Preprint

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Background. Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease that leads to severe disability and death in young men. DMD is caused by out-of-frame mutations in the largest known gene, which encodes dystrophin. The loss of DMD gene expression manifests in progressive degeneration and wasting of striated muscles aggravated by sterile inflammation. Current conventional treatments are palliative only, whereas experimental therapeutic approaches focus on the re-expression of dystrophin in myofibers. However, recent studies established that DMD pathology begins already in prenatal development prior to myofiber formation while, in adult muscle, it affects satellite (stem) cells and the proper development of myofibers. Regeneration defects that exacerbate muscle degeneration appear to be a good therapeutic target, as maintaining regeneration would counteract muscle wasting. It is also the only feasible treatment in advanced stages of the disease. Yet, it is unknown whether dystrophic myoblasts, the intermediary between satellite cells and myofibers and effectors of muscle growth and repair, are also affected. Therefore, we investigated whether DMD myoblasts show a dystrophic phenotype. Methods and Findings. Using a combination of transcriptomic, molecular, biochemical, and functional analyses we demonstrate, to our knowledge for the first time, convergent cell-autonomous abnormalities in primary mouse and human dystrophic myoblasts. In Dmdmdx mouse myoblasts lacking full-length dystrophin transcripts, expression of 170 other genes was significantly altered. Myod1 (p=2.9e-21) and key muscle genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signaling and fibrosis genes) were significantly downregulated. Gene ontology enrichment analysis indicated significant alterations in genes involved in muscle development and function. These transcriptomic abnormalities translated into increased proliferation (p=3.0e-3), reduced migration towards both sera-rich (p=3.8e-2) and cytokine-containing medium (p=1.0e-2), and significantly accelerated differentiation in 3D organotypic cultures. These altered myoblast functions are essential for muscle regeneration. The defects were caused by the loss of expression of full-length dystrophin as strikingly similar and not exacerbated alterations were also observed in dystrophin-null Dmdmdx-βgeo myoblasts. Furthermore, corresponding abnormalities were identified in human DMD primary myoblasts and in an established dystrophic mouse muscle (SC5) cell line, confirming universal, cross-species and cell-autonomous nature of this defect. Conclusions. These results, for the first time, demonstrate the disease continuum: DMD defects in satellite cells cause myoblast dysfunctions diminishing muscle regeneration, which is essential to counteract myofiber degeneration. Full-length dystrophins play a critical role in these processes. Contrary to the established belief, our data identify myoblasts as a novel and important therapeutic target for treatment of this lethal disease.

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Section: Discussionmentioning

confidence: 99%

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

Gosselin

Mournetas

Borczyk

et al. 2021

Preprint

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“…Astoundingly, there is even evidence that myofibres can function entirely without dystrophin, just with overexpression of specific genetic modifiers [26]. Furthermore, cellautonomous defects are noticeable in DMD cells, which, when healthy, express the 14 kb DMD transcript but do not produce detectable levels of full-length dystrophins [27][28][29][30]. These, undoubtedly surprising findings, shed a new light on DMD pathophysiology (see below).…”

Section: Dystrophin Structure and Presumed Functionsmentioning

confidence: 98%

“…Therefore, no abnormalities have been foreseen. Against this reasonable prediction, in proliferating myoblasts, mutations in the DMD gene cause multiple changes in calcium signalling [28,30]. Next-generation sequencing (NGS) and subsequent biochemical analyses revealed changes in the expression of a number of genes encoding calcium signalling and related proteins (e.g., STIM 1, SERCA1 and 2A, calsequestrin, PLC variants 3 and 4, NCX 1 and 2, PMCA, Gαq11, and IP 3 R) [28].…”

Section: The Loss Of the Absentmentioning

confidence: 99%

“…Against this reasonable prediction, in proliferating myoblasts, mutations in the DMD gene cause multiple changes in calcium signalling [28,30]. Next-generation sequencing (NGS) and subsequent biochemical analyses revealed changes in the expression of a number of genes encoding calcium signalling and related proteins (e.g., STIM 1, SERCA1 and 2A, calsequestrin, PLC variants 3 and 4, NCX 1 and 2, PMCA, Gαq11, and IP 3 R) [28]. Moreover, numerous functional abnormalities including elevated SOCE activity [30], altered responses of both metabotropic and ionotropic receptors [28,29], and reduced oxygen consumption, together with stimulated glycolysis [74] distinguish immortalised mdx myoblasts from their wild type (w/t) equivalents.…”

Section: The Loss Of the Absentmentioning

confidence: 99%

“…Next-generation sequencing (NGS) and subsequent biochemical analyses revealed changes in the expression of a number of genes encoding calcium signalling and related proteins (e.g., STIM 1, SERCA1 and 2A, calsequestrin, PLC variants 3 and 4, NCX 1 and 2, PMCA, Gαq11, and IP 3 R) [28]. Moreover, numerous functional abnormalities including elevated SOCE activity [30], altered responses of both metabotropic and ionotropic receptors [28,29], and reduced oxygen consumption, together with stimulated glycolysis [74] distinguish immortalised mdx myoblasts from their wild type (w/t) equivalents. As these cells were maintained for generations in culture, changes appear to be cell-autonomous rather than triggered by the dystrophic niche.…”

Section: The Loss Of the Absentmentioning

confidence: 99%

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Disrupted Calcium Homeostasis in Duchenne Muscular Dystrophy: A Common Mechanism behind Diverse Consequences

Zabłocka

Górecki

Zabłocki

2021

IJMS

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Duchenne muscular dystrophy (DMD) leads to disability and death in young men. This disease is caused by mutations in the DMD gene encoding diverse isoforms of dystrophin. Loss of full-length dystrophins is both necessary and sufficient for causing degeneration and wasting of striated muscles, neuropsychological impairment, and bone deformities. Among this spectrum of defects, abnormalities of calcium homeostasis are the common dystrophic feature. Given the fundamental role of Ca2+ in all cells, this biochemical alteration might be underlying all the DMD abnormalities. However, its mechanism is not completely understood. While abnormally elevated resting cytosolic Ca2+ concentration is found in all dystrophic cells, the aberrant mechanisms leading to that outcome have cell-specific components. We probe the diverse aspects of calcium response in various affected tissues. In skeletal muscles, cardiomyocytes, and neurons, dystrophin appears to serve as a scaffold for proteins engaged in calcium homeostasis, while its interactions with actin cytoskeleton influence endoplasmic reticulum organisation and motility. However, in myoblasts, lymphocytes, endotheliocytes, and mesenchymal and myogenic cells, calcium abnormalities cannot be clearly attributed to the loss of interaction between dystrophin and the calcium toolbox proteins. Nevertheless, DMD gene mutations in these cells lead to significant defects and the calcium anomalies are a symptom of the early developmental phase of this pathology. As the impaired calcium homeostasis appears to underpin multiple DMD abnormalities, understanding this alteration may lead to the development of new therapies. In fact, it appears possible to mitigate the impact of the abnormal calcium homeostasis and the dystrophic phenotype in the total absence of dystrophin. This opens new treatment avenues for this incurable disease.

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Investigating the Involvement of C−X−C Motif Chemokine 5 and P2X7 Purinoceptor in Ectopic Calcification in Mouse Models of Duchenne Muscular Dystrophy

Rumney,

Pomeroy,

Górecki

2024

J of Cellular Biochemistry

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Ectopic calcification of myofibers is an early pathogenic feature in patients and animal models of Duchenne muscular dystrophy (DMD). In previous studies using the Dmdmdx‐βgeo mouse model, we found that the dystrophin‐null phenotype exacerbates this abnormality and that mineralised myofibers are surrounded by macrophages. Furthermore, the P2X7 purinoceptor, functioning in immune cells offers protection against dystrophic calcification. In the present study, by exploring transcriptomic data from Dmdmdx mice, we hypothesised these effects to be mediated by C−X−C motif chemokine 5 (CXCL5) downstream of P2X7 activation. We found that CXCL5 is upregulated in the quadriceps muscles of Dmdmdx‐βgeo mice compared to wild‐type controls. In contrast, at the cell level, dystrophic (SC5) skeletal muscle cells secreted less CXCL5 chemokine than wild‐type (IMO) controls. Although release from IMO cells was increased by P2X7 activation, this could not explain the elevated CXCL5 levels observed in dystrophic muscle tissue. Instead, we found that CXCL5 is released by dystrophin‐null macrophages in response to P2X7 activation, suggesting that macrophages are the source of CXCL5 in dystrophic muscles. The effects of CXCL5 upon mineralisation were investigated using the Alizarin Red assay to quantify calcium deposition in vitro. In basal (low phosphate) media, CXCL5 increased calcification in IMO but not SC5 myoblasts. However, in cultures treated in high phosphate media, to mimic dysregulated phosphate metabolism occurring in DMD, CXCL5 decreased calcification in both IMO and SC5 cells. These data indicate that CXCL5 is part of a homoeostatic mechanism regulating intracellular calcium, that CXCL5 can be released by macrophages in response to the extracellular ATP damage‐associated signal, and that CXCL5 can be part of a damage response to protect against ectopic calcification. This mechanism is affected by DMD gene mutations.

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

Cited by 17 publications

References 48 publications

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

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

Disrupted Calcium Homeostasis in Duchenne Muscular Dystrophy: A Common Mechanism behind Diverse Consequences

Investigating the Involvement of C−X−C Motif Chemokine 5 and P2X7 Purinoceptor in Ectopic Calcification in Mouse Models of Duchenne Muscular Dystrophy

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