In muscle ATP is primarily known for its function as an energy source and as a mediator of the "excitation-transcription" process, which guarantees muscle plasticity in response to environmental ...stimuli. When quickly released in massive concentrations in the extracellular space as in presence of muscle membrane damage, ATP acts as a damage-associated molecular pattern molecule (DAMP). In experimental murine models of muscular dystrophies characterized by membrane instability, blockade of eATP/P2X7 receptor (R) purinergic signaling delayed the progression of the dystrophic phenotype dampening the local inflammatory response and inducing Foxp3
T Regulatory lymphocytes. These discoveries highlighted the relevance of ATP as a harbinger of immune-tissue damage in muscular genetic diseases. Given the interactions between the immune system and muscle regeneration, the comprehension of ATP/purinerigic pathway articulated organization in muscle cells has become of extreme interest. This review explores ATP release, metabolism, feedback control and cross-talk with members of muscle inflammasome in the context of muscular dystrophies.
Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in
lead to ...caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.
Limb-girdle muscular dystrophy R3, a rare genetic disorder affecting the limb proximal muscles, is caused by mutations in the α-sarcoglycan gene (Sgca) and aggravated by an immune-mediated damage, ...finely modulated by the extracellular (e)ATP/purinoceptors axis. Currently, no specific drugs are available. The aim of this study was to evaluate the therapeutic effectiveness of a selective P2X7 purinoreceptor antagonist, A438079. Sgca knockout mice were treated with A438079 every two days at 3 mg/Kg for 24 weeks. The P2X7 antagonist improved clinical parameters by ameliorating mice motor function and decreasing serum creatine kinase levels. Histological analysis of muscle morphology indicated a significant reduction of the percentage of central nuclei, of fiber size variability and of the extent of local fibrosis and inflammation. A cytometric characterization of the muscle inflammatory infiltrates showed that A438079 significantly decreased innate immune cells and upregulated the immunosuppressive regulatory T cell subpopulation. In α-sarcoglycan null mice, the selective P2X7 antagonist A438079 has been shown to be effective to counteract the progression of the dystrophic phenotype and to reduce the inflammatory response. P2X7 antagonism via selective inhibitors could be included in the immunosuppressant strategies aimed to dampen the basal immune-mediated damage and to favor a better engraftment of gene-cell therapies.
Elastin microfibril interface-located proteins (EMILINs) are extracellular matrix glycoproteins implicated in elastogenesis and cell proliferation.
Recently, a missense mutation in the EMILIN1 gene ...has been associated with autosomal dominant connective tissue disorder and motor-sensory neuropathy in a single family.
We identified by whole exome sequencing a novel heterozygous EMILIN1 mutation c.748C>T p.R250C located in the coiled coil forming region of the protein, in four affected members of an autosomal dominant family presenting a distal motor neuropathy phenotype.
In affected patient a sensory nerve biopsy showed slight and unspecific changes in the number and morphology of myelinated fibers. Immunofluorescence study of a motor nerve within a muscle biopsy documented the presence of EMILIN-1 in nerve structures. Skin section and skin derived fibroblasts displayed a reduced extracellular deposition of EMILIN-1 protein with a disorganized network of poorly ramified fibers in comparison with controls.
Downregulation of emilin1a in zebrafish displayed developmental delay, locomotion defects, and abnormal axonal arborization from spinal cord motor neurons. The phenotype was complemented by wild-type zebrafish emilin1a, and partially the human wild-type EMILIN1 cRNA, but not by the cRNA harboring the novel c.748C>T p.R250C.
These data suggest a role of EMILIN-1 in the pathogenesis of diseases affecting the peripheral nervous system.
•EMILINs are extracellular matrix proteins implicated in elastogenesis.•We report EMILIN1 mutation in a family with motor neuropathy•We localised EMILIN-1 protein in perineurum of motor nerve bundles•Patients fibroblasts displayed a reduced deposition of EMILIN-1•Emilin1a zebrafish morphants displayed altered locomotion and abnormal motorneurons
Duchenne muscular dystrophy (DMD) is a devastating X-linked muscular disease, caused by mutations in the DMD gene encoding Dystrophin and affecting 1:5000 boys worldwide. Lack of Dystrophin leads to ...progressive muscle wasting and degeneration resulting in cardiorespiratory failure. Despite the absence of a definitive cure, innovative therapeutic avenues are emerging. Myopathologic studies are important to further understand the biological mechanisms of the disease and to identify histopathologic benchmarks for clinical evaluations. We conducted a myopathologic analysis on twenty-four muscle biopsies from DMD patients, with particular emphasis on regeneration, fibro-adipogenic progenitors and muscle stem cells behavior. We describe an increase in content of fibro-adipogenic progenitors, central orchestrators of fibrotic progression and lipid deposition, concurrently with a decline in muscle regenerative capacity. This regenerative impairment strongly correlates with compromised activation and expansion of muscle stem cells. Furthermore, our study uncovers an early acquisition of a senescence phenotype by DMD-afflicted muscle stem cells. Here we describe the myopathologic trajectory intrinsic to DMD and establish muscle stem cell senescence as a pivotal readout for future therapeutic interventions.
The role of muscle biopsy in the diagnostic workup of floppy infants is controversial. Muscle sampling is invasive, and often, results are not specific. The rapid expansion of genetic approach has ...made the muscle histopathology analysis less crucial. This study aims to assess the role and efficacy of muscle histopathology in the diagnostic algorithm of hypotonia in early infancy through a retrospective analysis of 197 infants who underwent muscle biopsy in their first 18 months of life. Data analysis revealed that 92/197 (46.7%) of muscle biopsies were non-specific (80) or normal (12), not allowing a specific diagnosis. In 41/197 (20.8%) cases, biopsy suggested a metabolic or mitochondrial myopathy, while in 23/197 cases (11.7%), we found evidence of muscular dystrophy. In 19/197 cases (9.7%), histopathology characteristics of a congenital myopathy were reported. In 22/197 cases (11.7%), the histopathological study indicated presence of a neurogenic damage. Overall, 46 diagnoses were then achieved by oriented genetic tests. Muscle biopsy results were consistent with genetic results in 90% of cases. Diagnostic algorithms for the diagnosis of a floppy infant are largely missing. Muscle biopsy alone can lead to a diagnosis, help the clinician in the choice of a genetic test, or even modify a diagnosis made previously.
We present a 13-year-old patient with persistent increase of serum Creatine Kinase (CK) and myalgia after exertion. Skeletal muscle biopsy showed marked reduction of dystrophin expression leading to ...genetic analysis of DMD gene by MLPA, which detected a single deletion of exon 78. To the best of our knowledge, DMD exon 78 deletion has never been described in literature and, according to prediction, it should lead to loss of reading frame in the dystrophin gene. To further assess the actual effect of exon 78 deletion, we analysed cDNA from muscle mRNA. This analysis confirmed the absence of 32 bp of exon 78. Exclusion of exon 78 changes the open reading frame of exon 79 and generate a downstream stop codon, producing a dystrophin protein of 3703 amino acids instead of 3685 amino acids. Albeit loss of reading frame usually leads to protein degradation and severe phenotype, in this case, we demonstrated that deletion of DMD exon 78 can be associated with a functional protein able to bind DGC complex and a very mild phenotype. This study adds a novel deletion in DMD gene in human and helps to define the compliance between maintaining/disrupting the reading frame and clinical form of the disease.
Congenital titinopathies are an emerging group of a potentially severe form of congenital myopathies caused by biallelic mutations in titin, encoding the largest existing human protein involved in ...the formation and stability of sarcomeres. In this study we describe a patient with a congenital myopathy characterized by multiple contractures, a rigid spine, non progressive muscular weakness, and a novel homozygous TTN pathogenic variant in a metatranscript-only exon: the c.36400A > T, p.Lys12134*. Muscle biopsies showed increased internalized nuclei, variability in fiber size, mild fibrosis, type 1 fiber predominance, and a slight increase in the number of satellite cells. RNA studies revealed the retention of intron 170 and 171 in the open reading frame, and immunoflourescence and western blot studies, a normal titin content. Single fiber functional studies showed a slight decrease in absolute maximal force and a cross-sectional area with no decreases in tension, suggesting that weakness is not sarcomere-based but due to hypotrophy. Passive properties of single fibers were not affected, but the observed increased calcium sensitivity of force generation might contribute to the contractural phenotype and rigid spine of the patient. Our findings provide evidence for a pathogenic, causative role of a metatranscript-only titin variant in a long survivor congenital titinopathy patient with distal arthrogryposis and rigid spine.
Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by ...reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies.
Resource tableImage 1Unique stem cell lines identifierEURACi001-AEURACi002-AEURACi003-AAlternative names of stem cell linesB2CAV3 (EURACi001-A)L1CAV3 (EURACi002-A)N1CAV3 (EURACi003-A)InstitutionInstitute for Biomedicine, Eurac ResearchContact information of distributorAlessandra Rossini (alessandra.rossini@eurac.edu)Type of cell linesiPSCsOriginHumanCell sourcePeripheral blood mononuclear cells (PBMCs)Method of reprogrammingElectroporation of episomal vectors (pCXLE hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hUL)Multiline rationaleNon-isogenic cell lines obtained from patients with mutations in the same gene (CAV3)Gene modificationNOType of modificationSpontaneous mutationsAssociated diseaseCaveolinopathiesGene/locusHeterozygous CAV3 c.Δ184–192 (EURACi001-A)Heterozygous CAV3 c.303 TGG>TGC (EURACi002-A)Heterozygous CAV3 c.233 ACG>AAG (EURACi003-A)Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateJanuary 2016 (EURACi001-A)September 2016 (EURACi002-A)May 2016 (EURACi003-A)Cell line repository/bankN/AEthical approvalPeripheral blood was collected from patients after signing the informed consent provided by Cell Line and DNA Biobank from Patients Affected by Genetic Diseases, member of the Telethon Network of Genetic Biobanks, Istituto G. Gaslini, Genoa, Italy. The generation and use of iPSCs was reviewed and approved by Ethical Committee at Universita’ degli Studi di Milano (03.06.15, number 29/15).
Sarcoglycanopathies, limb-girdle muscular dystrophies (LGMD) caused by genetic loss-of-function of the membrane proteins sarcoglycans (SGs), are characterized by progressive degeneration of skeletal ...muscle. In these disorders, muscle necrosis is associated with immune–mediated damage, whose triggering and perpetuating molecular mechanisms are not fully elucidated yet. Extracellular adenosine triphosphate (eATP) seems to represent a crucial factor, with eATP activating purinergic receptors. Indeed, in vivo blockade of the eATP/P2X7 purinergic pathway ameliorated muscle disease progression. P2X7 inhibition improved the dystrophic process by restraining the activity of P2X7 receptors on immune cells. Whether P2X7 blockade can display a direct action on muscle cells is not known yet. In this study, we investigated eATP effects in primary cultures of myoblasts isolated from patients with LGMDR3 (α-sarcoglycanopathy) and in immortalized cells isolated from a patient with LGMDR5 (γ-sarcoglycanopathy). Our results demonstrated that, owing to a reduced ecto-ATPase activity and/or an enhanced release of ATP, patient cells are exposed to increased juxtamembrane concentrations of eATP and display a higher susceptivity to eATP signals. The purinoceptor P2Y2, which proved to be overexpressed in patient cells, was identified as a pivotal receptor responsible for the enhanced ATP-induced or UTP-induced Ca2+ increase in affected myoblasts. Moreover, P2Y2 stimulation in LDMDR3 muscle cells induced chemotaxis of immune cells and release of interleukin-8. In conclusion, a higher eATP concentration and sensitivity in primary human muscle cells carrying different α-SG or γ-SG loss-of-function mutations indicate that eATP/P2Y2 is an enhanced signaling axis in cells from patients with α-/γ-sarcoglycanopathy. Understanding the basis of the innate immune–mediated damage associated with the dystrophic process may be critical in overcoming the immunologic hurdles associated with emerging gene therapies for these disorders.
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