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Sporadic inclusion body myositis (sIBM) is the most common muscle disease of older people and is clinically characterized by slowly progressive asymmetrical muscle weakness, predominantly affecting the quadriceps, deep finger flexors, and foot extensors. At present, there are no enduring treatments for this relentless disease that eventually leads to severe disability and wheelchair dependency. Although sIBM is considered a rare muscle disorder, its prevalence is certainly higher as the disease is often undiagnosed or misdiagnosed. The histopathological phenotype of sIBM muscle biopsy includes muscle fiber degeneration and endomysial lymphocytic infiltrates that mainly consist of cytotoxic CD8+ T cells surrounding nonnecrotic muscle fibers expressing MHCI. Muscle fiber degeneration is characterized by vacuolization and the accumulation of congophilic misfolded multi-protein aggregates, mainly in their non-vacuolated cytoplasm. Many players have been identified in sIBM pathogenesis, including environmental factors, autoimmunity, abnormalities of protein transcription and processing, the accumulation of several toxic proteins, the impairment of autophagy and the ubiquitin–proteasome system, oxidative and nitrative stress, endoplasmic reticulum stress, myonuclear degeneration, and mitochondrial dysfunction. Aging has also been proposed as a contributor to the disease. However, the interplay between these processes and the primary event that leads to the coexistence of autoimmune and degenerative changes is still under debate. Here, we outline our current understanding of disease pathogenesis, focusing on degenerative mechanisms, and discuss the possible involvement of aging. [ABSTRACT FROM AUTHOR]

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed. [ABSTRACT FROM AUTHOR]

Missense mutations in MYOT encoding the sarcomeric Z‐disk protein myotilin cause three main myopathic phenotypes including proximal limb‐girdle muscular dystrophy, spheroid body myopathy, and late‐onset distal myopathy. We describe a family carrying a heterozygous MYOT deletion (Tyr4_His9del) that clinically was characterized by an early‐adult onset distal muscle weakness and pathologically by a myofibrillar myopathy (MFM). Molecular modeling of the full‐length myotilin protein revealed that the 4‐YERPKH‐9 amino acids are involved in local interactions within the N‐terminal portion of myotilin. Injection of in vitro synthetized mutated human MYOT RNA or of plasmid carrying its cDNA sequence in zebrafish embryos led to muscle defects characterized by sarcomeric disorganization of muscle fibers and widening of the I‐band, and severe motor impairments. We identify MYOT novel Tyr4_His9 deletion as the cause of an early‐onset MFM with a distal myopathy phenotype and provide data supporting the importance of the amino acid sequence for the structural role of myotilin in the sarcomeric organization of myofibers. [ABSTRACT FROM AUTHOR]

Down syndrome (DS) is a genetically based disease caused by triplication of chromosome 21. DS is characterized by multi‐systemic premature aging associated with deficit in motor coordination, balance, and postural control. Using a morphological, morphometrical, and immunocytochemical ultrastructural approach, this study investigated in vastus lateralis muscle of Ts65Dn mouse, a murine model of DS, the effect of an adapted physical training on the extracellular matrix (ECM) characteristics and whether the forecasted exercise‐induced ECM remodeling impacts on sarcomere organization. Morphometry demonstrated thicker basement membrane and larger collagen bundles with larger interfibrillar spacing as well as irregularly arrayed myofibrils and lower telethonin density on Z‐lines in trisomic versus euploid sedentary mice. In agreement with the multi‐systemic premature aging described in DS, these ECM alterations were similar to those previously observed in skeletal muscle of aged mice. Adapted physical training induced remodeling of ECM in both trisomic and euploid mice, that is, enlargement of the collagen bundles associated with hypertrophy of collagen fibrils and reduction of the interfibrillar spacing. A re‐alignment of the myofibrils and a higher telethonin density on Z‐line was found in trisomic mice. Altogether, our findings suggest that physical training is an effective tool in limiting/counteracting the trisomy‐associated musculoskeletal structural anomalies. The current findings constitute a solid experimental background for further study investigating the possible positive effect of physical training on skeletal muscle performance. Research Highlights: Vastus lateralis muscle of trisomic mice shows aging‐like alterations of extracellular matrix.Training promotes extracellular matrix remodeling.Training may be an effective tool to counteract trisomy‐associated alterations of skeletal muscle. [ABSTRACT FROM AUTHOR]

We aimed to verify whether the immune system may represent a source of potential biomarkers for the stratification of immune‐mediated necrotizing myopathies (IMNMs) subtypes. A group of 22 patients diagnosed with IMNM [7 with autoantibodies against signal recognition particle (SRP) and 15 against 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase (HMGCR)] and 12 controls were included. A significant preponderance of M1 macrophages was observed in both SRP+ and HMGCR+ muscle samples (p < 0.0001 in SRP+ and p = 0.0316 for HMGCR+), with higher values for SRP+ (p = 0.01). Despite the significant increase observed in the expression of TLR4 and all endosomal Toll‐like receptors (TLRs) at protein level in IMNM muscle tissue, only TLR7 has been shown considerably upregulated compared to controls at transcript level (p = 0.0026), whereas TLR9 was even decreased (p = 0.0223). Within IMNM subgroups, TLR4 (p = 0.0116) mRNA was significantly increased in SRP+ compared to HMGCR+ patients. Within IMNM group, only IL‐7 was differentially expressed between SRP+ and HMGCR+ patients, with higher values in SRP+ patients (p = 0.0468). Overall, innate immunity represents a key player in pathological mechanisms of IMNM. TLR4 and the inflammatory cytokine IL‐7 represent potential immune biomarkers able to differentiate between SRP+ and HMGCR+ patients. [ABSTRACT FROM AUTHOR]

Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and the accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at the one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes. which developed a myopathic phenotype consistent with that of human myofibrillar myopathy, including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets. [ABSTRACT FROM AUTHOR]

Two likely causative mutations in the RYR1 gene were identified in two patients with myopathy with tubular aggregates, but no evidence of cores or core‐like pathology on muscle biopsy. These patients were clinically evaluated and underwent routine laboratory investigations, electrophysiologic tests, muscle biopsy and muscle magnetic resonance imaging (MRI). They reported stiffness of the muscles following sustained activity or cold exposure and had serum creatine kinase elevation. The identified RYR1 mutations (p.Thr2206Met or p.Gly2434Arg, in patient 1 and patient 2, respectively) were previously identified in individuals with malignant hyperthermia susceptibility and are reported as causative according to the European Malignant Hyperthermia Group rules. To our knowledge, these data represent the first identification of causative mutations in the RYR1 gene in patients with tubular aggregate myopathy and extend the spectrum of histological alterations caused by mutation in the RYR1 gene. [ABSTRACT FROM AUTHOR]

Introduction/Aims: Stromal interaction molecule 1 (STIM1) is a reticular Ca2+ sensor composed of a luminal and a cytosolic domain. Autosomal dominant mutations in STIM1 cause tubular aggregate myopathy and Stormorken syndrome or its variant York platelet syndrome. In this study we aimed to expand the features related to new variants in STIM1. Methods: We performed a cross‐sectional study of individuals harboring monoallelic STIM1 variants recruited at five tertiary centers involved in a study of inherited myopathies analyzed with a multigene‐targeted panel. Results: We identified seven individuals (age range, 26‐57 years) harboring variants in STIM1, including five novel changes: three located in the EF‐hand domain, one in the sterile α motif (SAM) domain, and one in the cytoplasmatic region of the protein. Functional evaluation of the pathogenic variants using a heterologous expression system and measuring store‐operated calcium entry demonstrated their causative role and suggested a link of new variants with the clinical phenotype. Muscle contractures, found in three individuals, showed variability in body distribution and in the number of joints involved. Three patients showed cardiac and respiratory involvement. Short stature, hyposplenism, sensorineural hearing loss, hypothyroidism, and Gilbert syndrome were variably observed among the patients. Laboratory tests revealed hyperCKemia in six patients, thrombocytopenia in two patients, and hypocalcemia in one patient. Muscle biopsy showed the presence of tubular aggregates in three patients, type I fiber atrophy in one patient, and nonspecific myopathic changes in two patients. Discussion: Our clinical, histological, and molecular data expand the genetic and clinical spectrum of STIM1‐related diseases. [ABSTRACT FROM AUTHOR]

Brody disease is an autosomal recessive myopathy characterized by exercise-induced muscle stiffness due to mutations in the ATP2A1 gene. Almost 50 years after the initial case presentation, only 18 patients have been reported and many questions regarding the clinical phenotype and results of ancillary investigations remain unanswered, likely leading to incomplete recognition and consequently under-diagnosis. Additionally, little is known about the natural history of the disorder, genotype-phenotype correlations, and the effects of symptomatic treatment. We studied the largest cohort of Brody disease patients to date (n = 40), consisting of 22 new patients (19 novel mutations) and all 18 previously published patients. This observational study shows that the main feature of Brody disease is an exercise-induced muscle stiffness of the limbs, and often of the eyelids. Onset begins in childhood and there was no or only mild progression of symptoms over time. Four patients had episodes resembling malignant hyperthermia. The key finding at physical examination was delayed relaxation after repetitive contractions. Additionally, no atrophy was seen, muscle strength was generally preserved, and some patients had a remarkable athletic build. Symptomatic treatment was mostly ineffective or produced unacceptable side effects. EMG showed silent contractures in approximately half of the patients and no myotonia. Creatine kinase was normal or mildly elevated, and muscle biopsy showed mild myopathic changes with selective type II atrophy. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity was reduced and western blot analysis showed decreased or absent SERCA1 protein. Based on this cohort, we conclude that Brody disease should be considered in cases of exercise-induced muscle stiffness. When physical examination shows delayed relaxation, and there are no myotonic discharges at electromyography, we recommend direct sequencing of the ATP2A1 gene or next generation sequencing with a myopathy panel. Aside from clinical features, SERCA activity measurement and SERCA1 western blot can assist in proving the pathogenicity of novel ATP2A1 mutations. Finally, patients with Brody disease may be at risk for malignant hyperthermia-like episodes, and therefore appropriate perioperative measures are recommended. This study will help improve understanding and recognition of Brody disease as a distinct myopathy in the broader field of calcium-related myopathies. [ABSTRACT FROM AUTHOR]

Background: Chronic graft versus host disease (cGVHD) occurs in 20-30% of paediatric patients receiving haemopoietic stem cell transplantation (HSCT). Neuromuscular disorders such as polymyositis are considered a rare and distinctive but non-diagnostic manifestation of cGVHD and, in the absence of other characteristic signs and symptoms, biopsy is highly recommended to exclude other causes. Case report: We report a case of a 17-months-old child affected by hemophagocytic lymphohistiocytosis who underwent a matched unrelated donor haematopoietic stem cell transplantation (HSCT). She developed severe cGVHD-related polymyositis that was successfully treated with high-dose steroid therapy, rituximab and sirolimus. Conclusions: This is the first case of cGVHD-related-polymyositis described in a pediatric patient which was successfully treated with rituximab. [ABSTRACT FROM AUTHOR]

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2'-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules., 1. Introduction Duchenne muscular dystrophy (DMD) is an inherited X-linked degenerative muscle disorder mainly caused by frame-disrupting mutations following large rearrangements in the dystrophin gene [1]. DMD boys are affected [...]

Background: Several viruses have been described as causes of acquired inflammatory myopathies; however, the mechanisms by which they cause muscle disease are still unclear. The aim of this study was to describe the laboratory features of benign acute myositis in a small case series.Methods: A detailed pathological and serological analysis was performed in five African migrants who developed an acute viral myositis complicated by rhabdomyolysis.Results: Muscle biopsies clearly documented an inflammatory myopathy with histological features similar to polymyositis including CD8+ T cells surrounding and invading nonnecrotic muscle fibers, CD68+ macrophages and major histocompatibility complex class I antigen upregulation. In addition, positivity for myositis-specific antibodies (MSA), in particular anti-aminoacyl tRNA synthetases, was found in the serum of two patients.Conclusions: Our study demonstrated that T-cell mediated injury occurs in muscle of patients with acute viral myositis, and that MSA may be present in the serum of these patients. [ABSTRACT FROM AUTHOR]

The article focuses on the impact of adapted physical training on the extracellular matrix (ECM) in a murine model of Down syndrome (DS), showing that physical training can remodel the ECM in DS mice, potentially improving muscle structure and function.

Mitochondrial disorders are heterogeneous multisystemic disorders due to impaired oxidative phosphorylation causing defective mitochondrial energy production. Common histological hallmarks of mitochondrial disorders are RRFs (ragged red fibres), muscle fibres with abnormal focal accumulations of mitochondria. In contrast with the growing understanding of the genetic basis of mitochondrial disorders, the fate of phenotypically affected muscle fibres remains largely unknown. We investigated PCD (programmed cell death) in muscle of 17 patients with mitochondrial respiratory chain dysfunction. We documented that in affected muscle fibres, nuclear chromatin is condensed in lumpy irregular masses and cytochrome c is released into the cytosol to activate, along with Apaf-1 (apoptotic protease-activating factor 1), caspase 9 that, in turn, activates effector caspase 3, caspase 6, and caspase 7, suggesting the execution of the intrinsic apoptotic pathway. Whereas active caspase 3 underwent nuclear translocation, AIF (apoptosis-inducing factor) mainly stayed within mitochondria, into which an up-regulated Bax is relocated. The significant increase in caspase 2, caspase 3 and caspase 6 activity strongly suggest that the cell death programme is caspase-dependent and the activation of caspase 2 together with PUMA (p53 up-regulated modulator of apoptosis) up-regulation point to a role for oxidative stress in triggering the intrinsic pathway. Concurrently, in muscle of patients, the number of satellite cells was significantly increased and myonuclei were detected at different stages of myogenic differentiation, indicating that a reparative programme is ongoing in muscle of patients with mitochondrial disorders. Together, these data suggest that, in patients with mitochondrial disorders, affected muscle fibres are trapped in a mitochondria-regulated caspase-dependent PCD while repairing events take place. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that maternal peripheral blood leukocytes contribute to elevated levels of soluble TNF receptors (sTNFR) in preeclampsia (PE) with concomitant intrauterine growth restriction (IUGR). TNFR1 and TNFR2 were evaluated in a cross-sectional study comparing preeclamptic ( n = 15) with or without IUGR versus normotensive pregnant women (PREG, n = 30), and non-pregnant controls (Con; n = 20). Plasma levels of sTNFR1 were higher in PE (1675.0 ± 227.1 pg/mL) compared with PREG (1035.0 ± 101.1 pg/mL) and Con (589.3 ± 82.67 pg/mL), with the highest values observed in PE with IUGR (2624.0 ± 421.4 pg/mL; n = 6). Plasma sTNFR2 was higher during pregnancy (PE: 1836.0 ± 198.7 pg/mL; PREG: 1697.0 ± 95.0 pg/mL) compared with Con (598.3 ± 82.7 pg/mL). Urinary levels of sTNFR1 and sTNFR2 were higher in PE and PREG compared with the Con group. Abundance of TNFR1 mRNA in peripheral blood leukocytes was strongly correlated with plasma levels of sTNFR1 in PE. However, TNFR2 mRNA accumulation in leukocytes did not correlate with sTNFR2 plasma levels. The level of sTNFR1 in plasma was correlated with body weight of the newborn ( r = −0.56). The data suggest that maternal leukocytes contribute to sTNFR1 levels in plasma in association with decreasing newborn weight and PE with concomitant IUGR. [ABSTRACT FROM AUTHOR]

Autophagy has a large range of physiological functions and its dysregulation contributes to several human disorders, including autoinflammatory/autoimmune diseases such as inflammatory myopathies (IIMs). In order to better understand the pathogenetic mechanisms of these muscular disorders, we sought to define the role of autophagic processes and their relation with the innate immune system in the three main subtypes of IIM, specifically sporadic inclusion body myositis (sIBM), polymyositis (PM), dermatomyositis (DM) and juvenile dermatomyositis (JDM). We found that although the mRNA transcript levels of the autophagy-related genes BECN1, ATG5 and FBXO32 were similar in IIM and controls, autophagy activation in all IIM subgroups was suggested by immunoblotting results and confirmed by immunofluorescence. TLR4 and TLR3, two potent inducers of autophagy, were highly increased in IIM, with TLR4 transcripts significantly more expressed in PM and DM than in JDM, sIBM and controls, and TLR3 transcripts highly up-regulated in all IIM subgroups compared to controls. Co-localization between autophagic marker, LC3, and TLR4 and TLR3 was observed not only in sIBM but also in PM, DM and JDM muscle tissues. Furthermore, a highly association with the autophagic processes was observed in all IIM subgroups also for some TLR4 ligands, endogenous and bacterial HSP60, other than the high-mobility group box 1 (HMGB1). These findings indicate that autophagic processes are active not only in sIBM but also in PM, DM and JDM, probably in response to an exogenous or endogenous ‘danger signal’. However, autophagic activation and regulation, and also interaction with the innate immune system, differ in each type of IIM. Better understanding of these differences may lead to new therapies for the different IIM types. [ABSTRACT FROM AUTHOR]

Abstract: Brody disease is an inherited myopathy associated with a defective function of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) protein. Mutations in the ATP2A1 gene have been reported only in some patients. Therefore it has been proposed to distinguish patients with ATP2A1 mutations, Brody disease (BD), from patients without mutations, Brody syndrome (BS). We performed a detailed study of SERCA1 protein expression in muscle of patients with BD and BS, and evaluated the alternative splicing of SERCA1 in primary cultures of normal human muscle and in infant muscle. SERCA1 reactivity was observed in type 2 muscle fibers of patients with and without ATP2A1 mutations and staining intensity was similar in patients and controls. Immunoblot analysis showed a significant reduction of SERCA1 band in muscle of BD patients. In addition we demonstrated that the wild type and mutated protein exhibits similar solubility properties and that RIPA buffer improves the recovery of the wild type and mutated SERCA1 protein. We found that SERCA1b, the SERCA1 neonatal form, is the main protein isoform expressed in cultured human muscle fibers and infant muscle. Finally, we identified two novel heterozygous mutations within exon 3 of the ATP2A1 gene from a previously described patient with BD. [Copyright &y& Elsevier]

MDs (mitochondrial diseases) are a clinically heterogeneous group of disorders characterized by impairment of the respiratory chain function with altered oxidative phosphorylation. We tested the hypothesis that the function of vascular endothelium is affected by increased oxidative stress in MDs. A total of 12 patients with MDs and pair-matched controls were studied. Endothelial function was assessed by measuring FMD (flow-mediated vasodilation) of brachial and common femoral arteries. The test was repeated after vitamin C (500 mg, twice a day) and E (400 mg, once a day) supplementation for 30 days and 90 days after vitamin withdrawal. FMD was reduced in patients compared with controls [AUC/τ (time-averaged area under the curve) for the brachial artery, 1.05 ± 0.24 compared with 4.19 ± 0.59% respectively, P < 0.001; AUC/τ for the femoral artery, 0.98 ± 0.19 compared with 2.36 ± 0.29% respectively, P = 0.001; values are means ± S.E.M.] and correlated (brachial artery) with plasma lactate (r = -0.63, P < 0.01). Urinary 8-iso-PGF2α (8-iso-prostaglandin F2α) was higher in patients than controls (505.6 ± 85.9 compared with 302.5 ± 38.7 pg/mg of creatinine; P < 0.05) and correlated with plasma lactate (r = 0.70, P < 0.05). Immunohistochemical analysis showed 8-iso-PGF2α staining in MD-affected striated muscle cells and in blood vessels in muscle biopsies of patients. Antioxidant vitamins transiently restored FMD in patients [ΔAUC/τ (change in AUC/τ) for the brachial artery, +1.38 ± 0.49 %, P < 0.05; ΔAUC/τ for the femoral artery, +0.98 ± 0.24 %, P < 0.01] but had no effect on FMD in controls (brachial artery, -1.3 ± 0.63 %; and common femoral artery, -0.58 ± 0.30 %), thus abolishing the differences between patients and controls. The results of the present study indicate that oxidative stress is increased and is, at least partly, responsible for endothelial dysfunction in MDs. [ABSTRACT FROM AUTHOR]

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2′-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdxmice. Our data therefore demonstrate the long-termresidual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules. [ABSTRACT FROM AUTHOR]