Objective
Mutations of the selenoprotein N gene (SEPN1) cause SEPN1‐related myopathy (SEPN1‐RM), a novel early‐onset muscle disorder formerly divided into four different nosological categories. ...Selenoprotein N (SelN) is the only selenoprotein involved in a genetic disease; its function being unknown, no treatment is available for this potentially lethal disorder. Our objective was to clarify the role of SelN and the pathophysiology of SEPN1‐RM to identify therapeutic targets.
Methods
We established and analyzed an ex vivo model of SelN deficiency using fibroblast and myoblast primary cultures from patients with null SEPN1 mutations. DCFH assay, OxyBlot, Western blot, Fura‐2, and cell survival studies were performed to measure intracellular oxidant activity, oxidative stress markers, calcium handling, and response to exogenous treatments.
Results
SelN‐depleted cells showed oxidative/nitrosative stress manifested by increased intracellular oxidant activity (reactive oxygen species and nitric oxide) and/or excessive oxidation of proteins, including the contractile proteins actin and myosin heavy chain II in myotubes. SelN‐devoid myotubes showed also Ca2+ homeostasis abnormalities suggesting dysfunction of the redox‐sensor Ca2+ channel ryanodine receptor type 1. Furthermore, absence of SelN was associated with abnormal susceptibility to H2O2‐induced oxidative stress, demonstrated by increased cell death. This cell phenotype was restored by pretreatment with the antioxidant N‐acetylcysteine.
Interpretation
SelN plays a key role in redox homeostasis and human cell protection against oxidative stress. Oxidative/nitrosative stress is a primary pathogenic mechanism in SEPN1‐RM, which can be effectively targeted ex vivo by antioxidants. These findings pave the way to SEPN1‐RM treatment, which would represent a first specific pharmacological treatment for a congenital myopathy. Ann Neurol 2009;65:677–686
Cardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by increased myocardial fibrosis, which impairs left ventricular relaxation and predisposes to heart failure, ...and cardiac conduction abnormalities. While we previously discovered abnormally elevated extracellular signal-regulated kinase 1/2 (ERK1/2) activities in heart in LMNA cardiomyopathy, its role on the development of myocardial fibrosis remains unclear. We now showed that transforming growth factor (TGF)-β/Smad signaling participates in the activation of ERK1/2 signaling in LMNA cardiomyopathy. ERK1/2 acts on connective tissue growth factor (CTGF/CCN2) expression to mediate the myocardial fibrosis and left ventricular dysfunction. Studies in vivo demonstrate that inhibiting CTGF/CCN2 using a specific antibody decreases myocardial fibrosis and improves the left ventricular dysfunction. Together, these findings show that cardiac ERK1/2 activity is modulated in part by TGF-β/Smad signaling, leading to altered activation of CTGF/CCN2 to mediate fibrosis and alter cardiac function. This identifies a novel mechanism in the development of LMNA cardiomyopathy.
Short-term mechanical ventilation (MV) protects against sepsis-induced diaphragmatic dysfunction. Prolonged MV induces diaphragmatic dysfunction in non-septic animals, but few reports describe the ...effects of prolonged MV in sepsis. We hypothesized that prolonged MV is not protective but worsens the diaphragmatic dysfunction induced by a mild sepsis, because MV and sepsis share key signaling mechanisms, such as cytokine upregulation.
We studied the impact of prolonged MV (12 h) in four groups (n = 8) of male Wistar rats: 1) endotoxemia induced by intraperitoneal injection of Escherichia coli lipopolysaccharide, 2) MV without endotoxemia, 3) combination of endotoxemia and MV and 4) sham control. Diaphragm mechanical performance, pro-inflammatory cytokine concentrations (Tumor Necrosis Factor-α, Interleukin-1β, Interleukin-6) in plasma were measured.
Prolonged MV and sepsis independtly reduced maximum diaphragm force (-27%, P = 0.003; -37%, P<0.001; respectively). MV and sepsis acted additively to further decrease diaphragm force (-62%, P<0.001). Similar results were observed for diaphragm kinetics (maximum lengthening velocity -47%, P<0.001). Sepsis and MV reduced diaphragm cross sectional area of type I and IIx fibers, which was further increased by the combination of sepsis and MV (all P<0.05). Sepsis and MV were individually associated with the presence of a robust perimysial inflammatory infiltrate, which was more marked when sepsis and MV were both present (all P<0.05). Sepsis and, to a lesser extent, MV increased proinflammatory cytokine production in plasma and diaphragm (all P<0.05); proinflammatory cytokine expression in plasma was increased further by the combination of sepsis and MV (all P<0.05). Maximum diaphragm force correlated negatively with plasma and diaphragmatic cytokine production (all p<0.05).
Prolonged (12 h) MV exacerbated sepsis-induced decrease in diaphragm performance. Systemic and diaphragmatic overproduction of pro-inflammatory cytokines may contribute to diaphragm weakness.
Rapid advances in allele‐specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for ...autosomal‐dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele‐specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2‐mRNA harbouring the p.R465W mutation without affecting the wild‐type allele. Functional restoration was achieved in muscle from a knock‐in mouse model and in patient‐derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient‐derived cells is an essential breakthrough towards future gene‐based therapy for dominant centronuclear myopathy.
Synopsis
Autosomal dominant centronuclear myopathy (AD‐CNM) is a rare congenital myopathy due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele‐specific silencing of the mutant allele alleviates the phenotype in a CNM knock‐in mouse model and patient‐derived fibroblasts.
siRNA targeting the mutant allele leads to functional restoration of the impaired endocytosis in mutant human fibroblasts.
Early intra‐muscular administration of AAV1 expressing allele‐specific shRNA prevents the disease in the mouse model.
Late treatment in the disease's time course partially improve the phenotype due to a weaker transduction capacity of the muscle.
Allele‐specific silencing is a promising therapeutic strategy for AD‐CNM.
Autosomal dominant centronuclear myopathy (AD‐CNM) is a rare congenital myopathy due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele‐specific silencing of the mutant allele alleviates the phenotype in a CNM knock‐in mouse model and patient‐derived fibroblasts.
Emery-Dreifuss muscular dystrophy (EDMD) is a rare disorder characterized by early joint contractures, muscular dystrophy, and cardiac involvement with conduction defects and arrhythmias. So far, ...only 35% of EDMD cases are genetically elucidated and associated with
EMD or
LMNA gene mutations, suggesting the existence of additional major genes. By whole-genome scan, we identified linkage to the Xq26.3 locus containing the
FHL1 gene in three informative families belonging to our
EMD- and
LMNA-negative cohort. Analysis of the
FHL1 gene identified seven mutations, in the distal exons of
FHL1 in these families, three additional families, and one isolated case, which differently affect the three FHL1 protein isoforms: two missense mutations affecting highly conserved cysteines, one abolishing the termination codon, and four out-of-frame insertions or deletions. The predominant phenotype was characterized by myopathy with scapulo-peroneal and/or axial distribution, as well as joint contractures, and associated with a peculiar cardiac disease characterized by conduction defects, arrhythmias, and hypertrophic cardiomyopathy in all index cases of the seven families. Heterozygous female carriers were either asymptomatic or had cardiac disease and/or mild myopathy. Interestingly, four of the
FHL1-mutated male relatives had isolated cardiac disease, and an overt hypertrophic cardiomyopathy was present in two. Expression and functional studies demonstrated that the FHL1 proteins were severely reduced in all tested patients and that this was associated with a severe delay in myotube formation in the two patients for whom myoblasts were available. In conclusion,
FHL1 should be considered as a gene associated with the X-linked EDMD phenotype, as well as with hypertrophic cardiomyopathy.
Autosomal dominant centronuclear myopathy (AD-CNM) is due to mutations in the gene encoding dynamin 2 (DNM2) involved in endocytosis and intracellular membrane trafficking. To understand the ...pathomechanisms resulting from a DNM2 mutation, we generated a knock-in mouse model expressing the most frequent AD-CNM mutation (KI-Dnm2R465W). Heterozygous (HTZ) mice developed a myopathy showing a specific spatial and temporal muscle involvement. In the primarily and prominently affected tibialis anterior muscle, impairment of the contractile properties was evidenced at weaning and was progressively associated with atrophy and histopathological abnormalities mainly affecting mitochondria and reticular network. Expression of genes involved in ubiquitin–proteosome and autophagy pathways was up-regulated during DNM2-induced atrophy. In isolated muscle fibers from wild-type and HTZ mice, Dnm2 localized in regions of intense membrane trafficking (I-band and perinuclear region), emphasizing the pathophysiological hypothesis in which DNM2-dependent trafficking would be altered. In addition, HTZ fibers showed an increased calcium concentration as well as an intracellular Dnm2 and dysferlin accumulation. A similar dysferlin retention, never reported so far in congenital myopathies, was also demonstrated in biopsies from DNM2-CNM patients and can be considered as a new marker to orientate direct genetic testing. Homozygous (HMZ) mice died during the first hours of life. Impairment of clathrin-mediated endocytosis, demonstrated in HMZ embryonic fibroblasts, could be the cause of lethality. Overall, this first mouse model of DNM2-related myopathy shows the crucial role of DNM2 in muscle homeostasis and will be a precious tool to study DNM2 functions in muscle, pathomechanisms of DNM2-CNM and developing therapeutic strategies.
Selenoprotein N (SelN) deficiency causes a group of inherited neuromuscular disorders termed SEPN1-related myopathies (SEPN1-RM). Although the function of SelN remains unknown, recent data ...demonstrated that it is dispensable for mouse embryogenesis and suggested its involvement in the regulation of ryanodine receptors and/or cellular redox homeostasis. Here, we investigate the role of SelN in satellite cell (SC) function and muscle regeneration, using the Sepn1(-/-) mouse model. Following cardiotoxin-induced injury, SelN expression was strongly up-regulated in wild-type muscles and, for the first time, we detected its endogenous expression in a subset of mononucleated cells by immunohistochemistry. We show that SelN deficiency results in a reduced basal SC pool in adult skeletal muscles and in an imperfect muscle restoration following a single injury. A dramatic depletion of the SC pool was detected after the first round of degeneration and regeneration that totally prevented subsequent regeneration of Sepn1(-/-) muscles. We demonstrate that SelN deficiency affects SC dynamics on isolated single fibres and increases the proliferation of Sepn1(-/-) muscle precursors in vivo and in vitro. Most importantly, exhaustion of the SC population was specifically identified in muscle biopsies from patients with mutations in the SEPN1 gene. In conclusion, we describe for the first time a major physiological function of SelN in skeletal muscles, as a key regulator of SC function, which likely plays a central role in the pathophysiological mechanism leading to SEPN1-RM.
Obesity is associated with a decrease in mortality in the intensive care unit (ICU) (the "obesity paradox"). We hypothesized that obesity may paradoxically improve diaphragmatic function.
Diaphragm ...contractility was prospectively recorded in vitro in adult male Zucker lean (control), fatty, and diabetic fatty rats, at rest, after 12h mechanical ventilation and after fatigue. We analyzed diaphragm morphology, cytokines, and protein expression of the protein kinase signaling pathways.
Diaphragm active-force (AF) was higher in fatty (96±7mN.mm-2,P = 0.02) but not in diabetic fatty rats (90±17mN.mm-2) when compared with controls (84±8mN.mm-2). Recovery from fatigue was improved in fatty and diabetic fatty groups compared with controls. Ventilator-induced diaphragmatic dysfunction was observed in each group, but AF remained higher in fatty (82±8mN.mm-2,P = 0.03) compared with controls (70±8mN.mm-2). There was neutral lipid droplet accumulation in fatty and diabetic fatty. There were shifts towards a higher cross-sectional-area (CSA) of myosin heavy chain isoforms (MyHC)-2A fibers in fatty and diabetic fatty compared with control rats (P = 0.002 and P<0.001, respectively) and a smaller CSA of MyHC-2X in fatty compared with diabetic fatty and control rats (P<0.001 and P<0.001, respectively). The phosphorylated total-protein-kinase-B (pAKT)/AKT ratio was higher in fatty (182±58%,P = 0.03), but not in diabetic fatty when compared with controls and monocarboxylate-transporter-1 was higher in diabetic fatty (147±36%,P = 0.04), but not in fatty.
Diaphragmatic force is increased in Zucker obese rats before and after mechanical ventilation, and is associated with activation of AKT pathway signaling and complex changes in morphology.
Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force production. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains ...unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Emery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolymerizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations.
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•Phosphorylation of cofilin-1 on Thr25 by p-ERK1/2 protects it from degradation by the UPS•Phospho(T25)-cofilin-1 localizes on I-bands in sarcomeres•Phosphorylation of cofilin-1 on Thr25 alters sarcomeric organization•Phospho(T25)-cofilin-1 alter skeletal muscle force and lead to muscular dystrophy
Extracellular signal-regulated kinase (ERK) 1/2 has been shown to be important for the development of muscular dystrophy. Vignier et al. find that active ERK1/2 catalyzes phosphorylation of cofilin-1 on Thr25 and protects it from proteasomal degradation, which physically disrupts sarcomeric organization and causes loss of muscle force generation.
Selenium is an essential trace element and selenoprotein N (SelN) was the first selenium-containing protein shown to be directly involved in human inherited diseases. Mutations in the SEPN1 gene, ...encoding SelN, cause a group of muscular disorders characterized by predominant affection of axial muscles. SelN has been shown to participate in calcium and redox homeostasis, but its pathophysiological role in skeletal muscle remains largely unknown. To address SelN function in vivo, we generated a Sepn1-null mouse model by gene targeting. The Sepn1(-/-) mice had normal growth and lifespan, and were macroscopically indistinguishable from wild-type littermates. Only minor defects were observed in muscle morphology and contractile properties in SelN-deficient mice in basal conditions. However, when subjected to challenging physical exercise and stress conditions (forced swimming test), Sepn1(-/-) mice developed an obvious phenotype, characterized by limited motility and body rigidity during the swimming session, as well as a progressive curvature of the spine and predominant alteration of paravertebral muscles. This induced phenotype recapitulates the distribution of muscle involvement in patients with SEPN1-Related Myopathy, hence positioning this new animal model as a valuable tool to dissect the role of SelN in muscle function and to characterize the pathophysiological process.
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