Maud Beuvin

Mutations of the FHL1 Gene Cause Emery-Dreifuss Muscular Dystrophy

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.

Oxidative Stress in SEPN1-Related Myopathy: From Pathophysiology to Treatment

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.

A centronuclear myopathy-dynamin 2 mutation impairs skeletal muscle structure and function in mice

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-Dnm2(R465W)). 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.

Forelimb Treatment in a Large Cohort of Dystrophic Dogs Supports Delivery of a Recombinant AAV for Exon Skipping in Duchenne Patients

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

Satellite cell loss and impaired muscle regeneration in selenoprotein N deficiency

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.

Increased Muscle Stress-Sensitivity Induced by Selenoprotein N Inactivation in Mouse: A Mammalian Model for SEPN1-Related Myopathy

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.

DelK32-lamin A/C has abnormal location and induces incomplete tissue maturation and severe metabolic defects leading to premature death

The LMNA gene encodes lamin A/C intermediate filaments that polymerize beneath the nuclear membrane, and are also found in the nucleoplasm in an uncharacterized assembly state. They are thought to have structural functions and regulatory roles in signaling pathways via interaction with transcription factors. Mutations in LMNA have been involved in numerous inherited human diseases, including severe congenital muscular dystrophy (L-CMD). We created the Lmna(ΔK32) knock-in mouse harboring a L-CMD mutation. Lmna(ΔK32/ΔK32) mice exhibited striated muscle maturation delay and metabolic defects, including reduced adipose tissue and hypoglycemia leading to premature death. The level of mutant proteins was markedly lower in Lmna(ΔK32/ΔK32), and while wild-type lamin A/C proteins were progressively relocated from nucleoplasmic foci to the nuclear rim during embryonic development, mutant proteins were maintained in nucleoplasmic foci. In the liver and during adipocyte differentiation, expression of ΔK32-lamin A/C altered sterol regulatory element binding protein 1 (SREBP-1) transcriptional activities. Taken together, our results suggest that lamin A/C relocation at the nuclear lamina seems important for tissue maturation potentially by releasing its inhibitory function on transcriptional factors, including but not restricted to SREBP-1. And importantly, L-CMD patients should be investigated for putative metabolic disorders.

Dystrophin quantification: Biological and translational research implications

Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.

Can phrenic stimulation protect the diaphragm from mechanical ventilation-induced damage?

To the Editor: Mechanical ventilation is a prominent lifesaving treatment. It is, however, associated with an array of adverse effects, which include ventilator-associated pneumonias, volume-induced lung injury and, more recently identified, ventilator-induced diaphragm dysfunction (VIDD) [1–3]. VIDD combines diaphragm weakness with muscle fibre atrophy, remodelling and injury. Its mechanisms involve decreased protein synthesis, increased proteolysis, increased oxidative stress and mitochondrial dysfunction [2, 4]. Controlled mechanical ventilation appears to be the main, if not the sole, risk factor for VIDD, which in animal models is attenuated by the maintenance of respiratory efforts (assisted ventilatory modes) [2]. Although the corresponding human evidence is still lacking, this underlies the current notion that “clinicians should encourage persistent diaphragmatic activity” in patients receiving mechanical ventilation [2]. Diaphragm pacing has been proposed as a surrogate for spontaneous respiratory activity when the latter is not compatible with the condition of the patient [2, 5], but this approach has, seemingly, not yet been tested experimentally. Here we report a preliminary description of putative beneficial effects of diaphragm pacing in three mechanically ventilated sheep. Three female adult sheep (41, 32 and 34 kg), were anaesthetised (premedication: acepromazine 1.3 mg·kg−1 i.m. , 30 min before induction; induction: propofol 6 mg·kg−1 i.v. ; maintenance: continuous propofol 1–2 mg·kg−1·h−1, midazolam 0.3–2 mg·kg−1·h−1 and morphine 0.2–0.3 mg·kg−1·h−1; no paralysing agents), tracheotomised and mechanically ventilated with a minute ventilation ensuring normocapnia (Aisys, GE Healthcare, Datex Ohmeda, Madison, WI, USA). Additional oxygen was given to maintain transcutaneous-pulsed oxygen saturation >92%. Adequate fluid and nutritional support was provided and glycaemia controlled. Body temperature, heart rate and arterial pressure were monitored. Intradiaphragmatic phrenic nerve stimulation electrodes were inserted bilaterally in …

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy

Muscle contraction upon nerve stimulation relies on excitation–contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.