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

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

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies.

peer reviewedNemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation

A muscle hybrid promoter as a novel tool for gene therapy

International audienceGene therapy is a promising strategy to cure rare diseases. The lack of regulatory sequences ensuring specific and robust expression in skeletal and cardiac muscle is a substantial limitation of gene therapy efficiency targeting the muscle tissue. Here we describe a novel muscle hybrid (MH) promoter that is highly active in both skeletal and cardiac muscle cells. It has an easily exchangeable modular structure, including an intronic module that highly enhances the expression of the gene driven by it. In cultured myoblasts, myotubes, and cardiomyocytes, the MH promoter gives relatively stable expression as well as higher activity and protein levels than the standard CMV and desmin gene promoters or the previously developed synthetic or CKM-based promoters. Combined with AAV2/9, the MH promoter also provides a high in vivo expression level in skeletal muscle and the heart after both intramuscular and systemic delivery. It is much more efficient than the desmin-encoding gene promoter, and it maintains the same specificity. This novel promoter has potential for gene therapy in muscle cells. It can provide stable transgene expression, ensuring high levels of therapeutic protein, and limited side effects because of its specificity. This constitutes an improvement in the efficiency of genetic disease therapy

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

International audienceMechanical 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 the hemidiaphragms using the cervical incision thoraco-endoscopic approach (CITES) [6]. Diaphragm pacing (NeurRxDP4 stimulator, Synapse Biomedical Inc., Oberlin, OH, USA) was instituted within 2 h following the initiation of mechanical ventilation. Only one hemidiaphragm was stimulated throughout the experiments (right in two cases, left in one case), allowing comparison between mechanically ventilated hemidiaphragms (nonstimulated side) and mechanically ventilated with diaphragm pacing (MV+stim) ones (stimulated side). 30 min stimulation sessions were superimposed upon mechanical ventilation at 4 h intervals (18 breaths per minute, stimulation intensity 15 mA, stimulation frequency 20 Hz, inspiratory time 1.1 s and pulse width 150 ms, mechanical and electrical inspirations were synchronous). One animal suddenly died after 48 h of mechanical ventilation (#3) and the two others (#1 and #2) were sacrificed after 72 h of mechanical ventilation. Costal diaphragm strips measuring 1068 cm were taken through an abdominal incision, immediately post mortem in animal #3 and immediately before sacrifice in animals #1 and #2. Diaphragm samples were mounted on a small mound of 10% Gum Tragacanth placed on a cork disc and frozen in isopentane cooled with liquid nitrogen. Transverse serial cryosections (8 mm thick) of the costal diaphragm were stained with haematoxylin and eosin, and analysed qualitatively for structural abnormalities. Other sections were stained for adenosine triphosphatase (ATPase pH 9.4) and fibre types were identified, according to their histochemical reactions, as slow twitch type I or fast-twitch type II fibres using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). For each fibre type, an average cross-sectional area was determined from at least 150 fibres taken from six different fields in each hemidiaphragm, using NIS software (Nikon Instruments Europe B.V., Amsterdam, The Netherlands). Graphpad Prism (Graphpad Software, San Diego, CA) was used to calculate and plot the means and standard errors of the mean of measured quantities. Differences between the mechanically ventilated and MV+stim conditions were assessed using paired t-tests and were considered significant at p,0.05. In the three animals studied, mechanically ventilated hemidiaphragms showed signs of severe damage consisting of hypercontracted fibres with apparent lipid droplet accumulation and intense oedematous infiltrate of the interstitium leading to fibre disorganisation (fig. 1a). These structural abnormalities were not observed in the MV+stim hemidiaphragms, even after 72 h of mechanical ventilation (fig. 1a). The LETTERS 28

Gene therapy for striated muscle laminopathy

International audienceLMNA-related Congenital Muscular Dystrophy (L-CMD) is the most severe form of striated muscle laminopathies caused by mutations in LMNA. Our purpose is to development gene therapy for L-CMD to correct LMNA mutations at mRNA level and improve the function of affected organs. Homozygous Lmna-delk32 mice model L-CMD, pathology caused by both the expression of toxic mutant lamin A/C and absence of WT lamin A/C expression, while heterozygous mice, a model of dilated cardiomyopathy, present with reduced WT lamin A/C expression and residual mutant lamin A/C expression in the heart. Based on these facts, we aim at developing a therapeutic approach that both reduces the expression of the mutant proteins and restores the normal lamin A/C levels. To achieve such strategy, we developed AAV2/9 vector containing either human mature lamin A under control of a CMV promoter, or in combination with shRNA against either only mutant mouse Lmna mRNA or both alleles. These AAVs were injected intravenously at 1x10E11 viral genomes for WT, homozygous and heterozygous Lmna-delk32 mice at 2 days of age. All these treatments showed benefits in terms of max survival of homozygous mice. While, only transient benefit was shown for heterozygous mice. In addition, we could not find clear effects on cardiac function for heterozygous mice. Interestingly, 6 mice had liver nodule of total 50 treated mice, and three of them were diagnosed with hepatocellular carcinoma. From molecular analysis at end stage, expression of human lamin A mRNA was increased in both heart and liver. While, expression of endogenous mouse Lmna mRNA was decreased only in liver. Furthermore, we detected more quantity of viral genome in liver than heart. From this in vivo study, we will optimize and improve the therapeutic cassette to increase the efficacy of tissue targeting of these tools in the affected organs

Gene therapy for striated muscle laminopathy

International audienc