PAPERS THAT WILL BE PRESENTED IN WORLD MUSCLE SOCIETY ANNUAL MEETING 2009 - SWITZERLAND, SEPTEMBER 9-12.

M.I.1 New methods for assessing disease progression in neuromuscular disorders: Assessment of strength and function

E. Mercuri^a

^aCatholic University Rome, Pediatric Neurology, Rome, Italy

The number of clinical trials in children with hereditary neuromuscular disorders has steadily increased over the last decade. The issue of the lack of accepted gold standards for outcome measures in neuromuscular disorders has only recently been systematically addressed. A systematic review of the clinical trials published reveals that the existing measures are often not appropriate to detect functional changes in the cohorts assessed.

In the last few years there have been several advances in this field. The increasing collaboration between European and US centers has brought some consensus on the most suitable outcome measures for spinal muscular atrophy and Duchenne muscular dystrophy, highlighting the need for disease-specific scales.

Further discussion with regulatory agencies and with parents’ associations has also confirmed that outcome measures should measure changes that are’clinically meaningful’ for the patients and their families and the need for appropriate measures assessing quality of life of patients and caregivers.

M.I.2 Metabolic assessments of muscle disease

J. Vissing^a

^aRigshospitalet, University of Copenhagen, Department of Neurology, Copenhagen, Denmark

Assessment of metabolic function in muscle disease is a fundamental tool to understand the pathophysiology of these conditions, and to plan treatment strategies. The type of assessments differs between muscle conditions that are characterized by muscle wasting, and those that primarily have a deficiency in generating enough energy during exercise, due to inborn errors of muscle metabolism. Collectively, assessment of maximal work output, such as that which can be tested with an incremental test to exhaustion on a cycle ergometer, may provide important documentation of the functional limitation in any muscle disease. Stabile isotope methodology is another important tool to monitor metabolism in muscle diseases. In muscular dystrophies, stabile isotopes have been used to demonstrate the importance of skeletal muscle as a source of gluconeogenesis. Thus, in conditions in with severe muscle wasting, fasting induces pronounced hypoglycemia. Assessment of protein turnover, using stabile isotope-labelled amino acids, has also provided important insights into protein balance in conditions with muscle wasting, particularly in the context of exercise and protein supplement. In metabolic myopathies, stabile isotope methodology is pivotal in evaluating whether symptomatic carriers of single mutations in genes of intermediary metabolism are truly affected. Stabile isotope studies of carbohydrate and lipid oxidation in patients with respiratory chain deficiencies, and patients with enzyme defects of lipid or carbohydrate metabolism, have to a great extent been instrumental for the understanding of the pathophysiology underlying the limitations in exercise capacity, which characterize these conditions.

M.I.3 Quantitative assessment of skeletal muscle by NMR: Current and future approaches

P.G. Carlier^a and The NMR team

^aInstitute of Myology, NMR Laboratory, IdM – CEA, Paris, France

A range of NMR imaging, functional imaging and spectroscopy sequences and protocols are available for non-invasive investigation of skeletal muscle anatomy and structure, physiology and biochemistry in vivo. Their primary application is not for diagnostic purposes but rather to monitor the natural progression of muscle disorders as well as to assess the response to therapeutic interventions. To accomplish these missions, the NMR variables must be quantified with the highest possible reproducibility and exactitude.

Muscle trophicity can be measured very precisely, particularly using 3D anatomical protocols. In the future, tedious manual image processing will be replaced by segmentation algorithms, with minimum intervention of operators.

Based on a voxel by voxel analysis of the NMR signal, three main components can be identified in muscle: muscle tissue itself, fatty replacement and fibrosis. For a precise quantitation of the three components, one needs to correct for the bias introduced by magnetic field and radio-frequency field inhomogeneities. Newer approaches, from T1-rho or ultra-short TE sequences to Gd-molecules with high affinity for collagen, aim at a direct visualisation of the fibrotic component. It is possible to probe the sarcoplasmic membrane integrity by time-course studies of muscle T1w signal enhancement after injection of Gd-chelates, possibly coupled to albumin.

Functional and metabolic characterization of skeletal muscle with dedicated NMR sequences adds another dimension to the evaluation of disease progression and therapeutic responses. Muscle perfusion, capillary and intra-myocytic oxygenation and energy metabolism can be simultaneously determined using rapidly interleaved arterial spin labelled and BOLD imaging, deoxy-myoglobin proton spectroscopy and phosphorus spectroscopy during exercise protocols. They provide valuable information on the respective contributions of oxygen supply, extraction and utilization to particular conditions or diseases.

M.I.4 Discovering biomarkers for monitoring neuromuscular diseases: A turning point

A. Ferlini^a

^aUniversity of Ferrara, Experimental and Diagnostic Medicine, Genetics, Ferrara, Italy

Neuromuscular hereditary disorders (NMDs) represent heterogeneous phenotypes intensively studied both in terms of accurate genetic definition and pathophysiology. Since therapeutic approaches are now becoming reality in some NMDs, clinical trials have been designed to track down correction of a decline in muscle function, requiring large samples and lengthy times. In addition, clinical outcome measures may not always be sensitive enough to detect small changes in disease progression/regression and after short treatment periods. It is therefore imperative to be able to document benefits of the treatment at the individual level to justify demanding, risky, and expensive treatments. Biomarkers can effectively increase our capacity of studying and monitoring NMDs. They can be defined as diagnostic if able to implement the genotype–phenotype correlation, as well as to label the disease course; pathophysiological, when they disclose or integrate common pathways, therapeutic (pharmacogenetic, pharmacodynamic, pharmacokinetic and safety) when they can be translated into clinical trials. Other disciplines, such as oncology, routinely use biomarkers as surrogate or enrichment endpoints of treatments.

Examples of genomic (DNA and RNA) and proteomic biomarkers as gene-expression-based or genome signatures will be described in two pathologies (dystrophiniopathies and collagen VI myopathies) considered as paradigmatic examples for biomarkers discovery in NMDs. Particular emphasis will be put on less/non-invasive biomarkers, a crucial point in the selected disorders but more in general in many NMDs, where invasive sometimes multiple procedures are often required in order to demonstrate the efficacy of the treatment. Moving toward biomarkers in more accessible human cells or fluids will minimize the invasiveness of the follow up allowing us periodical measurements. This is a turning point in the research of NMDs since new therapies are emerging.

M.I.5 The dystrophic dogs as an excellent animal model of Duchenne muscular dystrophy (DMD)

S. Takeda^a

^aNational Institute of Neuroscience, National Center of Neurology and Psychiatry, Department of Molecular Therapy, Tokyo, Japan

 Introduction: Canine X-linked muscular dystrophy (CXMD) is caused by a point mutation at the splice acceptor site of exon 7 of the dystrophin gene, resulting in complete absence of dystrophin. To explore potential therapeutic approaches to DMD, we have established a beagle-based CXMD colony in Japan (CXMDJ). The phenotype of CXMDJ was similar, but a little milder than that of golden retriever CXMD. Evaluation of antisense morpholino-mediated exon skipping using dog models: Systemic delivery of morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine dystrophin gene, efficiently recovered functional dystrophin expression at the sarcolemma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol, in press). We have also tested antisense morpholinos targeting exon 51 of the mouse dystrophin gene in mdx 52 mice, in which gene targeting had disrupted exon 52 of the mouse dystrophin gene. Systemic delivery of a combination of two morpholinos showed an excellent restoration of sarcolemmal dystrophin and amelioration of the phenotypes in the mice. Significance of dystrophic dogs: Overall, the results using dystrophic mice are more stable than those in dystrophic dogs, partly because dystrophic dogs show a variation in the phenotypes even in a colony. Moreover, one must carefully evaluate the therapeutic effects within a limited number of dystrophic dogs, which is another disadvantage of dystrophic dogs. In addition, the gene mutation in CXMD is not common in DMD patients, therefore we cannot directly apply the morpholino sequences optimized in dogs to many human DMD patients. Dystrophic dogs, however, have a severe progressive course and clinical signs similar to DMD, whereas mdx mice exhibit mild clinical features. Thus, a combination of mouse and dog dystrophic models would be ideal to evaluate the therapeutic effects.

G.O.1 Diagnosis of muscular dystrophies at the nanometer scale

S. Puttini¹, M. Lekka², T. Kuntzer³, N. Mermod¹ and A. Kulik⁴

¹University of Lausanne, Lausanne, Switzerland

²Polish Academy of Sciences, Kraków, Poland

³Lausanne University Hospital (CHUV), Lausanne, Switzerland

⁴Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland

The diagnosis of muscular dystrophies or the assessment of the functional benefit of gene or cell therapies can be difficult, especially for poorly accessible muscles, and it often lacks a single-fiber resolution. In the present study, we evaluated whether muscle diseases can be diagnosed from small biopsies using atomic force microscopy (AFM). AFM was shown to provide a sensitive and quantitative description of the resistance of normal and dystrophic myofibers within live muscle tissues explanted from Duchenne mdx mice. The rescue of dystrophin expression by gene therapy approaches led to the functional recovery of treated dystrophic muscle fibers, as probed using AFM and by in situ whole-muscle strength measurements. Comparison of muscles treated with viral or non-viral vectors indicated that the efficacy of the gene transfer approaches could be distinguished with a single myofiber resolution. This indicated full correction of the resistance to deformation in nearly all of the muscle fibers treated with an adeno-associated viral vector that mediates exon-skipping on the dystrophin mRNA.

Having shown that AFM can provide a quantitative assessment of the expression of muscle proteins and of the muscular function in animal models, we assessed myofiber resistance in the context of human muscular dystrophies and myopathies. Thus, various forms of human Becker syndrome can also be detected using AFM in blind studies of small frozen biopsies from human patients.

Interestingly, it also allowed the detection of anomalies in a fraction of the muscle fibers from patients showing a muscle weakness that could not be attributed to a known molecular or genetic defect. Overall, we conclude that AFM may provide a useful method to complement current diagnosis tools of known and unknown muscular diseases, in research and in a clinical context.

M.P.1.03 Preliminary results from single subcutaneous administration of ACE-031, a form of the soluble activin type IIB receptor, in healthy postmenopausal volunteers

N.G. Borgstein^a, C.H. Condon^a, Y. Yang^a, D.M. Wilson^a, E. Haltom^a, J.L. Lachey^a, J. Seehra^a and M.L. Sherman^a

^aAcceleron Pharma, Cambridge, MA, United States

 ACE-031 is a fusion protein derived from the extracellular domain of the human activin receptor type IIB (ActRIIB) linked to the Fc portion of human IgG1. ACE-031 binds with high affinity to GDF-8 (myostatin) and other negative regulators of muscle mass. In numerous nonclinical studies using normal animals and various disease animal models, treatment with a soluble ActRIIB fusion protein results in increased lean mass and decreased adiposity. Pharmacologic responsiveness to ACE-031 has been observed in several species including non-human primates. Treatment with ACE-031 led to dose-dependent increases in lean body mass and strength.

The primary objective of the first-in-human randomized placebo-controlled study of ACE-031 was to evaluate the safety and tolerability of ACE-031 in healthy postmenopausal women. Forty-eight subjects were randomized in cohorts of 8 to receive either a single subcutaneous (SC) dose of ACE-031 or placebo (6 active: 2 placebo) at dose levels ranging from 0.02 mg/kg to 3 mg/kg. Subjects were followed for 57 days. Medications affecting muscle function were unchanged for at least 3 months prior to dosing, and subjects were asked to maintain their baseline physical activity for the duration of the study. Safety evaluations were conducted following each cohort to determine dose escalation. In addition to PK analyses, the pharmacodynamic activity of ACE-031 was also assessed by measurement of lean body mass (DXA), muscle volume (MRI), and quantitative muscle testing.

To date, the study remains blinded. All 48 subjects have been randomized and treated with ACE-031 or placebo;the follow-up phase is ongoing. Preliminary results including safety and efficacy data will be presented at the meeting.

M.P.1.04 Comparison of disease-causing mutations in Duchenne muscular dystrophy from the Cooperative International Neuromuscular Research Group with two large DMD mutation databases

L.P. Hache¹, E. Feingold², D. Escolar³, C. McDonald⁴ and P.R. Clemens¹

¹University of Pittsburgh, Neurology, Pittsburgh, United States

²University of Pittsburgh, Human Genetics, Pittsburgh, United States

³N/A, McLean, United States

⁴University of California, Davis, Sacramento, United States

 Objectives: Multiple dystrophin mutation types cause Duchenne muscular dystrophy (DMD). The recent development of DMD patient registries accentuates the need for global harmonization of mutation data collection. More consistent characterization of disease-causing mutations would enhance analyses of the distribution of such mutations from around the world. We investigated the mutations reported in DMD patients studied by the large international academic research group, the Cooperative International Neuromuscular Research Group (CINRG). We compared the types of mutations in the CINRG cohort to those reported in two large mutation databases, Leiden DMD mutation database and the French UMD-DMD. Methods: Diagnostic, strength, and medical history data were retrospectively reviewed for 374 DMD patients from 20 CINRG centers worldwide. The frequency of each type of mutation found in the CINRG data was compared with similar information abstracted from the Leiden and UMD-DMD mutation databases. The distribution of DMD-causing lesions in the CINRG data was also compared to data from the DuchenneConnect patient registry. Results: Of the 294 (78.6%) CINRG participants that had DNA testing, the majority had large deletions (72%), followed by no mutations identified (13%), point mutations (8%), large duplications (5%), and small deletions (2%). The distribution of dystrophin mutations within the CINRG database is similar to the two large published databases and the patient registry despite the dissimilar ways that these databases were created. The CINRG data is drawn solely from subjects entered into clinical trials. Furthermore, CINRG data may be susceptible to regional variations in dystrophin mutation type. Regional patterns in the CINRG data set will be presented. Conclusions: Harmonization of mutation data collection for DMD studies will benefit clinical trials and ultimately enhance pairing of eligible patients to specific molecular-based treatments.

M.P.1.05 Frequency of dystrophin gene mutations in the DuchenneConnect registry

V. Rangel Miller¹, G. Spinella² and P. Furlong³

¹Emory University, Department of Human Genetics, Atlanta, United States

²Parent Project Muscular Dystrophy, Falls Church, United States

³Parent Project Muscular Dystrophy, Middletown, United States

 Objective: DuchenneConnect was developed to link patients, providers, and researchers in the Duchenne/Becker muscular dystrophy community (DBMD). The program provides resources to learn about genetic testing, genetic counseling, management, and clinical trials. This abstract describes the frequency of reported mutations, diagnostic testing, and related questions posed by participants. Background: Numerous mutations have been reported in DBMD, and the level of characterization has improved with advances in molecular genetic testing technologies, however, many participants do not have results that provide well-characterized mutation information. Methods: DuchenneConnect is a self-report registry that captures clinical information and genetic test results. Design of the survey and review of the data follows guidelines employed by other North American DBMD registries and the TREAT-NMD Neuromuscular Network. A report tool enables participants to have their medical care provider review and verify the registry information. Results: As of March 2009, 1045/1403 registrants from 67 countries completed a survey, of which 559 had genetic testing, 295 had a muscle biopsy plus genetic testing, 69 had a muscle biopsy alone, 67 did not have testing, and 19 did not know. From 288/374 genetic test results received, 24% warrant additional testing to fully characterize the mutation. This abstract will present the aggregate mutation results (in a graphical table) and diagnostic studies performed. In addition, participants submitted 867 questions, from which 310 focused on genetic testing and/or results, and 89 arranged genetic testing. Conclusions: Currently, neuromuscular networks such as TREAT-NMD provide interim care guidelines which include confirmatory diagnostic testing. This abstract describes the frequency of mutations and test methods submitted to the registry, identifies questions commonly posed by participants, and emphasizes the importance of a molecular diagnosis.

M.P.1.06 An international registry for FKRP (fukutin-related protein) patients-the first international registry

T.A. Willis¹, M.C. Walter², K.M.D. Bushby¹, H. Lochmuller¹ and V. Straub¹

¹Newcastle University, Institute of Human Genetics, Newcastle, United Kingdom

²Institute of Neurology, Munich, Germany

 TREAT-NMD, a network of excellence for rare inherited neuromuscular diseases funded by the European Commission, together with patient organisations and clinicians from around the world has launched a global registry for patients with mutations in the fukutin-related protein gene (FKRP). This is the first international collection of data from both patients and professionals involved in the care of individuals with both limb girdle muscular dystrophy (LGMD2I) and the more severe end of the spectrum, congenital muscular dystrophy (MDC1C).

It aims to collect data on a longitudinal basis which can be used by health professionals and researchers for trials and prepare the patients for potential therapeutic interventions.

The registry is divided into two parts, a patient report part with consent for the second part, which is completed by the professional involved.

The patient part is available in a range of common languages and consists of easy drop down menus that can be completed on line. They include sections describing both current and previous best motor function achieved, with optional ‘pain’ and ‘quality of life’ questionnaires.

The professionals are required to confirm the genetic diagnosis and record respiratory and cardiac function. They also assess and record the patient’s motor function, including manual muscle testing on selected muscle groups, 6 min walk distances.

Conclusion: For clinicians and researchers this registry will provide an international database with precise information about the spectrum LGMD2I and CMD1C and its natural history, as well as collating a range of outcome measures that can be utilised in trials and potential therapies at a later date. For the patients this registry will enable specific groups of patients to take part in trials and to benefit potential therapies.

Trial readiness is essential in this evolving era of potential therapy.

Supported by the ENMC Consortium for Congenital Muscular Dystrophies.

M.P.1.07 The Smartnet Clinical Network – Creation of a national standardised assessment tool and natural history database for spinal muscular atrophy

A.G. Mayhew¹, E. Scott², F. Muntoni³ and A. Manzur³

¹Institute of Human Genetics, Newcastle, United Kingdom

²Muscular Dytrophy Campaign, London, United Kingdom

³Dubowitz Muscle Centre, Institute of Child Health, UCL, London, United Kingdom

 Objective: To develop a standard neuromuscular measurement tool for the assessment of children and young adults with Spinal Muscular Atrophy (SMA) in order to optimise their management and to create a national database to collect natural history data on type II and type III SMA. Background: The Smartnet Clinical Network UK was established in 2006 following a similar initiative standardising assessment procedures in ambulant Duchenne Muscular Dystrophy (DMD) boys. Increasingly there has been a need to establish standardised assessment procedures as clinical trials become more likely and regulatory authorities require more comprehensive measures of progress. Methods: Existing motor function scales were reviewed and the following scales chosen for inclusion: For non-ambulant children – Hammersmith Functional Motor Scale (HFMS). For ambulant children and adults – Modified North Star Ambulatory Assessment, originally designed for use in ambulant DMD. For non-ambulant children, teenagers and adults – Egen Klassifikation for SMA. An adjunct to this project was collaboration with the original author, Birgit Steffensen, to establish a revised scale for use in SMA that included questions on bulbar function, fatigue and hand function. This has resulted in the EK2. Results: Through the activities of the network, medical and physiotherapy assessment proforma’s have been produced. A web-based national clinical database has been developed in order to review the natural history of SMA, facilitate multicentre clinical audit and review services. The database is hosted on a joint website with the North Star project and went live in March 2009. Future developments: It is hoped that with further funding the work can be extended to cover the adult population more comprehensively and new outcome measures can be evaluated and correlations between measures assessed.

We gratefully acknowledge the financial support of Jennifer Trust for Spinal Muscular Atrophy and Muscular Dystrophy Campaign.

M.P.1.09 TREAT-NMD Clinical Trials Coordination Centre: Efficiency of networking

K. Gramsch^a, A. Pohl^a, J. Kirschner^a, R. Korinthenberg^a, S. Geismann^a and A. Tassoni^a

^aUniversity Medical Center Freiburg, Neuropediatrics and Muscle Disorders, Freiburg, Germany

 The Clinical Trials Coordination Centre (CTCC) is one part of the Network of Excellence TREAT-NMD (Translational Research in Europe – Assessment and Treatment of Neuromuscular Disease” www.treat-nmd.eu) funded under the 6th Framework Programme of the European Union since 2007. It is attached to the Clinical Trials Center and the Department of Neuropediatrics of the University Medical Center Freiburg.

The CTCC coordinates the clinical research and enables through methodical training, outcome research and performance of randomized controlled studies the promotion of evidence production after the criteria of Evidence based Medicine. It has implemented the Care and Trial Sites Registry (CTSR, www.treat-nmd.eu/trialsites) which shows now (03/09) 148 registered sites in 34 countries worldwide. Out of those countries 11 are non-European including 39 registered centres with 21 centres from North America. The overall amount of reported treated patients is 11342, 5890 with Duchenne/Becker Muscular Dystrophy, 2625 with Spinal Muscle Atrophy (Types 1–3) and 2827 with other rare phenotypes. According to a recent survey, the most important factors deciding on the success of a trial are good organisation through the responsible Contract Research Organisation and well-trained investigators (Hark et al. Applied Clinical Trials, 2009). 67% of the registered centres reported that there are familiar with ICH-GCP. So this network can stand for the realization of qualitatively high standard future trials.

As a network of centres caring for patients with neuromuscular disorders the CTSR will be used for dissemination and implementation of the latest evidence based treatment recommendation. The CTCC will use the CTSR to analyse in some exemplary countries how well the standards of care are implemented. In conclusion, the CTSR will not only facilitate multi-centre clinical trials but also help to improve clinical care for patients with neuromuscular disorders.

EM.P.1.01 Prevention of muscle fibrosis and myonecrosis in mdx mice by suramin, a TGF-β 1 blocker

M.J. Marques^a, A.P.T. Taniguti^a, A. Pertille^a and H. Santo Neto^a

^aUniversidade Estadual de Campinas – Unicamp, Anatomia, Campinas, Brazil

 Cycles of muscle degeneration in dystrophic muscles are associated with inflammation and fibrosis which affect the diaphragm and heart, leading to cardiorespiratory failure in patients with Duchenne muscular dystrophy and in mdx mice. We evaluated the effect of suramin, a transforming growth factor beta 1 (TGF-β1) blocker, on fibrosis in mdx. mdx mice (n = 24; 6 months old) received intraperitoneal injections of suramin (n = 12;60 mg/kg body weight) on alternate days for 7 weeks. Diaphragm (DIA), tibialis anterior (TA), biceps brachii (BB) and cardiac muscles were studied. Control mdx (n = 12) were injected with saline. Suramin-and saline-treated mdx performed exercise on a treadmill throughout the treatment period to worsen disease. Immunoblotting indicated a significant increase of TGF-β1 levels in mdx DIA, limb and cardiac muscles compared to C57Bl/10 mice. Suramin significantly decreased blood creatine kinase levels (saline-mdx:1540 ± 556 U/l vs suramin-mdx:474 ± 300 U/l; p < 0.05, Student’s t-test). Masson’s trichrome showed the highest level of fibrosis in DIA (13 ± 2.5% in saline-mdx vs 7.6 ± 3% in suramin-mdx; p < 0.05). The smallest fibrosis area was observed in TA muscle (0.9 ± 0.6% in saline-mdx vs 0.3 ± 0.2% in suramin-mdx; p < 0.05). Suramin significantly attenuated fibrosis in all muscles studied, except for the cardiac (1.4 ± 0.5% in saline vs 1.8 ± 0.3% in suramin). Suramin protected TA and BB muscles against myonecrosis and promoted regeneration, leading to a significant decrease in Evans blue dye-positive fibres with a concomitant increase in central nucleated fibres. Grip strength test showed that suramin reduced the loss of strength with time (loss of 0.03 g/g body weight in suramin-mdx vs 0.34 g/g body weight in saline-mdx; p < 0.05). These results indicate that suramin prevents fibrosis and myonecrosis in dystrophic mdx muscles and suggest that suramin might be a useful therapeutic alternative for the treatment of Duchenne muscular dystrophy.

EM.P.1.02 Long-term activin receptor type IIB inhibition improves strength and function of dystrophic muscle

J.L. Lachey¹, S. Bogdanovich², E.E. Pistilli², A.E. Pullen¹ and J. Seehra¹

¹Acceleron Pharma, Cambridge, United States

²University of Pennsylvania and Pennsylvania Muscle Institute, Philadelphia, United States

 The absence of the membrane stabilizing protein dystrophin characteristic of Duchenne muscular dystrophy increases vulnerability of muscle to contraction-induced damage. One strategy to prevent mechanical stress-induced damage in dystrophic muscle is to promote muscle fiber hypertrophy. Inhibition of activin receptor type IIB (ActRIIB) signaling induces skeletal muscle hypertrophy and results in increased muscle mass and strength in wild-type mice. Previous work also describes a functional benefit of increasing myofiber size in dystrophic muscle. To determine the efficacy of long-term ActRIIB inhibition in improving dystrophic muscle, 7-week old mdx mice were treated with vehicle or RAP-031, a fusion protein comprised of a form of the ActRIIB extracellular domain fused to a murine Fc, for 42 weeks. Body composition, grip strength and extensor digitorum longus (EDL) contraction force were assessed. Over the course of the study, RAP-031 significantly (p < 0.001) increased lean tissue mass compared to the vehicle cohort. Grip strength, normalized for lean tissue mass, was 28.3% higher (p < 0.01) in RAP-031 treated mice compared to the control group. EDL absolute twitch and tetanus contraction forces were increased 30% (p < 0.01) and 42.5% (p < 0.01) respectively in the RAP-031 group compared to controls. The forces generated during an eccentric contraction (ECC) protocol were significantly greater (p < 0.01) in the RAP-031 treated group in five trials compared to controls and there was no significant change in the force deficits resulting from the lengthening contractions. While the rate of force loss is similar, the finding that the absolute forces were greater throughout the ECC protocol could support a role for RAP-031 in increasing the degree of damage a muscle can withstand before contraction force is diminished to a critically low level. Overall, these data support a therapeutic benefit provided by chronic ActRIIB inhibition to dystrophic muscle.

EM.P.1.03 Potential involvement of angiotensin II in pathological signs of dystrophic muscle: In vivo and ex vivo outcome of a chronic treatment with enalapril, an angiotensin-converting enzyme inhibitor, in the exercise-aggravated mdx mouse model

A. Cozzoli¹, V. Sblendorio¹, R.F. Capogrosso¹, B. Nico², D. Conte Camerino¹ and A. De Luca¹

¹University of Bari, Dept. Pharmacobiology, Unit of Pharmacology, Bari, Italy

²University of Bari, Dept. Human Anatomy and Histology, Bari, Italy

Angiotensin (Ang)-converting enzyme inhibitors (ACE-I) have clinical value to treat cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients (Duboc et al. Am. Heart J., 2007). Also, Ang-II antagonist losartan was found to reduce advanced and induced muscle fibrosis in mdx mouse (Cohn et al., Nat. Med., 2007). We verified the involvement of Ang-II at an earlier stage of muscle pathology, by treating 4-5 week-old treadmill-exercised mdx mice with 1-5 mg/kg enalapril, an ACE-I (6 days/week i.p. for 4–8 weeks). In vivo, enalapril counteracted the exercise-induced decrease of maximal and normalized fore limb strength in a dose-dependent manner. A significant amelioration of resistance to exercise was also observed. Ex vivo, the treatment contrasted dose-dependently the exercise-induced reduction of the macroscopic chloride conductance, gCl, in EDL muscle. Enalapril might have blunted a direct effect of Ang-II on muscle chloride channel, since in vitro application of Ang-II (10 nM–300 nM) produced a concentration-dependent decrease of gCl in EDL muscle of wt mice, through an AT-1 receptor-mediated activation of PKC pathway. Enalapril at 5 mg/kg partially restored mechanical threshold and calcium-dependent contractile parameters of EDL muscle, suggesting a positive effect on the altered calcium homeostasis. No effect, at any dose, was observed on plasma creatine kinase and lactate dehydrogenase. An improvement of the histological profile was observed in both diaphragm and gastrocnemious muscle, along with a marked decrease of NF-kB positive fibres by immunohistochemistry. Thus, enalapril significantly ameliorates mechanical and inflammation-sensitive parameters in dystrophic muscle, thus reinforcing the interest of early treatment with ACE-Is in DMD patients. In addition the data disclose a possible direct role of Ang-II in some of the biophysical alterations observed in dystrophic muscles, which deserves further investigations (supported by Telethon – Italy Project No. GGP05130).

EM.P.1.04 Treatment approaches in laminin-α2-deficient congenital muscular dystrophy (MDC1A)

S. Meinen¹, S. Lin¹ and M.A. Rüegg²

¹Biozentrum, University of Basel, Neurobiology/Pharmacology, Basel, Switzerland

²Biozentrum, University of Basel, Neurobiology, Basel, Switzerland

MDC1A is a severe muscle wasting disease that affects young children and for which no curative treatment exists. Transgenic approaches have shown that overexpression of a miniaturized form of the extracellular matrix molecule agrin or inhibition of apoptosis can ameliorate disease progression in MDC1A mouse models. However, none of the treatments alleviates all of the symptoms. Here, we examined in a transgenic study whether a combined treatment with mini-agrin and inhibition of apoptosis has additive benefits in MDC1A mice. Our results confirm that inhibition of apoptosis by either overexpression of the anti-apoptotic protein Bcl2 or deletion of the pro-apoptotic gene Bax, ameliorates the disease, although with a much lower efficacy than mini-agrin. Remarkably, muscle-specific Bcl2 overexpression increases fibrosis in muscles, which was not the case upon body-wide deletion of Bax or application of the pharmacological apoptosis inhibitor omigapil. However, the fact that Bcl2 allows the muscle to maintain near-normal fiber numbers complements the effect of mini-agrin, which prevents the replacement of muscle with fibrotic tissue. In a parallel study we tested in MDC1A mice whether losartan, a validated angiotensin II type 1 receptor antagonist used to treat high blood pressure, would offer another entry point for the treatment of MDC1A patients. Losartan was shown to block TGF-β-activation in mouse models of Marfan syndrome and DMD and to restore muscle pathology and regeneration. In good correspondence to the fact that TGF-β levels are elevated and that fibrosis and failure of muscle regeneration are main reasons for the severe course of MDC1A, our data indicate that losartan helps to overcome some of the dystrophic phenotype. In conclusion, our studies suggest that a combined treatment with mini-agrin and apoptosis inhibition as well as prevention of fibrosis would be most beneficial for MDC1A patients particularly in regard to a markedly enhanced muscle function.

EM.P.1.06 Extracellular matrix metalloproteinase protein inducer (EMMPRIN/CD147) regulates myoblast differentiation through an MMP-mediated control of TGFb activity

M. Attia¹, E. Huet¹, S. Gawrzak², S. Menashi¹ and I. Martelly¹

¹Université Paris EST- Paris 12, Laboratoire CRRET, Créteil, France

²Warsaw University, Warsaw, Poland

 Skeletal muscle regeneration, mediated through the activation and differentiation of the myogenic stem cells called satellite cells, requires tissue remodeling where MMPs are thought to play an important role. In view of the MMP inducing function of EMMPRIN/CD147, we looked a possible role of EMMPRIN in in vitro myogenesis. As myoblasts, we used both primary cultures of rat satellite cells and the C2.7 cells, a sub clone from C2C12 myoblast cell line. These cells differentiate into myotubes in culture when shifted to low serum medium. Gelatin zymography of the cell lysates revealed that during differentiation there was a gradual decrease in MMP-9 and an increase in MMP-2 (both the proenzyme and active enzyme). There was a parallel increase in EMMPRIN protein level during differentiation which was associated with a marked increase in small molecular forms of this protein thought to correspond to the hypoglycosylated EMMPRIN. Treatment of myoblasts with recombinant EMMPRIN, increased EMMPRIN protein expression and reduced myoblast differentiation as evaluated by both morphological appearance and the expression of troponin T, a marker of myoblasts differentiation. On the contrary, treatment of cells with EMMPRIN siRNA, which significantly diminished EMMPRIN expression, accelerated myoblasts differentiation. Similarly acceleration of myogenesis was also observed in the presence of marimastat, a broad spectrum MMP inhibitor, suggesting an MMP-mediated inhibitory effect of EMMPRIN on differentiation. ELISA measurements of TGFb, a well known inhibitor of myoblast’s differentiation indicated that this cytokine may be the main target in the regulatory role of EMMPRIN in myogenesis. Our results suggest that EMMPRIN inhibits myoblasts differentiation by an MMP dependent activation of TGFb. EMMPRIN inhibition may therefore represent a novel strategy in the treatment of muscular degenerative disorders.

EM.P.1.07 Effects of muscle hypertrophy on individual myonuclear domain sizes in single muscle fibers from myostatin deficient or IGF-1 over-expressing mice

R. Qaiasr¹, K. Morine², E.R. Barton³, H.L. Sweeney² and L. Larsson^{4, 5}

¹Uppsala University, Department of Clinical Neurophysiology, Uppsala, Sweden

²University of Pennsylvania, Department of Physiology School of Medicine, Philadelphia, United States

³University of Pennsylvania School of Dental Medicine, Department of Anatomy and Cell Biology, Philadelphia, United States

⁴The Pennsylvania State University, Centre for Development and Health Genetics, USA

⁵Uppsala University, Clinical Neurophysiology, Uppsala, Sweden

 Myostatin deprived or IGF1 over-expressing mice are characterized by a 2–3-fold increase in muscle size compared to age- and sex-matched controls. Despite the similar increase in muscle size, these hypertrophic muscles show significant difference in the force generating capacity, i.e., maximum force normalized to muscle fiber cross-sectional area or specific force. That is, IGF1 overexpressing transgenic mice show a similar specific tension as in controls, while specific tension is significantly lower in the myostatin knock out mice. The mechanism underlying compromised muscle function in the myostatin knock out mice remains unknown. In attempt to explore these mechanisms we have investigated the size of the cytoplasmic volume (myonuclear domain, MND) supported by individual myonuclei in single muscle fiber segments from myostatin deficient, IGF1 overexpressing and control mice, using a novel algorithm to measure the MND size in 3D. The skinned fibers were mounted at a fix sarcomere length corresponding to optimal filament overlap for force generation, and stained with DAPI and rhodamine for myonuclei and actin respectively. Our image analysis algorithm was highly effective in determining spatial organization of myonuclei and distribution of individual MND along the length of the fiber. Preliminary results are encouraging and point towards an increased variability in MND size in animals with myostatin deficiency. This is suggested to be secondary to lack of satellite cells activation due to absence of myostatin. Using 3D laser scanning of fluorescently labelled skinned fibers, we also found myofibriller derangement with resultant force vectors partially negating each other. Thus, an impaired machinery for protein synthesis/degradation together with less optimal alignment of contractile elements are forwarded as probable mechanisms underlying the impaired force generation capacity in myostatin knock-out mice.

G.P.2.03 Partial reversal of ventilator-induced diaphragm muscle cell dysfunction

J. Ochala^a and L. Larsson^a

^aUppsala University, Clinical Neurophysiology, Uppsala, Sweden

 In critically ill patients, ventilator-induced diaphragm muscle fiber dysfunction is characterized by a severe atrophy and a decrease in maximal force production normalized to cross-sectional area, contributing to problems associated with weaning from the respirator. It remains unknown whether mechanical ventilation also disrupts other important contractile features such as Ca²⁺ activation. To address this question, we studied Ca²⁺ sensitivity of contraction using skinned diaphragm muscles fibers from piglets that were randomly sham-operated or mechanically ventilated and sedated for five days. Results emphasized severe impairments of Ca²⁺ sensitivities of force and stiffness in fibers from all mechanically ventilated piglets compared to sham-operated animals, suggesting a less efficient Ca²⁺ activation of cells, i.e., a lower relative number of strongly attached cross-bridges for a certain concentration of Ca²⁺. To further investigate whether this negative effect is reversible, we exposed the fibers to the EMD 57033 Ca²⁺ sensitizer. EMD 57033 improved the Ca²⁺ sensitivity of force and stiffness in fibers from animals that were mechanically ventilated for five days as well as in sham-operated piglets. Thus, EMD 57033 partially restored the Ca²⁺ activation of cells. This result offers a strong basis for evaluating the effect of EMD 57033 on diaphragm function in vivo.

G.P.4.02 The impact of silicone ankle foot orthosis (SAFO’s) in patients with Charcot-Marie-Tooth (CMT) disease on cadence, balance and falls

N. Cartwright¹ and S.L. Glover²

¹Queen Elizabeth Hospital Birmingham, Physiotherapy Department, Birmingham, United Kingdom

²Queen Elizabeth Hospital Birmingham, Physiotherapy Department, Birmingham, United Kingdom

 Patients with CMT frequently report difficulty maintaining their balance and have a history of falls. During detailed questionning into activities of daily living they commonly describe falling when changing direction and reduced cadence (gait speed). This can be related to the impaired motor/sensory activity within the lower limbs as a result of their diagnosis. The main focus of our attention as physiotherapists is to address the ankle instability using exercise and orthotics. Over a 14-month period, SAFO’s have been trialled to evaluate their impact on ankle stability, cadence and falls.

The timed 10-m step test has proven to be a reliable and sensitive measure to evaluate gait. The 360^° test was selected from the BERG Balance scale and amended to suit patients with CMT. This has proven to be an effective and time efficient balance assessment tool. Patients were questioned regarding their number of falls in the month previous to the SAFO assessment and a month following their application. The step and 360^° test were used pre- and post-application of bespoke SAFO’s.

Results from 22 patients with CMT indicate that SAFO’s increase patients cadence, improve lateral stability of the lower limb and eradicate or reduce the number of falls. Patients report good compliance and satisfaction in the use of this orthosis and we see this as an invaluable therapeutic resource in patients with neuromuscular disease.

M.P.2.04 Bioengineering and muscular dystrophy

M.G. d’Angelo¹, M. Romei¹, S. Gandossini¹, S. Bonato¹, D. Colombo², E. Marchi², A. Lo Mauro³, A.C. Turconi¹, G.P. Comi⁴, A. Aliverti³ and N. Bresolin⁵

¹IRCCS E Medea, NeuroRehabilitation, Bosisio Parini (Lecco), Italy

²IRCCS INRCA, Respiratory Rehabilitation, Casatenovo (Lecco), Italy

³Politecnico di Milano, BioEngineering Dept, Milano, Italy

⁴IRCCS Ospedale Maggiore, Neuroscience, Milano, Italy

⁵IRCCS E Medea, Bosisio Parini (Lecco), Italy

  Introduction: Symptoms of respiratory insufficiency can be subtle. Identification of early alteration is essential for therapeutical approach and quality of life of the patients with muscular dystrophies. Methods: 62 adult patients with MD (27 Limb-Girdle Muscular Dystrophy, LGMD, age: 35.2 + 14.7 years; 14 Becker Muscular Dystrophy, BMD, 35.8 + 12.1y; 21 Facio-Scapulo-Humeral Dystrophy, FSHD, 40.5 + 18.3y) and 20 healthy age-matched volunteers (Control Group, 32 + 9.2y) were evaluated. Patients were analysed through spirometry, maximal inspiratory/expiratory pressure measurements and through Optoelectronic Plethysmography (OEP). OEP is a technique that starting from 3D coordinates of 52 reflective markers positioned on trunk and acquired by an optoelectronic system allows to calculate ventilatory parameters, chest wall (CW) and its thoraco-abdominal compartments volume variations during breathing. Results: Spirometry data showed a mild restrictive respiratory pattern in all the three pathologic groups. During quiet breathing no significant differences were found in ventilatory and CW compartments parameters between pathological groups and controls. In Slow Vital Capacity trial, dystrophic subjects presented ventilatory parameters and breathing pattern significantly different from healthy subjects, particularly Vital Capacity values (LGMD: 3.5 ± 1.3 L, BMD:4.2 ± 0.9 L, FSHD: 3.5 ± 1.1 L, CG: 4.9 ± 0.8 L), more evident in non ambulant patients, suggesting expiratory muscles weakness. The pattern of CW compartments of FSHD patients strikingly deviated from normality especially during the inspiration phase, during which the diaphragm and the muscles of abdomen were recruited more than in controls and other pathologic groups, probably due to weakness of upper trunk muscles. OEP demonstrated to be able to characterize and quantify ventilatory parameters differences between dystrophic patients and healthy subjects and to identify specific breathing patterns.

M.P.2.05 Optoelectronic plethysmography in respiratory assessment in Duchenne muscular dystrophy

M. Romei¹, M.G. d’Angelo¹, S. Gandossini¹, S. Bonato¹, G.P. Comi², D. Colombo³, E. Marchi⁴, A. Lo Mauro⁵, A. Aliverti⁵ and N. Bresolin²

¹IRCCS E Medea, NeuroRehabilitation, Bosisio Parini (Lecco), Italy

²IRCCS Ospedale Maggiore, Neuroscience Dept., Milano, Italy

³IRCCS INRCA, Respiratory Rehabilitation, Casatenovo (Lecco), Italy

⁴IRCCS INRCA, Casatenovo (Lecco), Italy

⁵Politecnico di Milano, BioEngineering Dept., Milano, Italy

 Up to now respiratory problems in DMD have been mainly described in terms of reduction of vital capacity and inspiratory muscles weakness, even if these descriptors are not the unique determinants of the worsening of respiratory function in DMD. In fact, some patients with extremely low values of vital capacity can breathe autonomously, while others need constant ventilatory support. Methods: Respiratory function of 24 young-adult DMD (mean age: 18.5 + 3 years) was assessed through spirometry, night oxyhaemoglobin saturation and Optoelectronic Plethysmography (OEP). OEP is based on the measurement of the 3D motion of reflecting markers placed over the chest wall and provides a completely non-invasive breath-by-breath measurement of chest wall volume changes during breathing. The enrolled subjects (DMD patients and volunteers – CG), were asked to breath spontaneously for three minutes and to perform two Slow Vital Capacity (SVC) in supine position. Results and discussion: A severe restrictive respiratory pattern was evident in all DMD patients. Based on oxyhaemoglobin saturation, DMD patients were divided in two subgroups: 11/24 patients with at least 10% of night time with SpO2 < 95% (DeSat group) and the other 13/24 with most of the night time with SpO2 > 95% (Non-DeSat group). During quiet breathing, DeSat group presented values significantly different from CG. During SVC, the DMD subgroups showed similar inspiratory and expiratory capacity values that were statistically lower than CG. The contribution of abdominal compartment to the chest wall of DeSat group’s patients was significantly lower than Non DeSat group and than CG. This suggests a marked diaphragm weakness for patients of DeSat group and the involvement of accessory respiratory muscles in the inspiratory and expiratory phase. OEP demonstrated to be able to unravel early signs of respiratory muscle weakness that precede and anticipates the onset of nocturnal hypoxemia and respiratory failure.

M.P.2.06 External control of exhalation for cough assistance: a method for patients with glottis dysfunction

S.C. Lee¹, S.W. Kang² and S.H. Im³

¹Myongji Hospital, Kwandong University College of Medicine, Physical Medicine and Rehabilitation, Koyang, Kyunggi, Republic of Korea

²Yonsei University College of Medicine, Rehabilitation Medicine and Rehabilitation Institu, Seoul, Republic of Korea

³Jeju National University Hospital, Jeju National University School of Medicine, Physical Medicine & Rehabilitation, Jeju, Republic of Korea

 Objective: To investigate the effectiveness of our new method of assisting a cough by mimicking the functions of the glottis in patients with bulbar muscle weakness. Methods: The study subjects were patients diagnosed with neuromuscular disorders. They were 11 patients with bulbar muscle weakness as well as inspiratory and expiratory muscle weakness. We evaluated the subjects to confirm the effects of the external control method as follows. First, the unassisted peak cough flow (UPCF) was measured by having the subject cough as forcefully as possible through a commercial peak flow meter. Second, the assisted PCF (APCF) with the peak flow meter was evaluated. To assist with the inspiratory phase of the cough, the subjects were insufflated to their maximum insufflation capacity. To assist with the expulsive phase of the maximal voluntary cough, the subject was asked to cough as forcefully as possible with the help of a manual abdominal thrust. Third, the APCF with the external control method (APCFE) that assists the glottic function was measured using the same method as the APCF. We compared the PCFs under different conditions. Results: APCFEs were measured in all of the patients even when the UPCFs and/or APCFs could not be measured. For patients with measurable APCFs, the APCFEs were significantly higher than the APCFs. Conclusions: We showed the effectiveness of our method of assisting a cough by external control of the glottis in patients with bulbar muscle weakness who cannot effectively cough with the help of existing cough assistance methods.

M.P.2.07 The benefits of lung inflation training using positive end-expiratory pressure valve for patients with neuromuscular disorders

T. Matsumura^a, T. Saito^a, H. Fujimura^a and S. Shinno^a

^aNational Hospital Organization Toneyama National Hospital, Neurology, Toyonaka, Japan

Objective: Lung inflation training is important for neuromuscular patients to keep airway clearance and lung compliance, however, this is not easy for patients with respiratory and laryngeal dysfunction. We speculate that a positive end-expiratory pressure (PEEP) valve can overcome these problems. Thus, we evaluated the effect of lung inflation training with a resuscitation bag equipped with a PEEP valve. Methods: Participants included 83 neuromuscular patients (Amyotrophic lateral sclerosis: 24, Myotonic dystrophy: 20, Duchenne muscular dystrophy (DMD): 39). Lung inflation training with a PEEP valve was done as follows. Patients inspired air using a resuscitation bag with a PEEP valve of 20 cm H₂O until air leakage occurred from the PEEP valve. PEEP valve was then removed and patients were asked to expire as much as they could. Vital capacity (VC), maximum insufflations capacity (MIC) and lung inflation capacity using PEEP valve (PIC) was measured. PIC training was continued for 3–5 months in 6 DMD patients receiving tracheotomies to assess any sustained effect. Results: PIC was significantly larger than VC and MIC in three diseases noted earlier. MIC was larger than VC in only DMD. The difference between MIC and VC was negatively correlated to the difference between PIC and MIC. Mean PIC of 6 DMD patients receiving tracheotomies was increased from 344.7 ± 76.9–619.3 ± 205.3 ml. Although PIC did not exceed VC in the 7 patients complaining of strong discomfort from this training, no other adverse events were observed. Conclusions: Lung inflation training was simply and safely enabled using a PEEP valve. The PEEP valve was able to compensate for laryngeal dysfunction and this allowed PIC training to be effective in all diseases patients, even those receiving tracheotomies. Since the cost of PEEP valve is relatively low (approximately US $20), PIC training could be disseminated to outpatients.

M.P.2.09 Mdx diaphragm muscle as a target of dystrophin gene therapy

E. Kimura¹, T. Suga¹, M. Ishizaki¹, K. Uchino¹, T. Koide¹, Y. Uchida², Y. Maeda², J.S. Chamberlain³ and M. Uchino¹

¹Kumamoto University Graduate School of Medical Science, Neurology, Kumamoto, Japan

²Sojo University, Faculty of Pharmaceutical Sciences, Pharmacology, Kumamoto, Japan

³University of Washington School of Medicine, Neurology, Seattle, WA, United States

Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder, and there is currently no effective treatment. DMD causes respiratory or cardiac failure and results in death at about 20 years of age. Especially respiratory insufficiency is a major problem in the management of DMD patients. In mdx mouse, a DMD model, the pathological feature of diaphragm is severely affected, and then similar to the skeletal muscle of human DMD. Although, the muscle strength assay of the dystrophic diaphragm has been used to estimate mdx mouse respiratory impairment, systemic functional assessments compared with histopathological analysis have not been demonstrated. Here we report a sensitive procedure using whole-body plethysmography to monitor respiratory parameters detected during early insufficiency in the mdx mouse as well as an assessment of muscle tetanic force. In result, the dystrophic changes in the diaphragm lead to respiratory dysfunctions. Moreover, we propose gene therapy approaches targeting diaphragm with delivery of viral vectors. These total assessment system for rodent respiratory function, including whole-body plethysmography, may be useful to evaluate the therapeutic approaches for the neuro-muscular disease models.

EM.P.3.03 Differential expression of genes involved in muscular degeneration in four dystrophic mouse models

P.C.G. Onofre-Oliveira^a, P.C.M. Martins^a, V.L. Ferreira^a, D. Ayub-Guerrieri^a and M. Vainzof^a

^aHuman Genome Research Center – IB – USP, Genetics and Evolutive Biology, Sao Paulo, Brazil

 Muscle weakness in muscular dystrophies is the consequence of an imbalance between successive cycles of degeneration and regeneration, with further replacement of the degraded muscle fibers by adipose and connective tissues. One important marker for degeneration is the expression of TGFB1, which is an inflammatory cytokine with a possible role in the stimulation of fibrosis in the dystrophic muscle through the activation of genes related to the expression of collagen. Several mouse models for genetic muscular dystrophies are recognized. Among them, mdx, SJL/J, Lama2^dy−2J/J and Large^myd are models for Duchenne, LGMD2B, CMD1A and CMD1D muscular dystrophies, respectively, presenting variable histopathological and clinical findings. High expression of Tgfb1 and alpha 2 subunit of collagen 1 (Col1a2) genes has been found in the mdx mouse. Here we studied the expression of these two genes in three additional mouse models, carrying different defects in muscle proteins, to better understand the involved pathophysiological mechanisms, aiming future therapies. Using Real-Time PCR, we quantified expression of the Tgfb1 and Col1a2 genes in the mdx, SJL/J, Lama2^dy−2J/J and Large^myd strains and normal controls. The expression data was compared to histopathological alterations in the respective muscles. Tgfb1 was found to be highly expressed in all four models. However, the Col1a2 gene was upregulated in mdx and SJL/J, both of which showed the most preserved muscle histology, but exhibited lower expression in the more severely affected muscles from Lama2^dy−2J/J and Large^myd strains. The results suggest that Tgfb1 gene is activated in the dystrophic process in all types/stages of degeneration while activation of Col1a2 gene possibly occurs in earliest stages of this process. Funding by FAPESP, CNPq-INCT, FINEP, ABDIM.

EM.P.3.04 Muscle-derived Duchenne muscular dystrophy fibroblasts show altered production of extra-cellular matrix components

S. Zanotti^a, S. Gibertini^a, S. Saredi^a, F. Blasevich^a, R. Mantegazza^a, L. Morandi^a and M. Mora^a

^aFondazione IRCCS Istituto Neurologico C. Besta, Neuromuscular Diseases and Neuroimmunology Unit, Milano, Italy

 In order to increase understanding of pro-fibrotic mechanisms in dystrophic muscle, we isolated primary human fibroblasts from DMD and control muscle biopsies, and investigated their proliferating activity and soluble collagen production, as well as transcript and protein levels of decorin, myostatin, TGF-β1, MMP-1 (interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin), MMP-7 (matrilysin), and the MMP inhibitors TIMP-1, TIMP-2, TIMP-3 and TIMP-4.

We found significantly greater proliferation rate and soluble collagen production in DMD fibroblasts than controls. Decorin, myostatin and MMP-7 transcripts and proteins were significantly upregulated, and MMP-1 and TIMP-3 were significantly downregulated in DMD fibroblasts compared to controls.

The study has revealed several differences between fibroblasts from DMD muscle and those from normal muscle, particularly as regards factors likely to affect ECM turnover. Although the detailed significance of these differences is not clear, they show that DMD fibroblasts are characterized by a profibrotic phenotype.

Dystrophin absence itself could exert a direct influence on ECM homeostasis by allowing leakage of cellular components to the extracellular space, or abnormal cellular uptake of extracellular growth factors, cytokines, or enzymes. By such mechanisms dystrophin absence may influence muscle fibroblasts either directly by altering their adhesion properties or indirectly by interactions with molecules released into the ECM by muscle or inflammatory cells.

EM.P.3.05 Expression of fibulins 1–5 during myogenesis in vitro and in skeletal muscle regenerating in vivo, and in dystrophic mdx muscles

M.D. Grounds^a, T. Shavlakadze^a and J. Vukovic^a

^aThe University of Western Australia, School of Anatomy & human Biology, Perth, Australia

 Many myopathies are due to gene defects that result in absent or altered extracellular matrix (ECM) and numerous ECM molecules are in intimate contact with myofibres and with the quiescent muscle precursors (satellite cells). Complex interactions between ECM components and different cells play a crucial role in muscle function and all aspects of new muscle formation after damage. Fibulins (FBLN) are members of an adhesive glycoprotein family that are associated with fibrosis in some conditions. The role of FBLN 1–5 was investigated using immunostaining and qPCR in cultured myogenic C2C12 cells, in tissue sections of regenerating whole muscle grafts, in dystrophic (mdx) quadriceps and diaphragm, and in normal adult (2 month) and geriatric (23 month) mouse muscle. Cultured myoblasts expressed all fibulins and a subset was differentially regulated during myogenesis in myotubes. Immunostaining of regenerating myogenic cells (especially myotubes) in whole muscle grafts (at 6 days) generally confirmed this myogenic expression pattern. Dystrophic mdx quadriceps muscle (at 3 month) showed increased immunostaining for FBLN-1, -2 and -4 proteins around regenerating (small diameter) myofibres. While FBLN-3 and -5 were absent from dystrophic quadriceps aged up to 6 month, they were present in 3-month mdx diaphragm where they co-localised with fibronectin. These novel results indicate that different fibulins are differentially expressed and are involved in myogenesis and myofibre regeneration. A significant finding is that FBLN-3 and -5 are associated with the severe dystrophic pathology in the mdx diaphragm. Since fibrosis is a major complication resulting from progressive myofibre damage in dystrophic muscle, it is of especial interest to fully understand the roles of all ECM molecules, including fibulins, associated with this process.

EM.P.3.06 Transient upregulation of matrilin-2 gene expression suggests a role in early steps of skeletal muscle regeneration

É. Korpos¹, L. Mátés¹, L. Mendler², M. Kiricsi³, Á. Zvara⁴, F. Deák¹, Z. Rottenberger¹, A. Keller-Pintér², L. Puskás⁴, L. Dux² and I. Kiss¹

¹Biological Research Center of the Hungarian Academy of Sciences, Institute of Biochemistry, Szeged, Hungary

²University of Szeged, Faculty of General Medicine, Department of Biochemistry, Szeged, Hungary

³University of Szeged, Faculty of Natural Sciences, Department of Biochemistry and Molecular Biology, Szeged, Hungary

⁴Biological Research Center of the Hungarian Academy of Sciences, Laboratory of Functional Genomics, Szeged, Hungary

Adult skeletal muscle has a remarkable regenerative capacity in response to injuries. The regeneration is dependent on an orchestrated response between the inductive signals of cytokines, growth factors and extracellular matrix (ECM) components. Matrilin-2 is a modular, oligomeric protein deposited in the ECM of many cell types. Colocalization in human skin and in vitro interaction of matrilin-2 with collagen I, fibrillin-2, fibronectin and the laminin-1-nidogen-1 complex were previously shown. We used the notexin-induced in vivo model of muscle regeneration. Elevated level of matrilin-2 mRNA was observed already on day 2 of regeneration and reached maximum level between 4 and 7 days, similarly to myogenin gene expression. Matrilin-2 was localized in the matrix surrounding proliferating myoblasts on day 2 and around differentiating myoblasts and myotubes on day 4 of regeneration. In regenerating and control muscle partial colocalization of matrilin-2 was observed with fibronectin, the basement membrane laminin 2 and the costameric marker dystrophin. During in vitro differentiation of C2/7 myoblasts quantitative real-time RT-PCR showed a similar transient increase for matrilin-2, fibronectin, laminin A2 and collagen 6A1 gene expression. Immunostaining demonstrated deposition of matrilin-2 into extracellular filamentous network around proliferating, migrating and fusing myoblasts. We localized matrilin-2 in the epi-, peri- and endoneurium of the motoneurons. We co-localized matrilin-2 with alpha-bungarotoxin in the neuromuscular junctions between days 4 and 7, when the motor endplates are reformed. Detection of the targeted inactivation of Matn2 gene in transgenic mice on muscle regeneration is under progress. The observations suggest a role for matrilin-2 not only in the proliferation, migration, differentiation and fusion of myoblasts, but also in the assembly of the ECM-myofiber and nerve-myofiber contacts. Supported by grants OTKA T049608, ETT 008/2006 and RET-08/2004.

T.P.1.01 Pre-trial antisense screening of myogenic cells from boys with Duchenne muscular dystrophy and genomic and transcriptomic biomarkers discovery for treatment monitoring

M. Neri¹, M.S. Falzarano¹, M. Fabris¹, M. Bovolenta¹, E. Bassi¹, D. Perrone², A. Medici², P. Sabatelli¹, L. Merlini¹, F. Gualandi¹, P. Rimessi¹ and A. Ferlini¹

¹University of Ferrara, Dipartimento di medicina sperimentale e diagnostic, Ferrara, Italy

²University of Ferrara, Dipartimento di biologia evolutiva, Ferrara, Italy

Antisense oligoribonucleotides (AONs) are able to induce exon skipping in the dystrophin hnRNA; a successful first-in-man trial has recently been completed on DMD and a multicentric trial is approaching .The objectives of our project are (i) pre-screening characterization of myogenic cells from DMD boys with dystrophin deletions eligible for either exon 44 or 51 skipping approach to test their suitability to the trial; (ii) identification of genomic and transcriptomic biomarkers to be possibly used to monitor the treatment efficacy and safety.

We have set up myogenic cell cultures (both fibroblast and myoblasts) from 10 DMD patients and we forced fibroblasts into myogenic cells by using MyoD. Deletion mutations have been confirmed by MLPA. Intronic breakpoints have been defined by using our novel CGH-DMD array and rare SNPs within exon 44 and 51 have been excluded. Transcription studies on RNA from patients’ skeletal muscle confirmed the pathogenic nature of the deletion mutations as well as excluded inconsistent splicing behaviours.

We are transfecting myogenic cells with PRO051 (Prosensa Therapeutics B.V.) for the following studies: (a) Real-Time RT-PCR in order to detect and quantify skipped transcript amount and to correlate it with the intronic breakpoints, (b) Real-Time RT-PCR by using a TaqMan® Custom Array covering dystrophin exons in order to verify the correct dystrophin full mRNA composition after the treatment (c) functional effect of the AONs treatment by evaluating dystrophin protein rescue by ICC and WB; (d) dystrophin transcriptome atlas changes analysis through expression arrays before and after AONs treatment.

These studies will fully characterise AONs-treated cells of the selected Italian DMD patients ensuring their eligibility to multicentric AONs trials as planned by Prosensa Therapeutics B.V. in the next two years.

T.P.1.02 Importance of checking exon skipping events prior to clinical trials using systemically delivered antisense morpholino in Duchenne muscular dystrophy patients

S.Nakono¹, S. Ozasa¹, H. Kosuge¹, K. Nomura¹, I. Fujii², K. Ito², M. Matsukura² and S. Kimura¹

¹Kumamoto University Graduate School, Child Development, Kumamoto, Japan

²Sojo University, Laboratory of Clinical Pharmacology and Therapeuti, Kumamoto, Japan

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities of the dystrophin gene. DMD, which manifests into a severe muscle weakness phenotype, results from an out-of-frame deletion(s) in the dystrophin gene. In contrast, BMD, which results from an in-frame deletion(s) in the dystrophin gene, causes a milder muscle weakness. Antisense-mediated exon skipping, which changes out-of-frame deletions to in-frame ones, is a very promising therapeutic approach for DMD. One of the antisences, morpholino, is powerful tools, because it has higher affinity for their target nucleic acid sequences and greater resistance to degradation than conventional nucleic acids.

It is important to confirm whether the morpholino, which has same sequence used in the experiment with animal models, is effective for human, before clinical trial. Fibroblasts from DMD patients with a 48–50 exon deletion of the dystrophin gene were isolated, induced to differentiate to the myogenic lineage by AdMyoD (adenoviral vectors encoding MyoD regulated by CAG Promoter) and transfected with antisense morpholino B30, which is located at the splicing enhancer and/or I25 which is located at exon donor. The skipping event of exon 51 was correctly detected using B30 but not while using I25 (the band size was longer than the correct size). Sequence analysis showed that the fragment included only part of exon 51.

In this study, we developed a system that can easily screen effective sequence of antisense. The system was also used to identify patients who are eligible for the therapy.

T.P.1.04 Dual exon skipping in myostatin and dystrophin as a potential therapy for Duchenne muscular dystrophy

D.U. Kemaladewi¹, W.M.H. Hoogaars¹, S.H. van Heiningen¹, J.C.T. van Deutekom², J.T. den Dunnen¹, G.J.B. van Ommen¹, A.R. Aartsma-Rus¹, P. ten Dijke³ and P.A.C. ’t Hoen¹

¹Leiden University Medical Centre, Center for Human and Clinical Genetics, Leiden, The Netherlands

²Prosensa B.V., Leiden, The Netherlands

³Leiden University Medical Centre, Department of Molecular and Cell Biology, Leiden, The Netherlands

 Myostatin is a member of the transforming growth factor-β family that inhibits muscle growth. Mutations leading to non functional myostatin have been associated with hypermuscularity and enhanced muscle regeneration. In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in Duchenne muscular dystrophy (DMD) patients. We targeted myostatin exon 2 with antisense oligonucleotides (AON) in human primary myoblasts cell culture. We observed skipping of myostatin exon 2, which disrupted the open reading frame and specifically decreased myostatin mRNA expression level. Furthermore, upon myostatin downregulation, the expression levels of its target genes CDKN1A, PAX7, MYF5 and MYOG were found to be elevated. This suggests that myostatin downregulation leads to enhancement of satellite cells self renewal and differentiation of myoblasts towards myofibers. In addition, we combined two AON targeting dystrophin and myostatin in our in vitro culture system and showed that exon skipping in both target genes occurred without apparent interference. Therefore, we propose dual exon skipping in dystrophin and myostatin to simultaneously restore dystrophin synthesis and enhance muscle regeneration as a potential therapy for DMD.

T.P.1.05 Trans-splicing approaches to repair Duchenne dystrophin transcripts

S. Lorain^a, C. Peccate^a, G. Griffith^a, T. Voit^a and L. Garcia^a

^aInstitut de Myologie, UMR 7215 CNRS – U974 INSERM, Faculté de Médecine UPMC, Paris, France

Mutations in the dystrophin gene cause Duchenne muscular dystrophy, the most common severe childhood muscular pathology. With antisense sequences linked to a modified U7 small nuclear RNA, we achieved persistent exon skipping that removes the mutated exon and re-establishes the open reading frame on the dystrophin messenger RNA of the mdx mouse model for Duchenne dystrophy (Goyenvalle et al., Science. 2004). This strategy is promising but will always concern patients for which exon skipping restores a shorter but functional protein. We are therefore developing a novel RNA repair strategy by trans-splicing allowing the replacement of the mutated exon by its normal version.

The trans-splicing approach is based on the Spliceosome-mediated RNA trans-splicing (SmaRT, Intronn, Puttaraju et al., Nature Biotech. 1999), an emerging technology based on a trans-splicing molecule that forces the splicing to occur between the mutated endogenous pre-messenger RNA and an exogenous therapeutic RNA molecule which brings the normal sequence.

We applied this strategy in mdx dystrophic mouse to replace the mutated exon 23 by its normal version. After intramuscular injections of AAV1 vectors expressing trans-splicing molecules, we detected by RT-PCR analysis the presence of repaired transcripts. We are currently working on the optimization of the different elements of the trans-splicing molecule. Those results are promising as a novel therapeutical strategy to treat DMD patients that can not be included in any exon skipping strategies.

T.P.1.09 Oculopharyngeal muscular dystrophy (OPMD): Physiopathological mechanisms and gene therapy approaches

C. Trollet¹, O. Bales², Y. Anvar³, K. Foster⁴, K. Mamchaoui¹, P.A. ’t Hoen³, V. Raz³, S. van der Maarel³, M. Antoniou², V. Mouly¹, G. Butler-Browne¹ and G. Dickson⁴

¹UMRS 974 – Institut de Myologie, Pitie-Salpetriere University Hospital, Paris, France

²Department of Medical and Molecular Genetics, King’s College London School of Medicine, London, United Kingdom

³Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands

⁴Royal Holloway – School of Biological Sciences, Egham, United Kingdom

 A broad range of degenerative diseases is associated with intracellular inclusions formed by toxic, aggregation-prone mutant proteins. Intranuclear inclusions constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD), a rare inherited disease characterised by late-onset eyelid drooping (ptosis), swallowing difficulty (dysphagia) and proximal limb weakness. OPMD is caused by expansion of a trinucleotide (GCG) repeat in the gene that encodes for nuclear poly(A) binding protein (PABPN1). The mutation results in an extended polyalanine stretch that has been proposed to induce protein aggregation and formation of intranuclear inclusions.

We have available various experimental models (clones of conditionally immortalised primary mouse myoblasts stably transfected to express wild-type or mutant human PABPN1 under the control of the human desmin locus control region (DES-LCR); transgenic mouse expressing mutant bovine PABPN1 under the control of the human skeletal actin promoter) and myoblasts isolated from OPMD patients.

The aim of our study is to use these different materials to (1) improve the comprehension of the physiopathology of OPMD and to (2) develop gene therapy approaches to correct the mutation of PABPN1.

We are developing a gene therapy strategy by combining a RNAi knockdown and gene replacement approaches for PABPN1. We have designed and validated optimal siRNA sequences targeting PABPN1. We are currenly testing shRNA adeno-associated viral vectors (AAV-H1-shRNA) for persistent RNAi-mediated knockdown of PABPN1 mRNA. We also have generated a codon-optimised sequence for wild-type human PABPN1 with base changes at siRNA target sequences for a replacement strategy.

In parallel, we are using these genetic tools and models to work on a better characterisation of the disease, with the search for specific markers and deregulated pathways of OPMD.

T.P.1.10 Exon-skipping of dysferlin in CD133+ stem cells isolated from normal and patient affected with dysferlinopathies

C. Navarro¹, A. Farini¹, P. Razini¹, M. Krahn², L. Garcia³, N. Lévy² and Y. Torrente¹

¹University of Milan, Department of Neurological Science, Milano, Italy

²INSERM U910, Marseille, France

³UMR S 974, INSERM/UPMC, Institut de Myologie, Paris, France

Mutations in gene encoding Dysferlin are involved in LGMD-2B and MM. No treatment is available for these diseases. We characterized dysferlin expression in normal human CD133+ stem cells obtained from both circulating blood and muscle, either alone or in co-culture experiments with normal or dysferlin null mouse-derived myotubes. We found human dysferlin expression, by RT-PCR and immunostaining experiments. We designed oligonucleotides anti-sense nucleotides (AONs) targeting consensus regions important to direct splicing machinery, for patients carrying relevant mutations in dysferlin exons. The efficiency of skipping was tested by RT-PCR on CD133 + stem cell isolated from LGMD-2B or MM patients. We plan to test function of re-expressed dysferlin, after engraffing of human skipped CD133+ cells in a mouse model of dysferlinopathy (Dysf−/−) crossed with Scid immunodepressed mice. We believe that combined exon skipping and CD133+ stem cell therapy is a very promising approach that have to be deeply explored in the context of dysferlinopathies.

T.P.1.11 Correction of endogenous DMPK transcripts by chimeric U2 small nuclear RNA – artificial trans-splicing molecules as therapeutic strategy in dystrophia myotonica type 1

H.Y. Chen¹, P. Kathirvel¹, Q.B. Xiong¹, P.S. Lai² and W.C. Yee¹

¹Singapore General Hospital, Singapore, Singapore

²National University of Singapore, Singapore, Singapore

 Dystrophia myotonica type 1 (DM1) results from expansion of a CTG repeat in the 3’untranslated region of the dystrophia myotonica protein kinase gene (DMPK). Artificial trans-splicing molecules (ATMs), which correct DMPK transcripts by replacing the abnormal repeat sequence with an engineered wild type sequence, offer a potential therapeutic strategy in DM1. We had earlier developed ATMs targeting intron 14 of DMPK pre-mRNA which trans-spliced and corrected endogenous DMPK transcripts in myosarcoma cells with maximum trans-splicing efficiency of 7.41% (Chen HY, et al., Gene Ther 2009,16:211–7). To increase the efficiency of the ATMs, we incorporated modified U2 small nuclear RNA (snRNA) sequences into the ATM to mediate the trans-splicing process. In stable myosarcoma cells, compared to the ATM250 without U2 snRNA sequences, the chimeric construct U2-ATM250 carrying the U2 snRNA sequences increased trans-splicing of endogenous DMPK pre-mRNA transcripts from 11.85% to 30.33%. To evaluate the chimeric ATM as potential molecular therapy for DM1, U2-ATM250 molecules were transfected into DM1 fibroblasts that were immortalised using human telomerase (hTERT) and SV40 large T antigen and differentiated into myogenic cells using an inducible MyoD construct (pIND-hMyoD). Trans-splicing correction of mutant DMPK pre-mRNA transcripts with efficiencies of 36.00% and 22.61% respectively were achieved in duplicate experiments, as determined by real-time RT-PCR. A significant reduction in retained mutant DMPK mRNA was further observed in the nuclei of DM1 cells treated with the chimeric ATM. The findings confirm significant enhancement of DMPK transcript correction with incorporation of U2 snRNA sequences into the chimeric ATM, and support the application of the chimeric ATM as a potential therapeutic strategy for DM1.

T.P.2.01 Assessment of treatment efficiency using the mdx model: How to interpret the data?

M. Carre-Pierrat^a, L. Ségalat^a and L. Ségalat^a

^aCNRS, CGMC, Villeurbanne, France

 The literature on preclinical assays on the mdx mice is characterized by a very large number of assays and protocols that make comparisons between studies difficult, not to say often impossible. Although efforts have recently been made to standardize protocols, much remains to be done. This task is rendered complex by the lack of objective data as to which mice assays are the more relevant and the more predictive with respect to human treatment of DMD.

We have taken advantage of a large study of 20 approved drugs (see accompanying poster) that were tested using identical protocols for 22 histological, biochemical and functional parameters (readouts) commonly used in the mdx literature, to address the question of assay choice and measure parameter correlation.

This data analysis leads to several important conclusions: Relative treatment efficiency vary with muscle: some drugs seem quite beneficial to the diaphragm and not to the EDL, and vice-versa. More importantly, drug efficiency vary greatly with time: the drug ranking at 6 weeks, a time point that follows an extensive degeneration period, shows considerable discrepancies with that obtained at 18 weeks, a period of more stable condition. Finally, the correlation analysis between most used readouts shows lower than expected correlations.

These findings reinforce the need for a rigourous definition of preclinical testing procedures on the mdx model.

T.P.2.02 Effect of 20 approved drugs on a wide range of mdx parameters

M. Carre-Pierrat¹, A. Lafoux², F. Fougerousse³, C. Huchet-Cadiou² and L. Ségalat¹

¹CNRS, CGMC, Villeurbanne, France

²CNRS, UMR 6204, Nantes, France

³Genethon, Evry, France

 We have studied the effect of 20 approved pharmaceutical compounds on the muscles of dystrophin-deficient mdx5cv mice. These compounds were selected as the result of a prior screen of 800 molecules on the invertebrate model animal Caenorhabditis elegans. The compounds list includes several tricyclics (imipramine, trimipramine, amitryptiline, desipramine), non-tricyclic antidepressants (fluoxetine), muscle relaxants (carisoprodol, chlormezanone, cyclobenzaprine), channel blockers (nifedipine, mexiletine), antibiotics (cloxacillin), as well as Prednisone as a positive control. Drugs were tested through 2 feeding protocols of 4 weeks and 18 weeks of oral treatment. Each drug was tested at two concentrations. A total of 22 histological, biochemical and functional parameters, commonly used in the mdx literature, were recorded. An overall ranking of all treatments shows that Imipramine, a tricyclic antidepressant well tolerated in humans, is as good as Prednisone on the mdx model. Moreover, tricyclics increase the force output of the fast muscle EDL to level higher than wild-type. This extensive study also reveals that no drug behaves totally positively for the parameters tested.

T.P.2.03 Erythropoietin application in mdx mice: A pilot trial

A.A.S. Carvalho¹, M.R. Ugollini², A.C. Santomauro JR², L.Z.P. Oliveira², V.P.P. Lioi², R.A. dosSantos³, M.H.C. Carvalho³, L.G. Ferreira⁴, M.T. Nunes⁴, S.B. Zyngier² and D. Feder²

¹ABC Faculty of Medicine, Neurology, Santo Andre, Brazil

²ABC Faculty of Medicine, Pharmacology, Santo Andre, Brazil

³University of Sao Paulo, Pharmacology, Sao Paulo, Brazil

⁴University of Sao Paulo, Physiology, Sao Paulo, Brazil

Objective: To evaluate the effect of Erythropoietin application in an experimental model of dystrophy, the mdx mouse. Background: Apart from its erythropoietin function, erythropoietin (Epo) exerts also non-erythroid but tissue protective functions. The potential physiological role of the erythropoietin receptor (Epo-R) in skeletal muscle seems to be the control of proliferation and differentiation of myoblasts and satellites cells. Also, it has been suggested an important role of Epo in response to injury. Design/methods: The long term effects of intraperitoneal(IP) Epo injection on mdx mice, male, were analysed. Seven mdx mice received Epo (1000 IU/kg/IP) and six received saline. All animals were submitted to exercise in ergometer 5 days/week, at a speed of 20 cm/s, 10 min/day. The muscle strength was measured weekly for 12 weeks. After 12 weeks of treatment, all mdx mice were undergone to muscle biopsy and muscle samples were collected from right quadriceps and gastrocnemius and stained for histological evaluation. Left gastrocnemius was dissected, frozen in liquid nitrogen, and stored at −80 °C. The tissue was used for Real-time PCR and cytocines were measured. Results: The results did not show that 12 weeks of Epo treatment was beneficial for improving muscular strength in mdx mice but improved muscular strength during one week (week seven) in the treated group. The histological analysis did not detect differences in necrosis and regeneration between both groups. There were no difference in genic expression of TNF alpha and TGF beta 1 but a significant reduction of myostatin in treated mice. Conclusions/relevance: Myostatin reduction in erythropoietin treatment in dystrophic mice could explain some effects of erythropoietin in skeletal muscles. The lack of improvement in muscle strength for more than one week may reflect the amount of Epo dose used or the period of treatment. A higher dose and early use of Epo could be a potential application in our next trial.

T.P.2.04 Oral TNF alpha inhibitor in mdx dystrophic mice: A pilot trial

D. Feder¹, L.H. Brito¹, L.S. Saad², V. Oliveira¹, E.A.C. Barros¹, R.A. dosSantos³, L.G. Ferreira⁴, M.H.C. Carvalho³, M.T. Nunes⁴, S.B. Zyngier¹ and A.A.S. Carvalho⁵

¹ABC Faculty of Medicine, Pharmacology, Santo Andre, Brazil

²ABC Faculty of Medicine, Pharmacology, Sao Paulo, Brazil

³University of Sao Paulo, Pharmacology, Sao Paulo, Brazil

⁴University of Sao Paulo, Physiology, Sao Paulo, Brazil

⁵ABC Faculty of Medicine, Neurology, Santo Andre, Brazil

.

Objective: To evaluate the effect of Bkt-104, an oral inhibitor of TNF-α in mdx mouse. Background: Tumor necrosis factor (TNF)-α is elevated in inflammatory myopathies as well in DMD. The precise role of TNF-α is unknown, and it is possible involved in the pathogenesis of fibrosis in dystrophic muscle. Blunting TNF pharmacologically may be useful in preventing muscle fibrosis in dystrophic muscles. Design/methods: Long term effects of oral BKT-104 was analysed on mdx mice, male, 8 weeks old; five mice received BKT-104 30 mcg/kg 3 times a week and six received saline (double blinded, placebo). Mice were submitted to exercise in ergometer 5 days/week, at a speed of 20 cm/s, 10 min/day. The muscle strength was measured weekly for 12 weeks. After 12 weeks, all mdx mice undergone to muscle biopsy and right quadriceps and gastrocnemius were collected and stained with acid phosfatase and alkaline for histological evaluation. Left gastrocnemius was dissected, frozen in liquid nitrogen, and stored at −80 °C. The tissue was used for Real-time PCR and mRNA of cytocines were measured. Results: The results of this study did not show any improvement on muscular strength or histological alterations in BKT-104 treatment of mdx mice. There were no difference in genic expression of myostatin but a significant reduction of TNF-α and TGF beta 1 in treated mice. Conclusions/relevance: Presently, TNF inhibitors are only available as an injectable or infusion. BKT104 is a small molecule (sulfated disaccharide), an oral inhibitor of TNF-α (and consequential IL-8 production). BKT104 have show to be highly effective in modulating inflammatory disease in animal models, especially those regulated by TNF-α. This is a pilot study with the drug Bkt-104 in muscular dystrophy. The observed reduction in TNF-α and TGF beta 1 demonstrate the potential of the drug in reducing the fibrosis seen in muscular dystrophy. Acknowledge: Dr. Orly Eizenberg from Biokine Therapeutics.

T.P.2.05 Tamoxifen is more potent than raloxifene to ameliorate skeletal muscle function of adult mdx^5Cv mice

O.M. Dorchies^a, J. Reutenauer^a, O. Patthey-Vuadens^a and U.T. Ruegg^a

^aUniversity of Geneva, Laboratory of Pharmacology, Geneva, Switzerland

 We have shown recently that tamoxifen (TAM), a selective estrogen receptor modulator (SERM), given for 15 months to mdx^5Cv mice from 3 weeks of age on, markedly improved muscle function, decreased plasma creatine kinase levels, and corrected muscle hypertrophy. Here, we investigated whether TAM and raloxifene (RAL), another SERM, used at 0.01% in the chow given for 8–12 weeks to mdx^5Cv mice during the chronic stage of the disease (6-month-old), ameliorate dystrophic muscle condition. Both TAM- and RAL-treated dystrophic mice displayed an increased locomotor activity similar as normal mice and a 2.5-fold decrease in plasma creatine kinase activity. TAM-treated dystrophic mice regularly ameliorated their performance in the grid test, reaching values close to those of normal mice, while RAL-treated animals showed much less improvement. Electrically-evoked contractions of the triceps surae were recorded isometrically at the end of the treatment period. Phasic and tetanic tensions were respectively 30% and 45% lower in dystrophic mice compared to normal mice. TAM normalized the phasic tension and corrected 65% of the tetanic tension deficit. The triceps of the TAM-treated animals contracted and relaxed significantly slower than those of normal or dystrophic mice. Muscle fibres were gradually recruited by increasing the frequency of stimulation by steps of 10 Hz. The force-frequency curve of the TAM-treated animals displayed a leftward shift consistent with the slower kinetics of contraction and relaxation. TAM-treated animals were 30% more resistant to repetitive tetanization than normal or dystrophic mice. RAL-treated mice showed no alteration in any of these muscle contraction parameters. Collectively, our findings suggest that TAM is more potent than RAL in improving dystrophic muscle function. Additional examinations are in progress to determine the cause of the slower phenotype induced by TAM.

T.P.2.06 Exploring the role of phospholipase A₂ in Duchenne muscular dystrophy

A.K. Johansson^a, O. Petermann^a, O.M. Dorchies^a, E. Roulet^a and U.T. Ruegg^a

^aLaboratory of Pharmacology, School of Pharmaceutical Sciences, Univ. of Geneva, Geneva, Switzerland

 Duchenne muscular dystrophy (DMD) is an X-linked muscular disease caused by the absence of dystrophin and characterized by muscle membrane fragility and abnormally elevated levels of intracellular calcium. Increased activity of store-operated channels (SOC) has been proposed as the cause and entry point for the enhanced calcium influx observed in dystrophic muscles. Additionally, it has been shown that the enzyme calcium-independent phospholipase A₂ (iPLA₂) is involved in the regulation of SOC. By inhibiting iPLA₂, calcium entry in dystrophic fibers is greatly reduced.

This work has strived to further analyze the role of iPLA₂ as well as that of the cytosolic calcium-dependent PLA₂ enzyme (cPLA₂) in dystrophic muscle cells. Phospholipase A₂ activity has been assessed in both human and mouse muscle cells using either PED6 or arachidonoyl thio-PC as substrate. The results indicate that iPLA₂ activity is more prominent compared to cPLA₂. In addition, the influence of PLA₂ activity on calcium signaling has been investigated using Fura-2. Both pharmacological tools and gene silencing have been employed to enable the differentiation of iPLA₂ specific activity. In accordance with previous findings, it could be shown that modulating iPLA₂ activity has a direct influence on calcium entry in dystrophic muscle cells.

T.P.2.07 Attenuation of adverse effects of prednisolone on δ-sarcoglycan-deficient cardiomyopathy by mineralocorticoid-receptor-antagonism

A.M. Blain¹, R. Bauer¹, G. MacGowan², E. Greally¹, S. Laval¹, K. Bushby¹ and V. Straub¹

¹Newcastle University, Institute of Human Genetics, Newcastle Upon Tyne, United Kingdom

²Freeman Hospital, Newcastle Upon Tyne, United Kingdom

 An inflammatory component to Duchenne muscular dystrophy (DMD), Limb-girdle muscular dystrophy (LGMD) and other dystrophin–glycoprotein complex-associated muscular dystrophies has been well documented, and steroid therapy increases respiratory and skeletal muscle strength in DMD and sarcoglycan-deficient LGMD patients. In addition, retrospective studies in patients with DMD have suggested that steroids have beneficial effects on left ventricular function when assessed by echocardiography. We have however recently reported severe adverse myocardial effects of steroid treatment in the δ-sarcoglycan-deficient (Sgcd-null) mouse, a well characterised model of LGMD2F. We have therefore investigated the hypothesis that cross reactivity of prednisolone to cardiac mineralocorticoid receptors (MR) plays a role in the pathomechanism by co-administration of steroid-treated mice with the unselective MR-receptor antagonist spironolactone. Animals were treated for 8 weeks from 8 weeks of age (prior to the development of overt signs of cardiomyopathy) with clinically relevant doses of prednisolone (1.5 mg/kg/day) and spironolactone (3.0 mg/kg/day). At the end of the treatment period animals were assessed by in vivo heart conductance catheter and their hearts processed for histological analyses. Our data show that spironolactone has beneficial effects on cardiac remodelling and contractility in Sgcd-null mice and that it prevents steroid-induced deterioration of cardiac haemodynamics and acute sarcolemmal damage, although not cardiac fibrosis. The elucidation of mechanisms underlying steroid-induced cardiomyopathy may have implications for the design of future treatment regimens for LGMD and DMD providing proof of principle for combinational therapies to prevent steroid-induced cardiac damage in patients.

T.P.2.08 The stretch-activated calcium channel candidate proteins TRPC1 and 3 present a distinctly different expression pattern in mdx mice

L.H. Jorgensen¹, S.H. Laval¹, H. Brinkmeier², V. Straub¹, K. Bushby¹ and H. Lochmuller¹

¹Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, United Kingdom

²Ernst Moritz Arndt University of Greifswald, Institute of Pathophysiology, Karlsburg, Germany

 This study aims at identifying the primary causes of calcium dysregulation in Duchenne Muscular Dystrophy pathology. Recent studies have indicated that the stretch-activated calcium-channels (SACs) might be directly involved in DMD pathology. Specifically it has been shown that inhibition of these channels using either streptomycin, GsMTx4 or a dominant-negative approach ameliorates the pathological phenotype of the mdx mouse. The transient receptor potential (TRP) cation channels have been proposed as likely candidates for SACs and we therefore investigated expression level and localisation pattern of TRPC1, 3 and 6 in BL/10-mdx and BL/10 control mice at 6 weeks of age.

TA muscles were collected and stained for TRPC1, 3 and 6 using immunofluorescence on transverse sections. This revealed an expression pattern that was located both to the muscle membrane and in the cytoplasm of BL/10 and BL/10-mdx mice for TRPC1 and TRPC3. However, TRPC1 appeared to be differently expressed between BL/10 and BL/10-mdx with a decreased expression in the mdx mice and less expression at the muscle membranes as well. Single myofibers were isolated from EDL muscle from 6 weeks old BL/10 and BL/10-mdx mice and analyzed for expression of the calcium channel proteins TRPC1, 3 and 6 using immunofluorescence staining and confocal microscopy. Again there was a difference both in expression level and localisation of the TRPC proteins between wild type and mdx mice. TRPC1 appeared to be decreased whereas TRPC3 appeared to be increased in the mdx myofibers. TRPC6 was not expressed in individual myofibers.

This study indicates that not only is the activity of these calcium-channels disturbed in the mdx mouse, as previously suggested, also the protein expression level and localisation pattern within the muscle is affected in the dystrophic pathology.

T.P.2.09 Role of the cationic channel TRPC1 in Duchenne muscular dystrophy: Analysis of double knock-out TRPC1^−/− dystrophin⁻ mice

C. Gallo¹, G. Shapovalov¹, O.M. Dorchies¹, O. Petermann¹, N. Zanou², P. Gailly², U.T. Ruegg¹ and E. Roulet¹

¹Laboratory of Pharmacology, School of Pharmacy, University of Geneva, Geneva, Switzerland

²Université Catholique de Louvain, Louvain, Belgium

 Calcium dysregulation has been shown as one of the triggering events causing muscular degeneration in Duchenne muscular dystrophy (DMD). The increased Ca²⁺ influx could result from transient membrane lesions or from influx through store operated channels (SOC). Recent results indicate that this influx may be due to an over-activation of cationic channels belonging to the TRP family.

Since TRPC1 is one of the main candidates to SOC function (Allen et al., 2005; Ward et al., 2008), we have generated a double knock-out mouse TRPC1^−/− dys⁻ (mdx) to analyze the involvement of TRPC1 in Ca²⁺ regulation of dystrophic muscle.

Force production and muscle fatigue of TRPC1^−/− mice were characterized on excised muscles. A reduction of the maximal tetanic force and an increase of fatigability were found, indicating a probable involvement of TRPC1 in skeletal muscle function (Zanou et al., 2009, in preparation). Results on TRPC1^−/− dys⁻ mice will be shown.

Ca²⁺ imaging experiments were performed on isolated FDB muscle fibers, showing no significant contribution to SOC activity. However, preliminary results indicate a role of TRPC1 in basal Ca²⁺ influx.

Furthermore EDL-mdx myotubes were infected with AAV viruses containing shRNA against TRPC1 and analyzed by Ca²⁺ imaging. Results indicate no relevant SOC activity. Basal influx measurements are in progress.

In conclusion the molecular mechanism linking the functional and molecular properties is not fully clarified.

T.P.2.10 Identification of in-vivo TRPC1 activity in skeletal muscle at single-channel level; role in Ca²⁺ entry

G. Shapovalov¹, N. Zanou², C. Gallo¹, I. Anguish¹, E. Roulet¹, P. Gailly¹ and U.T. Ruegg¹

¹University of Geneva, Pharmacology, Geneve, Switzerland

²Institute of Neurosciences, Cell Physiology, Brussels, Belgium

 Store operated ion channels play an important role in regulation of Ca²⁺ homeostasis in skeletal muscle at rest as well as during contraction. Thus, a disregulation of SOC activity is considered an important factor contributing to cell degradation and death in many musculopathies, such as Duchenne or Becker muscular dystrophies. Transient receptor potential (TRP) proteins are a novel class of Ca²⁺ permeable ion channels exhibiting variety of regulatory functions. In particular, TRPC channels exhibit a mild selectivity for Ca²⁺ and are considered likely candidates to convey SOC activity. Using single-channel patch clamping of TRPC1 positive and negative isolated FBD fibers we have identified a mode of activity associated with TRPC1 protein. Currents with a characteristic conductance of 13 pS, reported in systems overexpressing TRPC1 could be observed in TRPC1+/+, where it was responsible for 12% of overal Ca²⁺ entry, but not in TRPC1−/− cells. This TRPC1 associated current was not upregulated by SR emptying, thus showing that TRPC1 is not responsible for SOC entry, nor did it exhibit mechanosensitive properties. Nonetheless, the absence of TRPC1 caused increased fatigability in TRPC1−/− as was evidenced by behavioral tests. Additionally, muscles from TRPC1−/− mice displayed a decrease in fibre cross-sectional area and generated less force than controls.

T.P.2.11 Comparison of subcutaneous injection and oral routes of administration of EGCG for ameliorating dystrophic lesions in a mouse model of Duchenne muscular dystrophy

Y. Nakae¹, O.M. Dorchies¹, P.J. Stoward² and U.T. Ruegg¹

¹University of Geneva, Laboratory of Pharmacology, Geneva, Switzerland

²University of Dundee, College of Life Sciences, Dundee, United Kingdom

  In 2006 Dorchies et al. reported that epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, given to mdx mice in their diet, starting when they were 3-weeks-old, limits their muscle degeneration. Nakae et al. (2008), using different assessment criteria, found that subcutaneous (s.c.) injection of 5 mg EGCG/kg b.w. into mdx mice 4 times a week for 8 weeks starting from the day after birth, also limits the degeneration. The aim of this study was to investigate whether s.c. EGCG administration was as effective for limiting the onset of dystrophic lesions once muscle degeneration has begun in 3-week-old mdx mice. The mice were injected subcutaneously with either saline or 5 mg or 10 mg EGCG/kg b.w. 4 times a week for 5 weeks. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effect of EGCG was assessed by measuring their serum creatine kinase (CK) activities continuously during the treatment. Integrated locomotor activities, isometric muscle contraction and the numbers of lipofuscin (LF) granules or clumps (a product of oxidative stress) per unit muscle-volume were determined at the end of the treatment. The CK activities of mdx mice decreased exponentially with age from 3- to 8-weeks-old. They were significantly lower by 60% in 8-week-old mice injected with 5 mg/kg EGCG and by 49% in 7-week-old mice given dietary EGCG. Specific phasic twitch and tetanic tensions of triceps surae muscles increased significantly after both oral and s.c. EGCG treatments. Locomotor activities increased significantly by 25–47% at the end of s.c. EGCG administration. Both oral and s.c. EGCG treatments reduced LF accumulation in diaphragm muscles. These results suggest that EGCG treatment by either route in mdx mice beginning at 3 weeks after birth leads to similar improvements to those observed previously for s.c. injection started immediately after birth before dystrophic lesions are apparent.

T.P.2.12 Nuclear factor-kappaB, mitogen-activated protein kinases and cyclooxygenase/5-lipoxygenase pathway inhibition by flavocoxid improves muscle function and morphology in mdx mice: a comparison study with methylprednisolone

S. Messina¹, M. Aguennouz¹, A. Mazzeo¹, A. Migliorato¹, A. Bitto², G.L. Vita¹, M. Russo¹, E. Rizzuto³, A. Musarò³, F. Squadrito² and G. Vita¹

¹Department of Neuroscience, Psychiatry and Anaesthesiology, University of Messina, Messina, Italy

²Department of Experimental Medicine and Pharmacology, University of Messina, Messina, Italy

³Department of Histology and Medical Embryology, Institute Pasteur Cenci-Bolognetti, Sapienza Univeristy of Rome, Rome, Italy

Several lines of evidence suggest that muscle degeneration in Duchenne muscular dystrophy (DMD) patients and mdx mice is exacerbated by the endogenous inflammatory response and increased oxidative stress with nuclear factor (NF)-κB playing key role in the disease process. We showed in previous studies that NF-κB inhibition with compounds which display also antioxidant properties has beneficial effects in mdx mice, but these compounds are not available for clinical studies. We report herein the effects of flavocoxid, a medical food containing two flavonoid extracts with anti-inflammatory, antioxidant and NF-κB inhibition properties, in mdx mice. We found that flavocoxid: (1) increased forelimb strength, strength normalized to body mass and decreased fatigue; (2) reduced serum creatine-kinase levels; (3) reduced the expression of oxidative stress markers and of inflammatory mediators cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and tumor necrosis factor-α) inhibited NF-κB and mitogen-activated protein kinases (MAPKs) signal pathways; (5) reduced muscle necrosis and enhanced regeneration. Our results emphasize the role of inflammation and oxidative stress in dystrophic muscle, involving NF-κB, MAPKs and the COX/5-LOX pathways, and suggest that flavocoxid, a safe medical food, has beneficial effects on muscle function and morphology in mdx mice superior to the effects of methylprednisolone, the gold standard treatment for DMD patients.

G.P.6.08 The evaluation of novel therapeutic strategies for the treatment of dysferlinopathy

M. Hornsey^a, Y. Chiu^a, L. Jorgensen^a, L. Klinge^a, S. Laval^a, R. Barresi^a, V. Straub^a, H. Lochmuller^a and K. Bushby^a

^aUniversity of Newcastle, Institute of Human Genetics, Newcastle, United Kingdom

 Mutations in the dysferlin gene cause limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi Myopathy and distal anterior compartment myopathy, collectively known as the dysferlinopathies. Dysferlin-deficient C57BL/10.SJL-Dysf mice exhibit adult onset muscular dystrophy, high serum creatine kinase (CK) levels and a prominent inflammatory infiltrate, providing an excellent model for the study of dysferlinopathy. Preliminary analyses indicate that pathology is limited to specific muscle groups even at late stages of disease, while in situ force measurement performed on pre-symptomatic tibialis anterior (TA) shows a delay in force recovery. However, dysferlin-deficient muscle does not show susceptibility to acute damage, but has an impaired ability to regenerate following muscle wounding using either the snake venom notexin or needle wounding. Histological analysis shows the delayed removal of necrotic myofibres with an initial poor recruitment of neutrophils. Our hypothesis is that the process of muscle regeneration in C57BL/10.SJL-Dysf mice can be viewed as a model for disease progression in man. We have evaluated the regenerative process in this model, especially force generation following notexin injection and frequency of necrotic fibres as markers for pathological progression and treatment efficacy.

We are currently evaluating a number of agents that modify neutrophil behaviour and/or recruitment to assess their ability to enhance the muscle regenerative process in dysferlin-deficient animals.

M.P.3.01 Gait steadiness and upper-body kinematics in DMD children

R. Ganea¹, N. Goemans², M. van den Hauwe², K. Aminian¹, A. Paraschiv-Ionescu¹ and P.Y. Jeannet³

¹Laboratory of Movement Analysis and Measurement, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

²Neuromuscular Center & Department of Child Neurology, Universitair Ziekenhuis Gasthuisberg, Leuven, Belgium

³Pediatric Neuromuscular Center, Department of Pediatrics, CHUV, Pediatrics, Lausanne, Switzerland

New tools that better assess life-limiting-events in neuromuscular disorders are needed as outcome measures in clinical trials. Quantitative assessment of movement patterns during walking in DMD patients is potentially an objective evaluation of disease severity. While lower limbs initiate and sustain locomotion, motion of the trunk is important for maintaining balance and may be characterized by a dynamic pattern that change with disease progression. Method: The gait pattern of 21 DMD children (age = 8.5 ± 1.7) and 17 age-matched healthy controls was analyzed during free walking over 200 m with one gyroscope on each leg and a 3D kinematical sensor (accelerometers and gyroscopes) on the trunk. Gait steadiness (an indicator of compensatory adaptation to the motor deficit) was quantified from (1) the range and step-to-step variability of trunk angular velocity norm (R-AVe), also quantified by median absolute deviation (MAD-AVe) and (2) the smoothness of trunk acceleration norm quantified by spectral entropy (SE-Acc). The MFM scale was used to assess disease severity. Results: The values of R-AVe, MAD-AVe and SE-Acc were significantly greater in DMD patients (62 ± 9°/s, 11.4 ± 3.7°/s, 0.75 ± 0.06, respectively) compared to healthy control (49.4 ± 7.4°/s, 8.2 ± 1.8°/s, 0.6 ± 0.07), independently of the walking speed which was lower in DMD (0.9 ± 0.17 m/s) compared to control (1.13 ± 0.12 m/s). Higher values were found in more severely affected patients. Discussion: This study shows that upper-body compensatory movement to maintain dynamic balance in DMD patients can be quantified during free-walking over 200 m. These in-lab validated parameters will allow the quantification of walking patterns recorded under long-term daily-life conditions while wearing a single kinematical sensor on the trunk. We hypothesize that the development of such new assessment parameters during gait may allow a better definition and quantification of functional limitation in neuromuscular disorders.

M.P.3.02 Detailed analysis of daily-life physical activity patterns in DMD children

P.Y. Jeannet¹, R. Ganea², C. Piot¹, N. Goemans³, M. van den Hauwe³, K. Aminian² and A. Paraschiv-Ionescu²

¹Pediatric Neuromuscular Center, Department of Pediatrics, CHUV, Lausanne, Switzerland

²Laboratory of Movement Analysis and Measurement, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

³Neuromuscular Center & Department of Child Neurology, Universitair Ziekenhuis Gasthuisberg, Leuven, Belgium

Activity monitors are now used as outcome measures in neuromuscular disorders, but they often monitor limited aspects of physical activity. The aim of this study was to provide a detailed analysis of daily-life physical activity (PA). In 20 ambulatory DMD boys, age 8.5 ± 1.7 y and 18 age-matched healthy controls age 8.3 ± 2.2 y. PA was monitored during 3 consecutive days, using a chest worn data-logger (Physilog^®) with a 3D inertial sensor (accelerometers and gyroscopes). Body posture and walking parameters were quantified. Gait smoothness was also assessed through quantification of spectral entropy of trunk acceleration pattern. The MFM scale was used to assess disease severity. Results: Time (in %) spent standing and walking in DMD vs. controls was 47.27 ± 9.48, 14.12 ± 5.13 vs. 55.12 ± 9.79, 19.40 ± 5.45. Time (in %) spent sitting and lying in DMD (45.4 ± 8.04, 7.31 ± 5.10) was increased compared to controls (38.91 ± 7.4, 5.95 ± 5.5). The number of walking episodes longer than 1 min and longer than 6 min were decreased compared to controls (7.61 ± 4.7, 0.11 ± 0.26 vs. 11.58 ± 6.5, 0.4 ± 0.6), the same was true for the maximum continuous walking duration and the maximum cadence, (217.39 ± 115.82, 180.27 ± 5.48 vs. 371.86 ± 211.56, 181.88 ± 3.34). Gait pattern was smoother in controls than in DMD (0.71 ± 0.08 vs. 0.61 ± 0.01). There was an overall significant correlation (p < 0.05) between the MFM scores and time spent walking, r = 0.48, number of walking episodes longer than 1 min, r = 0.5 and maximum cadence, r = 0.71. Discussion: This study shows that the type, duration, intensity and frequency of physical activity in DMD patients can be reliably determined if the appropriate algorithms are used. The high sampling rate (1 s) is particularly appropriate to characterize children’s PA which occurs in short bursts of rapidly changing activity. We hypothesize that such a detailed approach of PA assessment better reflects “life limiting events” which are relevant for patients with neuromuscular disorders.

M.P.3.03 6-minute walk test in Duchenne muscular dystrophy: Longitudinalobservations

C. McDonald¹, E. Henricson¹, R.T. Abresch¹, A. Nicorici¹, L. Atkinson², A. Reha², G.L. Elfring² and L.L. Miller²

¹University of California, Davis School of Medicine, Davis, CA, United States

²PTC Therapeutics, Inc., South Plainfield, NJ, United States

Background: Walking abnormalities are prominent in Duchenne/Becker muscular dystrophy (DMD/BMD). As a prelude to a Phase 2b 48-week registration trial of ataluren (PTC124™) in DMD/BMD, a short-term observational study established the feasibility of a 6-minute walk test (6MWT) in boys with DMD and documented ambulation differences relative to healthy controls. Methods: This long-term follow-up provides data on 6-minute walk distance (6MWD) (DMD n = 12; healthy n = 9) and stride length (DMD n = 9; healthy n = 7) for observational study participants who underwent repeat testing 1 year after initial testing. Results: Median [range] age at repeat testing was similar at 8.5 [5–12] years for boys with DMD and 8 [5–12] years for healthy controls. After 1 year, respective height and weight increased from median [range] of 129 [104–151] cm to 134 [112–169] cm and 35 [18–70] kg to 43 [20–77] kg for boys with DMD and from 136 [122–154] cm to 143 [128–159] cm and 35 [22–72] kg to 42 [28–79] kg for healthy boys. 6MWD decreased in 9/12 (75%) boys with DMD (median [range] from 350 [252–481] m to 304 [150–530] m) and increased in 7/8 (88%) healthy boys (median [range] from 616 [525–687] m to 620 [590–724] m). Stride length decreased in 7/10 (70%) boys with DMD (median [range] from 0.93 [0.69–1.21] m to 0.91 [0.63–1.25] m) and was maintained or increased in 7/7 (100%) healthy boys (median [range] from 1.50 [1.24–1.66] m to 1.51 [1.41–1.70] m). Conclusions: Most boys with DMD experience declines in ambulation over 1 year with changes that diverge from those in healthy control boys. Stabilization or improvement in 6MWD with 48 weeks of ataluren treatment in the ongoing Phase 2b registration trial may represent a clinically meaningful benefit to boys with DMD/BMD.

M.P.3.04 Assessment of StepWatch™ activity monitoring in phase 2b study of ataluren (PTC124™) in nmDMD/BMD

C. McDonald¹, K. Coleman², E. Henricson¹, R.T. Abresch¹, M. Eagle³, J. Florence⁴, E. Gappmaier⁵, A.M. Glanzman⁶, A. Reha⁷, G.L. Elfring⁷, L.L. Miller⁷ and L. Atkinson⁷

¹University of California, Davis School of Medicine, Davis, CA, United States

²Orthocare Innovations, Seattle, WA, United States

³Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom

⁴Washington University School of Medicine, St. Louis, MO, United States

⁵University of Utah, Salt Lake City, UT, United States

⁶Children’s Hospital of Philadelphia, Philadelphia, PA, United States

⁷PTC Therapeutics, Inc., South Plainfield, NJ, United States

 Background: A Phase 2b, international, multicenter, randomized, double-blinded, placebo-controlled, dose-ranging study is assessing the efficacy and safety of ataluren in 174 patients with nonsense mutation Duchenne/Becker muscular dystrophy (nmDMD/BMD). Methods: Pretreatment evaluations in this study included an in-clinic 6-minute walk test (6MWT) followed by 4–7 days of at-home StepWatch activity monitoring (SAM), each performed twice 6 weeks apart. Subjects wore the SAM device continuously from waking to bedtime. Results: 6MWT and SAM data are available for 164 subjects (ages 5–20 years) with 4 days of SAM recording. Mean [SD] average daily step count was 5236 [2012]. When comparing 5-11 year-old boys (n = 146) with nmDMD/BMD in this study vs historical healthy boys (n = 26) of the same age range, boys with nmDMD/BMD had lower mean [SD] average daily step count (5461 [1421] for nmDMD/BMD vs 7780 [2538] for controls); % of time spent at medium (15 [7] vs 31 [4]) and high (3 [5] vs 15 [5]) activity; and maximum continuous 20-minute effort (20 [5] vs 44 [9]), 30-minute effort (15 [3] vs 38 [9]), and 60-minute effort (8 [2] vs 28 [7]) steps/minute. Boys with nmDMD/BMD had higher % of time spent at low activity (82 [12] vs 51 [7]). For all SAM parameters, reliability was high for 137 subjects with test-retest data (range r = 0.73–0.81). Mean average daily step count correlated moderately (r = 0.53) with 6-minute walk distance (6MWD) (n = 162). When assessed by the study stratification factors, mean [SD] average daily step count was 4211 [2077] and 5935 [1640] for boys with baseline 6MWD <350 m vs 350 m, respectively; 4159 [1890] and 6073[1690] for boys 9 vs <9 years, respectively; and 4904 [1732] and 5345 [2109] for corticosteroid non-users vs users, respectively. Conclusion: At-home activity is impaired in boys with nmDMD/BMD, can be reliably assessed by SAM, and correlates with factors known to affect disease severity.

M.P.3.05 Use of a novel system for grading timed function test performance in phase 2b study of ataluren (PTC124™) in nonsense mutation Duchenne and Becker muscular dystrophy (nmDMD/BMD)

M. Elfring¹, R.T. Abresch², J. Florence³, E. Gappmaier⁴, A.M. Glanzman⁵, E. Henricson², A. Reha⁶, G.L. Elfring⁶, L.L. Miller⁶ and L. Atkinson⁶

¹Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom

²University of California, Davis School of Medicine, Davis, CA, United States

³Washington University School of Medicine, St. Louis, MO, United States

⁴University of Utah, Salt Lake City, UT, United States

⁵Children’s Hospital of Philadelphia, Philadelphia, PA, United States

⁶PTC Therapeutics, Inc., South Plainfield, NJ, United States

 Background: A Phase 2b, randomized, double-blinded, placebo-controlled study is assessing the efficacy and safety of ataluren in 174 patients (ages 5–20 years) with nmDMD/BMD. Screening and baseline evaluations (6 weeks apart) included 6-minute walk distance (6MWD); timed function tests (TFTs) (10-meter walk/run, 4-stair climb, 4-stair descent, stand from supine); and myometry (shoulder abduction, elbow and knee flexion/extension). In addition, a new method was used to grade how TFTs were performed. Methods: Based on recognized movement patterns reflective of disease progression and the clinical effects of corticosteroid usage in DMD/BMD, a TFT method grading system was devised. Grades ranged from non-performance (0) to normal performance (6). Clinical evaluators were given in-person training, written manuals, and video examples. TFT method grading was performed simultaneously with TFTs. Results: Median [range] TFT method grades were 5 (able to pick up speed but runs with a double stance phase) [2–6] for 10-meter walk/run; 3 (climbs 1 foot at a time, with both feet on a step before moving to the next step, using 1 arm on 1 handrail) [1–5] for 4-stair climb; 2 (descends 1 foot at a time, with both feet on a step before moving to the next step, requires both arms on 1 or both handrails) [1–5] for 4-stair descent; and 4 (half Gowers’) [1–6] for stand from supine. TFT method grade test-retest reliability was high (range r = .75 to.83). Lesser performance (lower grade) correlated with higher TFT times (range r = −.69 to −.53). However, grading differentiated ability in boys with similarly fast TFT times. TFT method grade correlated better with 6MWD (range r = .63 to.70) and knee extension (range r = .54 to.67) than with other myometry parameters (range r = .10 to.49). Conclusion: TFT method grades are reliable and correlate appropriately with other clinical measures, while providing additional clinically meaningful information about functional ability in boys with DMD/BMD.

M.P.3.06 Quantification of upper limb activity in non ambulant boys with Duchenne muscular dystrophy using accelerometry: A proof-of-concept study

L. Servais^a, A. Canal^a, J.Y. Hogrel^a and T. Voit^a

^aHôpital de la Pitié Salpétrière, Institut de Myologie, Paris, France

Disease progression in ambulant boys with Duchenne muscular dystrophy (DMD) is frequently assessed by the 6-minutes walk test, which is also used as a primary outcome measure of ongoing therapeutic trials. There is currently no standardized method to assess upper limb function in non-ambulatory patients, which complicates trials in these cohorts.

Upper limb function may be assessed by direct muscular strength measurement, either by clinical test that still have to be standardized and validated, or by activity monitoring. This latter approach is probably the more strongly correlated with patient’s quality of life.

Measurement of upper limb activity by accelerometry in non-ambulatory patients presents different technical requirements. The accelerometer must be as light as possible, wireless, able to indicate its spatial orientation in order to discriminate acceleration from rotation movements that modify gravity projection, able to discriminate active patient’s movements from wheelchair or caregiver induced movements.

We tested Movea Motionpod^® accelerometer, that matches the above mentioned criteria in three non-ambulatory patients with DMD and one patient with myotubular myopathy who serves as pathological control, this patient being unable to produce any spontaneous wrist movements. These tests demonstrate that in the range of activity of non-ambulatory DMD boys, Movea Motionpod^® accelerometer with concomitant angle measurement reliably allows to discriminate acceleration from gravity projection change. In addition, wheelchair movement could easily be identified by the rapid angular change of the yaw angle. Most of caregiver-induced accelerations could also be discriminated since they were widely out of the range of the patient’s maximal accelerations.

In conclusion, this preliminary study demonstrates that Movea Motionpod^® accelerometer could constitute a useful tool for qualitative and quantitative monitoring motor activity of non-ambulatory patients with DMD.

M.P.3.07 Isometric dynamometry with a Biodex system for the follow-up of patients suffering from LGMD2A (calpainopathy)

J.Y. Hogrel^a, G. Ollivier^a, A. Canal^a and Calpain Study Group

^aInstitut de Myologie, Paris, France

 Strength is one of the more frequently used variables to follow the patients suffering from neuromuscular disorders by the mere fact that muscles are force generators. Several evaluation tools for measuring muscle strength are available. Among the quantified muscle testing methods, isokinetic systems have rarely been applied to neuromuscular disorders because they are not a priori designed for such patients. The objective of this work was to evaluate the feasibility of measuring maximal isometric contractions in patients using a Biodex System 3. A specific isometric protocol was setup to take into account patient weakness. Isokinetic evaluation was not chosen because the attachments were generally too heavy to be mobilized by the patients. The attachments were positioned in order to cancel the effect of gravity. Twenty-one patients suffering from LGMD 2A (calpainopathy) were regularly followed from 2005 to 2009 by a standardized isometric protocol. Elbow and knee flexion and extension were assessed using this particular protocol but also using manual muscle testing and quantified muscle testing (QMT).

The patient positions during Biodex testing were easily repeated from session to session. Since the Biodex is limited in precision (0.7 Nm), the strength of very weak patients could not be measured with high reliability. This limit can be however circumvented using the Biodex research software and the analog outputs toward an acquisition system. When strength was lower than about 2 Nm, all measurement methods were prone to be variable. The correlations between QMT and Biodex results were good to excellent depending on the muscle function: the weaker functions led to lower correlation coefficients and reflected measurement variability due to muscle weakness.

Although expensive, Biodex can be used for the follow-up of patient strength in isometric conditions.

M.P.3.08 How can measurement of muscle torque in children by hand-held dynamometry enhance clinical decision making

J. Saulnier¹, L.J. Hébert², C. Lepage³ and D.B. Maltais⁴

¹Rehabilitation Institute of Quebec City, Neuromuscular Diseases Program, Quebec City, Canada

²National Defence of Canada, Laval University, Faculty of Medicine, Department of Radiology, Quebec City, Canada

³Centre for Interdisciplinary Research in Rehabilitation and Social Integration, Rehabilitation Institute of Quebec City, Department of Education and Scientific Support, Quebec City, Canada

⁴Centre for Interdisciplinary Research in Rehabilitation and Social Integration, Laval University, Faculty of Medicine, Department of Rehabilitation, Quebec City, Canada

In a previous study, we confirmed the feasibility, intra- and inter-rater reliability, and concurrent validity with the Cybex dynamometer of a standardised protocol for maximal isometric muscle torque (MIMT) in a paediatric population using a hand-held dynamometer (HHD). Using normative reference values obtained from a large cohort of healthy children (n = 365), three case studies are presented to demonstrate how quantitative muscle strength can be used to monitor the stages of a disease progression and positively influence clinical decisions. The three cases chosen were a 17-year-old male with a transverse myelitis secondary to chemotherapy (C1), a 13-year-old male with spinal muscular atrophy type III (SMA III)(C2), and a 9-year-old male presenting with a mitochondrial myopathy (C3). The MIMT of the following muscles, according to each case, were measured: shoulder abduction, elbow flexion and extension, hip flexion and abduction, and knee flexion and extension. MIMT of each subject was assessed at several occasions over a period of 15, 9, and 39 months for C1, C2, and C3, respectively. Using the inferior limit of the 95% confidence interval of MIMT of healthy children for the same age group and gender, the three cases showed strength deficits between 3.7% and 14.2% (C1), 0.6% and 83.9% (C2), and 19.6% and 44.1% (C3). For each case, the localization, severity, and progression of muscle impairments were specific to certain muscle groups, which would explain why, as reported by the children, the parents, and the educators, it was possible or not to complete specific functional activities. This quantitative data provided by the physiotherapist has brought a unique added value to the rehabilitation team. The assessment of MIMT using HHD allows to enhance clinical decision making by clarifying patient clinical status, make better prognosis, identifying specific individual needs, develop a well-aimed treatment plan, and establish specific individual recommendations.

M.P.3.09 Relation between total power estimated from 3D accelerometric data and distance covered during the six-minute walk test

I. Ledoux^a, V. Decostre^a, A. Canal^a and J.Y. Hogrel^a

^aInstitut de Myologie, Paris, France

The six-minute walk test (6MWT) is used as a measure of functional exercise capacity. Initially developed for patients with heart or lung diseases, the 6-minute walk distance (6MWD) is chosen as an outcome measure in more and more clinical protocols involving neuromuscular patients. The aim of this study was to study the relation between total power estimated from 3D-accelerometric data and distance covered during six-minute walk test.

Fifty subjects participated to this pilot study. They were healthy or suffering from various neuromuscular disorders; children or adults; men or women.

Accelerometric data were recorded along the three axis of space using a specific device (Locometrix) during a 6MWT performed in a 25 m long corridor. The device was placed over the middle of the low back, near the centre of gravity of the subjects. Total power and powers in each direction of the device were computed from each lap using Fourier transform integrals.

The relation between total power and 6MWD was well fitted by a parabolic regression equation, probably due to the relation between power and speed. In healthy subjects, the distribution of the power was almost stable in each direction. However, in some patients, total power was decreased during walking because these patients were either reducing their speed of walk or losing their efficiency to walk. The analysis showed that accelerometry can bring objective information on patient fatigability or warm-up effect during the 6MWT. Moreover 3D-accelerometry allows analysing the re-distribution of power in the medio-lateral, antero-posterior and cranio-caudal directions with respect to time.

M.P.4.02 Perioral skin biopsy to study skeletal muscle protein expression

L. Santoro¹, C. Fiorillo², M. Nolano³, V. Provitera⁴, S. Faraso⁴, C. Vitiello⁴, S. Aurino⁴ and V. Nigro⁵

¹University Federico II, Italy, Neurological Sciences, Naples, Italy

²University Federico II, Naples, Italy, Department of Neurological Sciences, Naples, Italy

³Fondazione Clinica del Lavoro, Salvatore Maugeri, Telese Terme, Benevento, Italy

⁴Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy

⁵Seconda Università degli Studi di Napoli, Naples, Italy, Dipartimento di Patologia Generale, Naples, Italy

 Background: Specific diagnosis of muscle dystrophy can be challenging because of the constantly expanding classification of this group of pathology, and not so infrequently a second muscle biopsy is required. Clinical trials for molecular treatment of muscular dystrophies also require multiple sampling of skeletal muscle to monitor the protein rescue. This practice is invasive and could arise ethical problems. A less invasive tool to obtain sequential muscle sampling is necessary. Methods: We performed 2 mm punch skin biopsies from the perioral region of 6 healthy subjects and 6 patients with genetically defined forms of muscular dystrophy and analysed sample with immunofluorescence for specific skeletal muscle proteins. Results: Large intradermal bundles of orbicularis oris muscle were constantly present in all skin biopsies. Controls had a typical muscular antigenic pattern, while muscular dystrophy patients exhibited the expected protein defect. Conclusions: Skeletal muscle protein expression can be examined using perioral skin biopsy. This is a rapid, easily repeatable and minimally invasive technique. We propose skin biopsy as a new method to eventually confirm the diagnosis in unspecific muscular dystrophies, to assess the disease progression and to follow up the response to genetic or conventional therapies.

 T.P.3.01 Glucocorticoids influence therapeutic efficacy of idebenone (Catena^®) on peak expiratory flow in patients with Duchenne muscular dystrophy (DMD)

G.M. Buyse¹, N. Goemans¹, M. van den Hauwe¹, I.J.M. de Groot², U. Schara³, B. Ceulemans⁴ and T. Meier⁵

¹University Hospitals Leuven, Leuven, Belgium

²UMC St Radboud, Nijmegen, Netherlands

³University of Essen, Essen, Germany

⁴University Hospital Antwerp, Antwerp, Belgium

⁵Santhera Pharmaceuticals, Liestal, Switzerland

 Background & aims: In DMD respiratory complications are a predominant cause of morbidity and mortality. A Phase IIa randomized controlled trial (DELPHI) in 21 DMD boys (8–16 years) showed efficacy of idebenone on cardiac and respiratory parameters. Patients treated with idebenone improved on peak expiratory flow (PEF) and PEF % predicted (PEF-%p) whilst patients receiving placebo deteriorated over the 12 months study period. We have now performed a post-hoc subgroup analysis to verify any potential interference of glucocorticoids on these early markers of respiratory weakness in DMD patients. Results: In the complete study population (irrespective of glucocorticoid use) idebenone treatment improved PEF by 30.8 ± 54.4 L/min and PEF-%p by 2.8 ± 13.8% during the study period, whilst patients on placebo deteriorated in PEF by −13.8 ± 51 L/min (p = 0.04) and PEF-%p by −8.5 ± 13.8% (p = 0.04). The total effect size (difference between idebenone treatment and placebo) was 11.3% for PEF-%p. Baseline values of PEF-%p were significantly higher in the patients receiving glucocorticoids (86.9 ± 18.7%; N = 13) versus glucocorticoid-naive patients (53.9 ± 23.6%; N = 8; p = 0.001), and glucocorticoids had a marked impact on the difference in change from baseline between the placebo and idebenone treatment arms. In the glucocorticoid-naïve patients, idebenone caused a 8.0 ± 12.1% increase in PEF-%p, whilst patients on placebo declined by −12.3 ± 17.9% (p = 0.05), resulting in a total effect size of 20.3%. In the patients receiving concomitant glucocorticoids, PEF-%p declined by − 0.4 ± 14.6% in the idebenone group compared to −6.2 ± 12.4% (p = 0.24) in the placebo group, resulting in a total effect size of 5.8%. Conclusions: Glucocorticoids slow the decline of early markers of respiratory muscle weakness (PEF and PEF-%p) in DMD. Furthermore, the effect size of idebenone on these parameters was significantly larger in glucocorticoid-naïve patients compared to glucocorticoid users.

T.P.3.02 Use of glucocorticoids in Duchenne MD: Consensus report of the CDC Duchenne care considerations neurology panel

R. Finkel¹, D. Biggar², C. Bonnemann¹, C. Constantin³, D. Escolar⁴, E. Massey⁵, T. Miller⁶, J. Pascual⁷, J. Sladky⁸, K. Wagner⁹, B. Wong¹⁰ and K. Bushby¹¹

¹Children’s Hospital of Philadelphia, Pediatrics, Philadelphia, United States

²Bloorview Kids Rehab Hospital, Pediatrics, Toronto, Canada

³Centers for Disease Control and Prevention, Center on Birth Defects & Developmental Disability, Atlanta, United States

⁴Children’s National Medical Center, Pediatric, Washington, United States;

⁵Duke University, Pediatrics, Durham, United States

⁶University of Arizona, Neurology, Tuscon, United States

⁷University of Texas Southwestern Medical Center, Neurology, Dallas, United States

⁸Emory University, Pediatrics, Atlanta, United States

⁹Johns Hopkins Medical Institutions, Neurologoy, Baltimore, United States

¹⁰Cincinnati Children’s Hospital Medical Center, Pediatrics, Cincinnati, United States

¹¹Newcastle University, Genetics, Newcastle, United Kingdom

 Background: The US Centers for Disease Control (CDC) convened a panel of 84 experts to address various aspects of the diagnosis and management of Duchenne MD (DMD). The neuromuscular panel was charged with reviewing pharmacologic topics. Use of glucocorticoids (GC: prednisone/prednisolone [pred] and deflazacort [dflz]) and related adverse side-effects (AEs) were addressed and is summarized here. Methods: Guided by the RAND/UCLA Appropriateness Method, available scientific evidence was augmented with expert opinion to develop rating materials. Eleven clinical experts in DMD independently rated thirty clinical scenarios. Each assessment and intervention was rated as necessary, appropriate, inappropriate, or uncertain and also captured disagreement among the group. The expert panelists reviewed and interpreted the ratings to develop the care management recommendations. Results: A trial of GC therapy was deemed necessary in all boys with DMD. Pred and dflz were judged similar in effectiveness. Dflz may be preferable when pre-existing obesity or behavioral issues are present (though not currently approved for use in the US). Daily treatment with pred (0.75 mg/kg/day) or dflz (0.9 mg/kg/day) should be started when the boy is in a plateau or decline phase of motor function, focusing upon ambulation skills. The dosage is typically increased as the child grows until a weight of 40 kg is reached. Should AEs arise which are unmanageable or intolerable, despite utilizing aggressive management strategies, then reduction in dosage is necessary. Persistence of such AEs should then prompt consideration of other dosing regimes: alternate day or high-dose weekend. Conclusions: GC therapy is a mainstay of pharmacological management of DMD at present. These care consideration guidelines provide a framework for the initiation of GC treatment, dose adjustment and regular monitoring for AEs. This requires a coordinated effort of health care providers.

T.P.3.03 Long-term, low-dosage, early steroid therapy in Duchenne muscular dystrophy prolongs ambulation and preserves respiratory and cardiac function. 13 years follow-up

L. Merlini¹, E. Malaspina², A. Cicognani², E. Franzoni², A.R. Armaroli³, B. Talim⁴, F. Gualandi⁵, M.E. Michelini⁶ and A. Ferlini⁵

¹IRCCS - IOR, Laboratory of Biology, Bologna, Italy

²University of Bologna, Department of Pediatrics, Bologna, Italy

³Ospedale S. Anna, Sezione di Genetica, Ferrara, Italy

⁴Hacettepe University, Department of Pediatrics, Ankara, Turkey

⁵University of Ferrara, Dip. di Medicina Sperimentale e Diagnostica, Ferrara, Italy

⁶S. Anna Hospital, Department of Pediatrics, Ferrara, Italy

 Duchenne muscular dystrophy (DMD) is an incurable disease of childhood. In the natural history course of DMD, loss of walking ability at the mean age of 9.5 years (range 6–13) is followed by restrictive respiratory syndrome, cardiomyopathy, and eventually death. The major aim of steroids in the ambulant phase of DMD is to prolong the ability to walk. RCT have shown that steroids improved muscle strength and function for six months to two years, but the overall long-term benefit remains unclear, and has to be weighed against the long-term side effects of steroids. In 1996 we started a prospective long-term open, parallel group, double consent trial of low-dosage prednisone in five early cases of DMD. Prednisone was given 0.75 mg/kg for 2 weeks, then, 1.25 mg/kg on alternate days. Ability to rise from the floor was prolonged comparing with the three controls. Four of the boys on prednisone could be followed up regularly for 13 years. At the last control (mean age 15.8 ± 1.2 years) the four boys could walk independently. One, aged 17 years, lost the ability to rise from the floor at 12 years, and to climb stairs at 16 years. Another one, aged 16.9 years lost the ability to rise from the floor at 15.8 years. Forced vital capacity ranged from 80 to 104 percent of predicted. All had a left ventricular ejection fraction of more than 50% indicating absence of cardiomyopathy. Bone mineral density assessed by dual energy X-ray absorptiometery (DEXA) scans ranged from 0.637 to 0.704 g/cm². While body mass index was normal (22 ± 2.3), total fat % was 50 ± 6.1 as determined by DEXA total body. Growth failure with short stature was present in all with decline below the third centile from the age of 12 years. Cataracts was not observed.

Low dosage steroid therapy started before the age of 4 years in these patients with DMD has prolonged ambulation and preserved pulmonary and cardiac function moving their clinical phenotype toward the outliers Duchenne and Becker muscular dystrophy.

T.P.3.04 Clinical trial using nitric oxide releasing drug and nonsteroidal antiinflammatory drugs in muscular dystrophy: Design of a study

S. Gandossini¹, M.G. d’Angelo¹, S. Bonato¹, G.P. Comi², F. Magri², M. Moggio², M. Sciacco², A.C. Turconi¹, C. Sciorati³, N. Bresolin⁴ and E. Clementi⁵

¹IRCCS E.Medea, NeuroRehabilitation Dept., Neuromuscular Unit, Bosisio Parini (Lc), Italy

²Dino Ferrari Centre, IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena Foundation, Dept. of Neurological Sciences, Milan, Italy

³IH San Raffaele Scientific Institute, Milan, Italy

⁴IRCCS E.Medea;Dino Ferrari Centre, IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena Foundation, Milan, Italy

⁵IRCCS E.Medea;Clinical Pharmacology Unit, Dept. Preclinical Sciences, L. Sacco University Hospital, University of Milano, Milan, Italy

 Introduction: Nitric Oxide (NO) acts as a protective agent in muscle by regulating excitation-contraction coupling and enhancing vasodilation and glucose uptake during exercise. In addition, NO prevents the muscle from being damaged during contractile activity and contributes to myogenesis and maintenance of the satellite stem cell pool. In mouse models of muscular dystrophy HCT 1026, a NO releasing derivate of flurbiprofen, that combines the action of NO and of nonsteroidal antiinflammatory drugs (NSAID), slows the progression of the disease and maintains muscle integrity. Our study assesses whether these encouraging data can be translated into patients. Methods: An open-controlled therapeutic trial in 50 adult subjects affected with Limb girdle (LGMD), Becker (BMD) and Duchenne dystrophy (DMD) to assess tolerability and efficacy of the drug combination is designed. One branch of patients received ibuprofene (200 mg²/day) and isosorbide dinitrate retard (40 mg/day) and other branch received no drug. Inclusion and exclusion criteria are established. Serial clinical assessment are planned. Tolerability and adverse events are assessed by physical examinations, blood tests, instrumental evaluations (echocardiograms, ECG, spirometry, oxyhaemoglobin saturation). Primary outcome measure is represented by MFM scale (Motor Function Measures); secondary outcome measures by the MRC scale (Medical research Council scale), pulmonary and cardiac tests, quality of life scale. Results: The clinical trial started in April 2008. Up to now we enrolled 31 patients (16 LGMD, 4 BMD and 11 DMD). The mean age of patients’ is 30 ± 2.8 years. Overall the compliance is good. Some of the patients complained of low blood pressure and slight headache (at the beginning of the treatment) and transient gastric pain. Five patients dropped out due to long lasting and severe headache. We plan to end the pharmacological treatment and have conclusive data for statistical analysis by the middle of 2010.

T.P.3.05 Reproducibility and correlation of pretreatment outcome measures in phase 2b study of ataluren (PTC124^™) in nonsense mutation Duchenne and Becker muscular dystrophy (nmDMD/BMD)

J. Florence¹, R.T. Abresch², M. Eagle³, E. Gappmaier⁴, A.M. Glanzman⁵, E. Henricson², A. Reha⁶, G.L. Elfring⁶, L.L. Miller⁶ and L. Atkinson⁶

¹Washington University School of Medicine, St. Louis, MO, United States

²University of California Davis School of Medicine, Davis, CA, United States

³Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom

⁴University of Utah, Salt Lake City, UT, United States

⁵Children’s Hospital of Philadelphia, Philadelphia, PA, United States

⁶PTC Therapeutics Inc., South Plainfield, NJ, United States

Background: Ataluren is an investigational, orally bioavailable, small molecule with potential clinical utility in treating genetic disorders through induction of ribosomal readthrough of nonsense (premature stop codon) mutations in mRNA and production of full-length, functional proteins. Methods: A Phase 2b, randomized, double-blinded, placebo-controlled, dose-ranging study is assessing the efficacy and safety of 48 weeks of ataluren in 174 patients with nmDMD/BMD enrolled at 37 centers. Screening and baseline evaluations (6 weeks apart) included 6-minute walk distance (6MWD); timed function tests (TFTs) (10-meter walk/run, 4-stair climb, 4-stair descent, and stand from supine); and myometry (shoulder abduction, elbow and knee flexion/extension). Correlations and predictive factors were explored. Results: Pretreatment data from screening and baseline visits are available for 174 patients (median [range] age = 8 [5–20] years, height = 123 [97–174] cm, weight = 27 [15–80] kg, corticosteroid usage = 123/174 [71%], cardiac medication usage = 20/174 [12%]). The median [range] between-test interval was 42 [0–91] days. Mean [SD] 6MWD was 357 [93] m at screening and 356 [96] m at baseline. Test–retest correlations were high: 6MWD (r = .91), TFTs (range r = .78 to .90), and myometry (range r = .78 to .92). 6MWD correlated better with TFTs (range r = − .79 to −.67) than with myometry (range r = .35 to .68). As expected, younger age and use of steroids, as predefined by study stratification factors of age (<9 versus >9 years) and corticosteroid usage (yes versus no), predicted for improved 6MWD and performance on other tests. Conclusions: Pretreatment data show that clinical evaluations are reproducible in this multicenter, international study. 6MWD correlates with other functional measures of disease severity and with factors known to predict for disease severity.

T.P.3.06 Metformin improves weight gain and body mass index in Duchenne muscular dystrophy

M.M. Rutter¹, B.L. Wong², S.R. Rose¹ and D.J. Klein¹

¹Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Cincinnati, United States

²Cincinnati Children’s Hospital Medical Center, Division of Pediatric Neurology, Cincinnati, United States

 Background: Glucocorticoids prolong ambulation and survival in Duchenne Muscular Dystrophy (DMD), but increase risk for developing obesity, insulin resistance and abnormal carbohydrate metabolism. Weight gain is exacerbated by loss of ambulation. Obesity may increase risk for cardiovascular disease, and impair pulmonary function, mobility and quality of life in DMD. Metformin improves weight and insulin resistance in patients with obesity and type 2 diabetes. We hypothesized that metformin may prevent metabolic complications of chronic glucocorticoid therapy in DMD. Objective: To determine whether metformin improves weight and body mass index (BMI) in glucocorticoid-treated DMD boys with excessive weight gain and insulin resistance. Methods: Case series of DMD boys with excessive weight gain and insulin resistance treated with metformin. Primary outcomes were rate of weight change and BMI. Results: Eighteen boys were treated with metformin. To date, 10 have been followed for 5–26 (mean 12) months. Mean age was 13.8 yr (median 12, range 8.2–24). All were on chronic steroids (deflazacort, prednisone); nine had lost ambulation. Metformin doses ranged from 1000–2000 mg daily. All patients had baseline normal fasting glucose; three had impaired glucose tolerance. Oral glucose tolerance tests showed mean fasting insulin 27 μU/ml and peak insulin 254 μU/ml (range 91–521). Mean rate of weight gain on metformin was +0.5 kg/yr (median −1.55, range − 7.9 to +18.8), significantly improved compared to pre-treatment gain (+8.2 kg/yr, range +2.0 to +12.9, p = 0.009). Six boys lost weight, three had stabilization, and one gained weight. Nine boys had improved BMI (mean change −2.5 kg/m², range −6.5 to +0.6, p = 0.004). Five boys experienced gastrointestinal symptoms, but insufficient to discontinue treatment. Conclusions: Metformin results in weight loss, slowed weight gain and improved BMI in DMD boys with excessive weight gain and insulin resistance related to chronic glucocorticoid treatment.

T.P.3.07 Design of an international phase III study with idebenone (Catena^®) in patients with Duchenne muscular dystrophy (DMD) – The DELOS study

G.M. Buyse¹, T. Meier² and R.S. Finkel³

¹University Hospitals Leuven, Leuven, Belgium

²Santhera Pharmaceuticals, Liestal, Switzerland

³Children’s Hospital of Philadelphia, Philadelphia, United States

 Respiratory complications cause early morbidity and mortality in patients with DMD. The use of glucocorticoids slows the decline in respiratory function, but their long-term use is hampered by significant side effects. Idebenone, a small molecule ATP production modulator and antioxidant, has shown efficacy on cardiac and respiratory parameters in a Phase IIa randomized controlled study in DMD (DELPHI study). Amongst other findings, patients receiving idebenone at 450 mg/d for 12 months improved in peak expiratory flow (PEF), whilst patients receiving placebo declined on this early marker of respiratory involvement in DMD.

Based on these results a Phase III clinical study with idebenone in DMD was planned. This study, termed DELOS, has a randomized, double-blind, placebo-controlled design with 25 participating centers in Europe, USA and Canada. In total 240 patients, both ambulatory and non-ambulatory, aged 10–18 years will be enrolled and equally distributed to receive either 900 mg/d idebenone (CATENA®, 150 mg film-coated tablets) or matching placebo for 12 months.

The DELOS study is designed to demonstrate improvement in a clinically meaningful early parameter of respiratory function in DMD patients in their second decade. The primary endpoint of the study is change from baseline to Week 52 in PEF % predicted, supported by secondary respiratory endpoints (FVC, maximal mouth pressures, peak cough flow), measures of muscle strength, quality of life and assessment of safety and tolerability. The DELPHI study showed that the effect size of idebenone on the primary endpoint is smaller in patients on concomitant glucocorticoids compared to glucocorticoid-naïve patients. The DELOS study is powered based on the effect size for PEF % predicted seen in the preceding DELPHI study and allows detecting a significant difference between idebenone and placebo treatment in the ITT population as well as in both subgroups of the study (i.e. glucocorticoid users and non-users).

T.P.3.08 Clinical trial of epigallocatechin-3-gallate in Duchenne muscular dystrophy

A. von Moers¹, F. Paul¹, M. Schülke¹, S. Ohlraum¹, Y. Nakae², O.M. Dorchies² and U.T. Ruegg²

¹NeuroCure Clinical Research Center NCRC, Charité, Berlin, Germany

²University of Geneva, Pharmacology, Geneva, Switzerland

We reported that epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, given to mdx mice via their diet or by subcutaneous injection limits muscle degeneration. Encouraged by these results and those with EGCG in autoimmune encephalomyelitis, a model of multiple sclerosis, we decided to initiate a trial using EGCG administration to Duchenne muscular dystrophy (DMD) patients.

This prospective, multicentric pilot study is aimed to assess safety and tolerability of a 12 months treatment with oral EGCG in increasing doses of up to 200 mg daily (max. 10 mg/kg body weight) in ambulatory DMD children aged 5 years or more. Furthermore, the effect of EGCG on disease-related functional scores will be assessed in this randomized, double-blind, placebo-controlled clinical trial. The study will be followed by biomarkers including serum creatine kinase (CK) and locomotor activity will be determined.

We expect that similar improvements as those observed in dystrophic mice will be found in DMD patients.

T.P.4.01 Effects of idebenone on mitochondrial function in cells from muscular dystrophy patients and healthy individuals

R. Haefeli¹, A. Gemperli² and N. Güven²

¹University of Basel, Biozentrum, Basel, Switzerland

²Santhera Pharmaceuticals, Liestal, Switzerland

Impairment of mitochondrial function is observed in multiple neurodegenerative and neuromuscular disorders as well as in aging. The small molecule idebenone has been reported by numerous studies to protect mitochondrial function. Although its mode of action is still under investigation, multiple possibilities have been proposed. First, idebenone decreases oxidative damage and mitochondrial swelling both in vitro and in vivo and potentiates the antioxidant effect of Vitamin E both directly and supportively by recycling of oxidized Vitamin E. Second, as short chain CoQ10 analogue it functions also as electron carrier in the mitochondrial respiratory chain where it is among the most efficient substrates for complex II and III and efficiently drives the generation of a membrane potential [3]. Finally, idebenone is also a very effective electron acceptor for glycerophosphate dehydrogenase (G3PDH) and strongly increases G3PDH activity in brown adipose tissue mitochondria. Since elevated enzyme activity is indicative of increased mG3PDH protein levels it could suggest that idebenone might also influence protein expression. To gain a deeper understanding of its molecular mechanism, we investigated the effect of idebenone and similar compounds such as CoQ10 and Vitamin E on levels of oxidative stress, ATP levels, mitochondrial membrane potential (ΔΨm), electron flux and mitochondrial content in normal cells as well as cells from patients with muscular dystrophy. We observed distinct characteristics of idebenone that differentiate idebenone against other ubiquinone analogues which could account for its observed benefitial effects and serve as a rationale for the use of idebenone in the treatment of dystrophic disorders such as DMD.

T.P.4.02 Effects of rituximab treatment in two patients affected by dysferlin-deficient muscular dystrophy

A. Lerario¹, F. Cogiamanian¹, C. Marchesi¹, S. Bonfiglio¹, M. Belicchi¹, L. Porretti², N. Bresolin¹ and Y. Torrente¹

¹Department of Neurological Sciences, Fondazione IRCCS Ospedale Maggiore Policlinico, Milan, Italy

²Centro Interdipartimentale di Citometria, Department of Regenerative Medicine, Milan, Italy

 To investigate whether the B-cell depletion influence the disease course, two patients with Miyoshi myopathy (MM) were treated with four weekly infusions of Rituximab (RTX). Two patients were treated with four weekly dose of 375 mg/m²/infusion of RTX. To check the improvement of muscle strength after treatment, the isometric hand grip Maximal voluntary contraction (MVC) have been measured by load cell four times during treatment and once a year later. In order to assess the reproducibility of our grip assessment we determined the hand MVC analysis in a group of 16 healthy subjects. Moreover, we have measured the number of B cell by flow cytometric analysis during the course of treatment. The analysis of B cells number during the course of treatment shows how the number of cells CD20 and CD19 positive have been deleted to 0–0.01%. The decrease of B cells was followed by an improvement of the mobility of the pelvic and shoulder girdles as MRC% shows. The values of Maximal voluntary contraction (MVC) of both patients started from values lower than normal while during treatment, patients have an improvement percentage of muscle strength. The peak of strength of both patients matched with the minimum values of B cells. The development of muscle strength presents fluctuations over time, although we can still observe that muscle strength is greater than the first survey a year later. There were no severe adverse events associated with infusion of RTX. We can consider the increase in muscle strength observed in both treated patients as a consequence of the treatment with RTX. To our knowledge, this is the first case of increase in muscle strength in patients with MM. Furthermore, the results of this study indicate that B cell depletion with RTX appears to be useful in the treatment of patients affected by MM suggesting a possible role of B cells in the pathophysiology of this muscle disorder.

T.P.4.03 Treatment with Bortezomib (PS-341) of Golden Retriever Muscular Dystrophy (GRMD): Analysis of proteasome inhibition and morphology of dystrophic skeletal muscle

K.P.C. Araújo¹, D.F. Moreira², T.P. Gaiad¹, M.A. Miglino¹, S.L. Gorniak³, D. Feder⁴, J.E. Belizário² and C.E. Ambrósio¹

¹University of Sao Paulo, Department of Surgery, Sao Paulo, Brazil

²University of Sao Paulo, Department of Pharmacology, Sao Paulo, Brazil

³University of Sao Paulo, Department of Pathology, Sao Paulo, Brazil

⁴ABC Foundation, Physiology and Morphology, Sao Paulo, Brazil

.

Golden Retriever Muscular Dystrophy (GRMD) is a degenerative myopathy homologue to Duchenne Muscular Dystrophy (DMD) in humans, caused by the absence of dystrophin protein. It results in muscle fibers necrosis followed by connective tissue deposition lead to atrophy and fibrosis. Recent studies support the fact that degradation of dystrophin through the proteasomal dependent pathway is involved in the pathogenesis of DMD. The proteasome inhibitors such as Bortezomib might rescue the structure of the dystrophin and improve muscle condition. The aim of this study was evaluated the treatment of GRMD dogs with Bortezomib. During 9 weeks, two GRMD dogs (TD – treated dogs) received intravenous injections of Bortezomib (1.3–1.65 mg/m²) twice a week and three other dogs (CD – control dogs) have not received any drug. Blood samples were obtained at 1, 4 and 24 h after each cycle of Bortezomib administration and the enzymatic activity of proteasome was assessed. Statistical analysis considered p < .05. The femoral biceps muscle was collected by biopsy before (T0) and after treatment (T1) for microscopic analysis. Maximal inhibition of the proteasome in blood was observed 1 h after Bortezomib administration in last cycle (p < .05), keeping up with satisfactory values for 4 h. After 24 h the levels of inhibition were similar to the CD. At T1 the skeletal muscle from TD revealed more uniform fiber diameter and less lymphocytic invasion than CD, suggesting that the inflammatory process was reduced and that signs of muscular dystrophy were greatly reduced. In conclusion, the inhibition of proteasome was dosage-dependent, the maximal dosage at the last cycle showed maximal proteasome inhibition. To confirm the morphological results, an immunohistochemical analysis of dystrophin is necessary. Although, these preliminary results suggest that Bortezomib can improve the histopathological appearance of dystrophin-deficient skeletal muscle.

T.P.4.04 Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts

H. Vandenburgh¹, J. Shansky¹, F. Benesch-Lee¹, K. Skelly¹, J. Spinazzola¹, Y. Saponjian² and B.S. Tseng²

¹Myomics Inc., Providence RI, United States

²Massachusetts General Hospital, Providence RI, United States

Identification of epigenetic factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx mouse myoblasts, the genetic homologue of DMD, were tissue engineered in 96 micro-well plates into three-dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Twenty-five compounds of interest as potential treatments for patients with DMD were tested at 3–6 concentrations. Nine of the compounds (insulin-like growth factor-1, creatine, trichostatin A, and six from the glucocorticoid family) significantly increased tetanic force relative to placebo-treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC₅₀ of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort, prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since in vivo mdx muscle and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new treatments for muscle weakness in DMD and other muscle disorders.

T.P.4.05 Jaw exercise in patients with Duchenne muscular dystrophy (multicenter study) – Therapeutic intervention to the masseter muscle

S. Nozaki¹, M. Kawai², R. Shimoyama³, N. Futamura⁴, T. Matsumura⁵ and Y. Kikuchi⁶

¹Hyogo University of Health Science, Kobe, Japan

²Higashisaitama National Hospital, Hasuda, Japan

³Matsue National Hospital, Matsue, Japan

⁴Hyogochuo National Hospital, Sanda, Japan

⁵Toneyama National Hospital, Toyonaka, Japan

⁶Nagasaki University, Nagasaki, Japan

 Objective: We evaluated the efficacy of the intervention for decreased bite force in the oral phase dysphagia in DMD. Method: This study was designed as open intervention study by jaw exercise with hot pack in DMD patients. Subjects were 20 patients with DMD in five hospitals who were unable to sit up by themselves, aged 21.0 ± 4.1 years. Outcome measurements were bite force and jaw opening. In bite force measurement, Occlusal Force Meter GM10 (Nagano Keiki) was used. Five consecutive measurements were made on the same target teeth. The maximum distance between the upper and lower incisors was measured with a slide caliper as jaw opening. The jaw exercise training assisted by a therapist and self exercise was done by the patient. The exercise program included 15 min hot pack and additional massage on masseter muscle given by a therapist for 15 min, once a day, twice weekly. The self exercise consisted of 10 repetitions of mouth opening and closing in maximum range was done three times daily before meal. Baseline bite force and jaw opening were compared with those at 2, 4 and 6 months of exercise training. Statistic analysis: The change of the bite force/jaw opening measurement from the baseline to that of 2 or 4 or 6 months were analyzed by Friedman analysis. Results: The mean bite force at baseline and 2, 4, 6 months of exercise training were 80.3 ± 45.1 N, 82.6 ± 39.5 N, 93.0 ± 40.3 N, 102.6 ± 22.3 N. The change of the bite force from the baseline to 6 months was significant (p = 0.005). The change of jaw opening from baseline to that at 2 or 4 or 6 months were not significant. Conclusion: Jaw exercise with hot pack is significantly effective for improvement of bite force in DMD patients after 6 months of exercise training.

T.P.4.06 Does physiotherapy change the fibrosis amount on canine dystrophic muscle and dynamical features of gait?

T.P. Gaiad¹, T.M. Guimarães², K.P.C. Araujo¹, F.A. Caromano³, J.C. Serrão², M.A. Miglino¹ and C.E. Ambrosio⁴

¹University of São Paulo, Department of Surgery – FMVZ, São Paulo, Brazil

²University of São Paulo, Department of Biodynamics of Movement – EEFE, São Paulo, Brazil

³University of São Paulo, Department of Physiotherapy – School of Medicine, São Paulo, Brazil

⁴University of São Paulo – FZEA, Department of Basic Science, Pirassununga, Brazil

 Physiotherapy has been widely used as support treatment for muscular dystrophies. Its effect on dystrophic muscle and global function should be better understood to guide treatments. This study aims to understand the role of motor physical therapy on collagen deposition and some dynamical parameters of gait of this canine model for Duchenne Muscular Dystrophy. Five GRMD dogs had fragments of biceps femoris muscle collected by biopsy. Data of Ground Reaction Forces (GRF) in craniocaudal (Fx) and vertical (Fy) direction were collect using a Force Plate Kistler AG (9287 A/100 Hz) and normalized for body weight. Two animals (therapy dogs: TD) underwent a protocol which consisted of velocity controlled walking activity in an area of 288 meters total length, 3 times/week per 12 weeks. Control dogs (CD) maintained their daily routine. Zero time (T0) is considered at collect I (n = 5) and time after therapy (T1) – collect II. Statistical analysis considered p < .05. Immunohistochemistry anti-collagen types I and III (Calbiochem®) were performed. At T1, thicker tracts of collagen type I was observed at the endomysium of TD compared to T0. Fy of thoracic and pelvic limbs at T1 of TD (10.50 ± 1.82/6.05 ± 0.45) and CD (10.19 ± 0.92/ 6.68 ± 4.40) was higher than T0 (6.91 ± 2.14/ 4.01 ± 1.47). Only CD presented increase of support time of thoracic limbs at T1 (49–53%). At T0, dogs presented Fx+ ratio pelvic/thoracic (P/T) of 0.88 and at T1 the TD 1.6 (p = .002) e CD 0.9 (p = .481). Fx- at T1 showed decrease values for TD (p = .013) compared to T0. At this intensity of therapy, the muscular amount of collagen type I increase. However, the vertical force distribution between pelvic and thoracic limbs is maintained and the propulsive force of the thoracic ones decreases.

T.P.4.07 The opening of pulmonary respiration resulted in muscle degeneration of diaphragm in canine X-linked muscular dystrophy

A.N. Nakamura¹, M.K. Kobayashi¹, K.Y. Yuasa², N.Y. Yugeta³ and S.T. Takeda¹

¹National Institute of Neuroscience, National Center of Neurology and Psychiatry, Department of Molecular Therapy, Kodaira, Japan

²Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Nishi-tokyo, Japan

³School of Veterinary Medicine, Azabu University, Department of Surgery I, Sagamihara, Japan

 Background: Canine X-linked muscular dystrophy dogs in Japan (CXMDJ), an excellent animal model for Duchenne muscular dystrophy (DMD), showed a prominent increase in serum creatine kinase (CK) levels at birth and their mortality rate was 35% during the neonatal period. Objective and hypotheses: Delivery stress such as compression through birth canal may affect the serum CK and mortality rate. We, therefore, tried Caesarean section to reduce the rate of neonatal death of affected pups. Methods: We compared serum CK between natural (71 normal dogs, 37 carriers, 41 CXMDJ) and Caesarean deliveries (39 normal dogs, 26 carriers, 34 CXMDJ). We then compared CK values between cord and venous blood after (23 normal dogs, 18 carriers, and 22 CXMDJ), or before a beginning of pulmonary respiration (5 normal dogs, 3 carriers and 6 CXMDJ). We also examined diaphragm of the CXMDJ before breathing. Result: CK values were decreased in normal and carrier dogs but not in CXMDJ by Caesarean deliveries, whereas the mortality rate was not improved by the procedure in three groups. After a beginning of pulmonary respiration, CK values of venous blood was not different from that of cord blood in normal and carrier dogs, while values of venous blood in CXMDJ was increased at 40-fold of their cord blood. Before breathing, the difference in CK values between cord and venous blood was not significant in three groups. The histopathology of CXMDJ diaphragm showed slight degeneration before pulmonary respiration, but extensive degeneration was noticed after the opening of pulmonary respiration. Utrophin expression in diaphragm of CXMDJ before breathing was much lower than that of 3-month-old CXMDJ. Conclusions: An opening of pulmonary respiration resulted in severe degeneration of diaphragm and respiratory distress in neonatal CXMDJ through mechanical load and utrophin expression cannot compensate the lack of dystrophin at the period.

T.P.4.09 Measuring restored dystrophin in treated muscle: An immunohistological intensity measurement method

V. Arechavala-Gomeza¹, L. Feng¹, A. Malerba², I.R. Graham², S.C. Brown³, C. Sewry⁴, J. Morgan¹ and F. Muntoni¹

¹UCL-Institute of Child Health, Dubowitz Neuromuscular Centre, London, United Kingdom

²Royal Holloway University of London, London, United Kingdom

³Imperial College London, London, United Kingdom

⁴Robert Jones and Agnes Hunt NHS Trust, Owestry, United Kingdom

The quantification of sarcolemma-associated protein levels in muscle biopsies is of particular importance to the disease process of several muscular dystrophies, but is complicated by the relative low levels of protein present. We have developed a comparative method to evaluate differences in expression of these proteins by using immunohistological techniques and analysis software readily available. Captured images are analysed by the operator using an imaging system to measure intensity corresponding to the expression of these proteins. We used this method in transverse cryosections of skeletal muscles taken from patients suffering from Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD) and normal controls immunostained with antibodies to dystrophin, β-dystroglican, α-sarcoglycan, utrophin and spectrin. While DMD patients’ biopsies are characterised by the lack of dystrophin protein, BMD patients show intermediate amount compared to normal controls. We were able to accurately distinguish between the different sets of patients with this technique. We are confident that this method will add value to the techniques routinely used in diagnostic laboratories, in particular in assessing the efficacy of potential treatments designed to increase amounts of dystrophin protein. To test this hypothesis, and to confirm the sensitivity of the technique to detect an increase in amount of dystrophin protein expected in such trials, we studied with this method muscle biopsies from mdx mice treated with different doses of morpholino antisense oligonucleotides and normal controls.

T.P.4.10 Towards a better understanding of truncated dystrophin instability

V. Robin¹, C. Beley¹, M. Reboud², T. Voit¹ and L. Garcia¹

¹UMRS974 INSERM, UMRS7215 CNRS, Institut de Myologie, UPMC, Paris, France

²Institut Jacques Monod, FRE2865, Paris, France

 Duchenne Muscular Dystrophy and Becker Muscular Dystrophy are both genetic neuromuscular disorders due to mutations in the dystrophin gene. DMD patients are characterized by a lack of dystrophin in muscles and present a severe phenotype, while BMD display a much milder phenotype and express either lower amounts of dystrophin or still semi-functional truncated dystrophins. In some cases, such as in patients with 45–47 and 45–48 deletions (about 50% of the BMD population), resulting truncated dystrophins are unstable.

Currently, therapeutic strategies for DMD are extensively explored, whereas the comprehension of the mechanisms responsible of BMD remains largely poorly investigated. Our aim is to understand the instability of the truncated dystrophins when exons 45–47 and 45–48 are deleted, in order to identify therapeutic avenues for these patients.

We have collected skin fibroblasts from BMD patients. These cells were grown ex vivo and converted into myogenic progenitors by using a lentivector coding for an inducible MyoD with doxycycline. This allowed us to obtain a biological material to test the effect