T.P.5.06 Pharmacological characterization of phospholipase A2 activities in primary cultures of mouse dystrophic muscle

Abstracts / Neuromuscular Disorders 19 (2009) 543–660

been shown by various studies to protect mitochondria from oxidative stress and to improve mitochondrial function. We therefore tested if idebenone could also alleviate some of the mitochondrial impairment in DMD. Primary myocytes from DMD patients showed markedly increased levels of ROS compared to control cells, which was associated with a reduction in mitochondrial membrane potential. Consistent with this, DMD myocytes only showed about 60% of cellular ATP levels compared to control cells. Treatment with idebenone was able to reduce ROS and more importantly also restored ATP levels in DMD cells in a dose dependent manner with a maximum increase of 100 % at a dose range between 0.1 and 5 lM. This effect was associated with improved mitochondrial membrane potential. Using the mdxmouse model of DMD, treatment with idebenone for 3 days also showed improved mitochondrial electron flow and a correction of ATP levels in the m. soleus comparable to levels in wild type muscle tissue. Although mice at this age (8 weeks) do not show cardiac pathology, we also observed marked increase of ATP in heart and diaphragm extracts. These results are in line with our previous results demonstrating that idebenone corrected cardiac diastolic dysfunction in older mice and improved voluntary running performance in the mdx-mouse. Our results show that idebenone can protect against the mitochondrial dysfunction associated with dystrophin deficiency in vitro and in vivo and serves as a rationale for the use of idebenone in the treatment of DMD. doi:10.1016/j.nmd.2009.06.228

T.P.5.04 Effect of a mild exercise regime on disease parameters in the mdx mouse model de Winter, W.M.C. van Roon-Mom, M. van Putten, C.L. G.J.B. van Ommen, P.A.C. ’tHoen, A.M. Aartsma-Rus Leiden University Medical Center, Human Genetics, Leiden, The Netherlands Duchenne Muscular Dystrophy (DMD) is a severe, progressive, muscle wasting neuromuscular disorder, characterized by the synthesis of truncated non-functional dystrophin proteins. One of the most promising therapeutic approaches for DMD involves the restoration of the genetic defect by antisense oligonucleotide (AON) mediated exon skipping that results in the synthesis of shortened but partly functional dystrophin proteins. To assess the therapeutic effects of AON treatment in more detail and to determine which levels of dystrophin restoration are required for improved muscle function in mdx mice, we have set up several functional tests for monitoring muscle strength and condition. These tests consist of grip strength, rota-rod and 2 and 4 limb hanging wire tests. Where possible, standardized operating procedures (from the TREAT-NMD website) were implemented. Since there is some debate on the effect of exercise on disease progression, we assessed whether there is a difference in histology and in previously identified fibrotic and immunologic RNA biomarker levels between exercised and nonexercised mice. Male mdx mice (n = 5) underwent a 12-week functional test regime starting at the age of 4 weeks. Mice performed different functional tests on consecutive days on a weekly basis. Creatine kinase levels were determined once a week. After sacrifice the percentage of fibrotic/necrotic areas throughout different skeletal muscles and heart were determined using a computer-automated image analysis system. We found no indications for differences in fibrosis between the exercised and non-exercised mice for heart and skeletal muscle,

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nor did the amount of fibrosis vary between different locations within muscles. Gene expression levels of disease-related genes did not differ between the groups, while exercise-related genes did. Based on these results the functional test regime was found not to affect the natural disease progression in mdx mice. doi:10.1016/j.nmd.2009.06.229

T.P.5.05 Muscle-specific expression of myotubularin ameliorates the phenotype of targeted muscles in mouse models of X-linked myotubular myopathy T. Jamet 1, F. Fougerousse 2, N. Guerchet 2, A. Douar 2, J.L. Mandel 1, M. Montus 2, A. Buj-Bello1 1

IGBMC, Illkirch, France, 2 Genethon, Evry, France

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital disease that affects skeletal musculature, which is characterized by the presence of small myofibres with frequent occurrence of central nuclei. The disease is due to mutations in the MTM1 gene, encoding a phosphoinositide phosphatase named myotubularin, and specific treatment is currently unavailable. We have recently shown that a single intramuscular injection of an adeno-associated virus (AAV) vector expressing myotubularin under the cytomegalovirus (CMV) in symptomatic muscle-specific Mtm1-deficient mice ameliorates the phenotype in targeted muscles. We have now extended these gene transfer assays also to constitutive Mtm1 mutants and used a novel serotype 1 AAV vector that contains the desmin promoter for muscle-specific expression of myotubularin. We show that intramuscular injection of this vector leads to an important improvement of the pathology and function of targeted muscles. Myotubularin replacement in mice largely ameliorates nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. Our results indicate that AAV-mediated myotubularin gene transfer may represent a therapeutic approach for the disease. doi:10.1016/j.nmd.2009.06.230

T.P.5.06 Pharmacological characterization of phospholipase A2 activities in primary cultures of mouse dystrophic muscle O.M. Dorchies, A.K. Johansson, U.T. Ruegg University of Geneva, Laboratory of Pharmacology, Geneva, Switzerland Using primary cultures of mouse dystrophic muscle, we investigated the regulation of phospholipases A2 (PLA2), enzymes suspected to aggravate the dystrophic phenotype through facilitation of calcium entry. Compared to normal individuals, patients with Duchenne muscular dystrophy exhibit a several-fold elevation of PLA2 activity in their muscles. Upon muscle contraction, calcium depletion from the sarcoplasmic reticulum triggers the activation of the membrane-bound calcium-independent PLA2 b (iPLA2 b) through a still elusive mechanism, resulting in the formation of lysophosphatidyl choline that in turn activates plasma membrane channels responsible for store-operated calcium entry (SOCE). We have previously shown that muscles from mdx5Cv mice over-express iPLA2 b, and that SOCE in dystrophic muscle fibres is partly inhibited by the iPLA2 inhibitor bromo-enol-lactone. The contribution to SOCE of other PLA2 such as the cytosolic iPLA2 c and cPLA2 a is not established. Here, using PED6, a fluorogenic substrate for

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Abstracts / Neuromuscular Disorders 19 (2009) 543–660

T.P.5.07 Substantial improvement of laminin a2 chain deficiency by laminin a1 chain overexpression persists throughout life K.I. Gawlik, M. Durbeej

NADPH oxidase (NOX). In the present study, we found that primary cultures of dystrophic myotubes generated more ROS than normal myotubes upon plasma membrane depolarization or phorbol 12myristate 13-acetate stimulation. This increase was associated with a greater NOX activity in dystrophic myotubes. Using RT-PCR, we confirmed the presence of mRNA transcripts for plasma membrane (p22phox) and cytosplasmic (p40phox, p47phox, p67phox) subunits, as well as NOX1, NOX2 and NOX4 isoforms in cultured myotubes. Preliminary results showed an increased mRNA level of these subunits (p22phox, in particular) and NOX2 protein levels in dystrophic myotubes. Moreover, we found that ROS generated by the NOX contribute to the abnormal Ca2+ transients after plasma membrane depolarization of dystrophic myotubes as two NOX inhibitors (diphenylene iodonium and apocynin) restored these transients to those of normal myotubes. Finally, as Ca2+ entry has been described to be increased in dystrophic muscle cells and could explain the abnormal Ca2+ transients observed in dystrophic myotubes, we tested the effects of NOX inhibitors on Ca2+ influx in normal and dystrophic myotubes. Interestingly, NOX inhibitors inhibited Ca2+ influx in both basal conditions and upon plasma membrane depolarization in dystrophic myotubes, restoring it to the one of normal myotubes.

Lund University, Department of Experimental Medical Science, Lund, Sweden

doi:10.1016/j.nmd.2009.06.233

PLA2, we have determined that, (i) the contribution of iPLA2 b and iPLA2 c to total PLA2 activity were similar in dystrophic as in normal cultures, (ii) removal of external calcium resulted in activation of PLA2 activity; thapsigargin used for further store depletion reinforced this effect, (iii) PLA2 activity was increased by c-Jun N-terminal kinase (JNK), glutathione depletion, prednisolone, and to a lesser extent protein kinase C activation and TNFa, (iv) PLA2 activity was decreased by inhibitors of the mitochondrial electron transport chain, the mitochondrial uncoupler FCCP, and to a lesser extent by the glutathione precursor N-acetylcystein, and (v) overall, oxidants, antioxidants, and inhibitors of the membrane-associated superoxide generating enzymes NADPH oxidase had a minor impact on PLA2 activity. doi:10.1016/j.nmd.2009.06.231

Despite advances in genetic studies, laminin a2 chain deficient congenital muscular dystrophy (MDC1A) remains an incurable disease. Several approaches to treat MDC1A in mouse models have been designed. Most of them show promising results in young animals, but older animals have only been characterised to some extent. However, in order to design an efficient therapy for patients, longterm effects of treatment should be estimated in animal models. Previously, we demonstrated that laminin a1 chain functionally replaces laminin a2 chain in multiple tissues in mice. Here, we analyse twelve 1.5–2-year-old animals. Transgenic expression of laminin a1 chain in laminin a2 chain deficient mice normalized life span, enabled normal activity, improved skeletal and heart muscle morphology, normalized creatine kinase levels and led to near normal muscle strength. Such an improvement of dystrophic phenotype that persists to old age has not been demonstrated in mice before and it holds a promise for efficient treatment for MDC1A patients in the future. In conclusion, our data strengthen the argument that laminin a1 chain is an excellent candidate to be tested in clinical studies. doi:10.1016/j.nmd.2009.06.232

T.P.5.08 Contribution of reactive oxygen species generated through NADPH oxidase to abnormal Ca2+ signals in dystrophic skeletal muscle cells Y. Hibaoui1, K. Bedard 2, K.H. Krause 2, U.T. Ruegg 1 1

Laboratory of Pharmacology, Geneva Lausanne School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland, 2 Departments of Pathology and Immunology, Centre Medical Universitaire, University of Geneva, Geneva, Switzerland Although the molecular mechanisms leading to muscle degeneration in Duchenne muscular dystrophy are not fully understood, numerous studies point to a crucial role of deregulation of Ca2+ homeostasis and increased generation of reactive oxygen species (ROS) in the pathogenesis of the disease. In this regard, there are a number of candidate sources of ROS, such as mitochondria and

T.P.5.09 Reduced mitochondrial copy number in mdx diaphragm – An adaptive response to reduce oxidative stress? M. Erb, L. Sumanovski, I. Courdier-Fruh, N. Güven Santhera Pharmaceuticals, Liestal, Switzerland Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex, leading to pathological calcium (Ca2+) homeostasis in dystrophic muscles and altered mitochondrial function [1]. To gain a deeper understanding on the mechanisms influencing the muscle mitochondria, we first measured ATP levels and the level of oxidative stress in the diaphragm of mdx mice. At 7 weeks of age we found normal ATP levels, but reduced levels of the oxidative stress marker nitrotyrosine. To evaluate if the reduced oxidative stress goes along with a reduced number of mitochondria, we analyzed the mitochondrial DNA (mtDNA) copy number by qPCR. We found a 50% reduction of the mtDNA copy number in the diaphragm of 7-weeks-old mdx mice. To assess if the reduced mtDNA qPCR signal could reflect the presence of major mtDNA deletions, we amplified the full length mitochondrial genome by long range PCR but were unable to detect any deletions. The reduced mtDNA copy number was paralleled by reduced transcript levels of mitochondrially encoded genes as described earlier [2]. The analysis of the pgc-1alpha and pgc-1beta transcriptional levels showed that the reduced mtDNA copy number is not caused by a reduced transcription of these 2 factors, which are known to play a key role in the control of mitochondrial biogenesis. Interestingly, while pgc-1alpha levels were unchanged compared to control animals, pgc-1beta transcript levels were 200% of control levels. The finding that a reduced mitochondrial number and reduced levels of oxidative stress are found in the diaphragm of 7-weeks-old mdx mice, could reflect an adaptive response to reduce the oxidative stress resulting from dysfunctional mitochondria [3]. doi:10.1016/j.nmd.2009.06.234