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DECEMBER
29 - (The Journal of Gene Therapy, 11(1): 46-56) By-passing the nonsense mutation in the 4CV mouse model of muscular dystrophy by induced exon skipping
Chalermchai Mitrpant, Sue Fletcher, Patrick L. Iversen, Steve D. Wilton - Australia
Background: Duchenne muscular dystrophy (DMD), a
severe neuromuscular disorder, is caused by protein-truncating mutations in the
dystrophin gene. Absence of functional dystrophin renders muscle fibres more
vulnerable to damage and necrosis. We report antisense oligomer (AO) induced
exon skipping in the B6Ros.Cg-Dmdmdx-4Cv/J (4CV)
mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin
exon 53. Both exons 52 and 53 must be excised to remove the mutation and
maintain the reading frame.
Methods: A series of 2-O-methyl
modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and
evaluated for the removal of each exon, and the most effective compounds were
then combined to induce dual exon skipping in both myoblast cultures and in
vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino
oligomers (PMOs) was evaluated both in vitro and in vivo at the
RNA and protein levels.
Results: Compared to the original mdx mouse studies, induction of
exon skipping from the 4CV dystrophin mRNA was far more
challenging. PMO cocktails could restore synthesis of near-full length
dystrophin protein in cultured 4CV myogenic cells and in
vivo, after a single intramuscular injection.
Conclusions: By-passing the protein-truncating mutation in the 4CV
mouse model of muscular dystrophy could not be achieved with single oligomers
targeting both exons and was only achieved after the application of AO cocktails
to remove exons 52 and 53. As in previous studies, the stability and efficiency
of PMOs proved superior to 2OMeAOs for consistent and sustained protein
induction in vivo.
25 - (Neuromuscular Disorders, 2008)Treatment with inhibitors of the NF-κB pathway improves whole body tension development in the mdx mouse
Ashley L. Siegel, Cathy Bledsoe, Jesse Lavin, Francesca Gatti, Jonas Berge, Gregory Millman, Eric Turin, W. Tyler Winders, John Rutter, Beniamino Palmeiri and C. George Carlson - USA
The whole body tension (WBT) method was used to evaluate the hypothesis that long term treatment with NF-κB inhibitors improves the total forward pulling tension exerted by the limb musculature of the mdx mouse. Mdx mice exhibited significantly reduced WBT values and more profound weakening during the course of generating multiple forward pulling movements than age-matched nondystrophic mice. Long term treatment with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) did not significantly reduce nuclear p65 activation in the costal diaphragm, but increased WBT by 12% in mature (12 month) mice. Daily treatment (30 days) of 1 month old mdx mice with the inhibitor ursodeoxycholic acid (UDCA) reduced costal diaphragm nuclear p65 activation by 40% and increased WBT by 21%. These results indicate that treatment with NF-κB inhibitors improves WBT in the mdx mouse and further establishes the utility of the WBT procedure in assessing therapeutic efficacy
20 - (Muscle Nerve 39: 3-9, 2009) Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029
Lisa S. Krivickas, Ronan Walsh, MD , Anthony A. Amato - USA
Myostatin inhibitors are being investigated as treatments for myopathies. We assessed single muscle fiber contractile properties before and after 6 months of study drug in 6 patients with facioscapulohumeral, Becker, and limb-girdle muscular dystrophy. Five of the patients received MYO-029, a myostatin inhibitor, and 1 received placebo. The chemically skinned single muscle fiber preparation was used to measure single fiber force, specific force, maximum unloaded shortening velocity, power, and specific power in type I and IIa fibers from each subject. In 4 of 5 patients who received MYO-029, improvement was seen in single muscle fiber contractile properties; thus, there may be a beneficial effect of myostatin inhibition on muscle physiology at the cellular level. No improvement was seen in the patient who received placebo. This finding may be clinically relevant in spite of the fact that quantitative muscle strength measurements in our patients did not improve. Further studies of myostatin inhibition as a treatment for muscular dystrophy are warranted, and single muscle fiber contractile studies are a useful assay for muscle function at the cellular level.
20 - (Biotechnol. Bioeng. 2009;102: 624-63) Fabrication of skeletal muscle constructs by topographic activation of cell alignment
Yi Zhao , Hansong Zeng , Jin Nam , Sudha Agarwal - USA
Skeletal muscle fiber construction for tissue-engineered grafts requires assembly of unidirectionally aligned juxtaposed myotubes. To construct such a tissue, a polymer microchip with linearly aligned microgrooves was fabricated that could direct myoblast adaptation under stringent conditions. The closely spaced microgrooves fabricated by a modified replica molding process guided linear cellular alignment. Examination of the myoblasts by immunofluorescence microscopy demonstrated that the microgrooves with subcellular widths and appropriate height-to-width ratios were required for practically complete linear alignment of myoblasts. The topology-dependent cell alignment encouraged differentiation of myoblasts into multinucleate, myosin heavy chain positive myotubes. The monolayer of myotubes formed on the microstructured chips allowed attachment, growth and differentiation of subsequent layers of linearly arranged myoblasts, parallel to the primary monolayer of myotubes. The consequent de position of additional myoblasts on the previous layer of myotubes resulted in three-dimensional multi-layered structures of myotubes, typical of differentiated skeletal muscle tissue. The findings demonstrate that the on-chip device holds promise for providing an efficient means for guided muscle tissue construction.
1 - (PNAS,
Dec 2008)
HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a
common target in Duchenne muscular dystophy treatment
Claudia Colussi, Chiara Mozzetta, Aymone Gurtner, Barbara Illi, Jessica Rosati,
Stefania Straino, Gianluca Ragone, Mario Pescatori, Germana Zaccagnini, Annalisa
Antonini, Giulia Minetti, Fabio Martelli, Giulia Piaggio, Paola Gallinari,
Christian Steinkulher, Emilio Clementi, Carmela Dell'Aversana, Lucia Altucci,
Antonello Mai, Maurizio C. Capogrossi, Pier Lorenzo Puri, and Carlo Gaetano -
Italy
The overlapping histological and biochemical features
underlying the beneficial effect of deacetylase inhibitors and NO donors in
dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO
signaling, and the histone deacetylases (HDACs). Higher global deacetylase
activity and selective increased expression of the class I histone deacetylase
HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in
vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was
sufficient to replicate the morphological and functional benefits observed with
deacetylase inhibitors and NO donors. We found that restoration of NO signaling
in vivo, by adenoviral-mediated expression of a constitutively active
endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived
satellite cells to NO donors, resulted in HDAC2 blockade by cysteine
S-nitrosylation. These data reveal a special contribution of HDAC2 in the
pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition
by NO-dependent S-nitrosylation is important for the therapeutic response to NO
donors in MDX mice. They also define a common target for independent
pharmacological interventions in the treatment of Duchenne muscular dystrophy.
NOVEMBER
17 - (48th Annual Meeting of The American Society for Cell Biology, December 2008 - San Francisco)
1) Myostatin reduction in Erythropoietin application in mdx dystrophic mice. A Pilot trial
DAVID FEDER; A. S. Carvalho; S. B. Zyngier; J. P. Ferraz; L. S. Souza; A. C. Santomauro; L. P. Oliveira; M. R. Ugollini; V. P. Lioi; L. G. Ferreira; M. T. Nunes; M. C. Carvalho; R. A. dosSantos - Brazil
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.
Material and methods: We analyzed the effects of Epo injection on skeletal
muscle of mdx mice to study the effects of long term (12 weeks). Seven mdx mice
male received Epo injection of 1000 IU/kg/IP and six mdx mice male received
placebo (double blinded, placebo). All animals were submitted to exercise 5 days/week,
20 cm/s, 10 min. 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 with acid phosfatase and alkaline phosfatase for histological evaluation.
Left gastrocnemius was dissected, frozen in liquid nitrogen, and stored at -80
C. Total tissue RNA were extracted with Trizol Reagent. Total RNA (2 ug) was
used for first-strand cDNA synthesis. 200 ng of cDNA was used for Real-time PCR
and mRNA of TNF alpha, TGF beta1 and myostatin were measured.
Results. The results of this study 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 group taking Epo. The histological
analysis did not show differences in necrosis and regeneration between placebo
group and group receiving Epo. There were no difference in genic expression of
TNF alpha and TGF beta 1 but a significant reduction of myostatin in treated
mice.
Conclusion: 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.
2) Vivo-Morpholino Oligomers Induce Potent Exon-skipping
of Dystrophin in Cardiac and Skeletal Muscles of Mice
S. Jiang; P. A. Morcos - USA
Duchene muscular dystrophy (DMD) is a severe muscle disorder caused by frameshift and nonsense mutations in the dystrophin gene. Morpholino antisense oligomers have been shown to induce exon skipping and partially restore the function of dystrophin in the skeletal muscles of animal models of DMD, particularly in mdx mice. However, exon-skipping mediated by morpholino oligomers in the heart is moderate due to reduced delivery efficiency in cardiac muscles. Vivo-Morpholinos, non-peptidic transporter-conjugated morpholino oligomers, exhibit high delivery efficiency in vivo with no detectable toxicity. We examined the efficiency of exon skipping mediated by a Vivo-Morpholino oligomer, V-Mor23, targeting dystrophin exon 23 in normal mice. Intraperitoneal (I.P.) injection of V-Mor23 achieved higher efficiency of exon skipping than intravenous (I.V.) injection in the diaphragm, while I.V. injection achieved more efficient for most other tissues. Two daily I.V. injections of 30 mg/kg V-Mor23 induced potent exon-skipping in the heart (62%), diaphragm (66%) and extensor digitorum longus (70%), which are critical muscles in the treatment of DMD. The effect is dose-dependent and is achieved as rapid as one day after injection. This is the first report of rapid and robust dystrophin exon 23 skipping in the heart of normal mice injected with oligomers. V-Mor23 also exhibits high levels of exon skipping in the cardiac muscles of mdx mice. The high degree of exon skipping achieved in critical muscle types including the heart and diaphragm suggest Vivo-Morpholinos represent a potential treatment for DMD patients.
3) Block Myostatin via AAV2-Delivered Myostatin Propeptide
in Dystrophic Muscle Improves Dystrophic Pathology and Muscle Cell
Transplantation
J. Zhu; J. Ma; C. Qiao; J. Li; Y. Li; X. Xiao; J. Huard -USA
Myostatin (MSTN) is a potent negative regulator of muscle
growth. We have found that like TGF-β1, MSTN also contributes to the formation
of fibrosis in injured skeletal muscle. In the current study we utilized an
adeno-associated viral vector carrying the MSTN propeptide gene (AAV-MPRO), an
inhibitor of MSTN, to specifically inhibit the action of MSTN in the skeletal
muscles of mdx/SCID mouse, a model of muscular dystrophy with immune-deficiency.
Our objective was to investigate whether AAV-mediated MPRO attenuated the
severity of muscular dystrophy and improved the success of cell transplantation.
In the current study we injected AAV-MPRO/GPF particles in 50 µl of PBS into the
gastrocnemius muscles (GMs) of mdx/SCID mice (4 weeks of age) 4 weeks prior to
muscle progenitor cell (MPC) transplantation (300,000 cells per GM). Two weeks
after cell transplantation, mice were sacrifice, and muscle samples were
harvested and cyrosectioned. Histology (H&E and Masson’s trichromine stain) and
immnunohistochemstry (dystrophin) were performed.
Our results revealed that MPRO improved the dystrophic pathology of mdx mice by
promoting muscle growth and reducing collagen deposition. After AAV2-MPRO
transfer, a significant increase in muscle weight was observed in comparison to
the AAV2-GFP transduced control. Moreover, the mean diameter of the muscle
fibers in the AAV2-MPRO transduced muscles was significantly larger than that of
AAV2-GFP transduced muscles. And the number of degenerative foci was decreased
in the former muscles. Masson’s trichrome stain indicated a significant
reduction of fibrous scar tissue in the AAV2-MPRO transduced GMs. Normal MPCs
injected into AAV2-MPRO transduced dystrophic muscles surpassed the regeneration
capacity of cells injected into AAV2-GFP transduced control muscle, evidenced by
significantly more dystrophin-positive myofibers in the former. In conclusion,
AAV2-MPRO ameliorated dystrophic pathology of mdx/SCID mice, and improving the
milieu in the host dystrophic muscle prior to cell transplantation is an
alternative approach to enhance the efficiency of cell transplantation.
10 - (American Heart Association Meeting, 2008; Circulation Volume 118, Issue 18 Supplement; October 28, 2008):
1 Cincinnati Childrens Hosp, Cincinnati, OH;2 The Christ Hosp, Cincinnati, OH;3 Cincinnati Childrens Hosp, Cincinnati, OH
Patients with Duchenne Muscular Dystrophy (DMD), a dystrophinopathy, universally develop dilated cardiomyopathy, which is associated with abnormal myocardial strain, as well as heterogeneous development of fibrosis as demonstrated by pathology and more recently by cardiac MRI methods. We sought to determine the presence of systolic dyssynchrony in this population using cardiac MRI tagging methods. We analyzed tagged MRI images for the presence of dyssynchrony in 61 males (age 12.5 ± 4.4 y) with a dystrophin mutation undergoing clinical cardiac MRI. Tagged MRI images were analyzed using HARP® analysis of regional strain and strain-time curves. The mid-myocardial slice was specifically analyzed, by dividing it into 6 coronary perfusion regions. Dyssynchrony was defined by the presence of either of 2 previously published indexes modified for use with MRI data: 1) time difference of 1st to last regional peak strain > 100 ms; 2) standard deviation of time differences to peak strain for each of the six regions > 33 ms. Additional indexes evaluated included heart rate, LV ejection fraction, mid-myocardial composite circumferential strain, and presence of delayed myocardial hyperenhancement (MDE). Among the 61 subjects analyzed, 28 (46%) exhibited dyssynchrony indexes 1 and 2, while 8 additional subjects met dyssynchrony index 2 but not index 1. Only 4 subjects, all of whom met both dyssynchrony criteria, had positive MDE and abnormal EF <55%. The regions of slowest activation were highly dispersed and not clustered to the areas of positive MDE. One additional subject with dyssynchrony by either of the critieria also had abnormal EF but did not have MDE. There was no statistically significant difference between mean EF (61 vs 62%), age (12.58 vs 12.63 yrs), heart rate (105 vs 108 bpm) or mid-myocardial composite circumferential strain (–13.3 vs –12.9%) between those subjects with dyssynchrony (by either criteria) and those without. DMD patients frequently exhibit systolic dyssynchrony even in the presence of normal EF. However, the dispersed nature of the dyssynchrony suggests that resynchronization therapy once EF becomes abnormal is unlikely to be of benefit in DMD cardiomyopathy.
Univ of Missouri, Columbia, MO
Duchenne muscular dystrophy (DMD) is a fatal genetic disease with no effective treatment. While heart failure is a leading cause of death in DMD, gene and cell therapies have largely focused on skeletal muscle therapies. The most promising skeletal muscle gene therapy treatments involve truncated mini-dystrophin genes delivered with adeno-associated viral vectors (AAV). It is currently unclear whether these mini-dystrophin genes developed for skeletal muscle can ameliorate cardiomyopathy. Here we evaluated whether cardiac specific expression of mini-dystrophin can normalize heart function in mdx mice, a model for DMD. A series of transgenic mdx mice were generated to express the skeletal muscle proven mini-dystrophin gene in the heart. Heart pathology and function were examined in adult and very old mice. Mini-dystrophin enhanced cardiomyocyte sarcolemmal strength and prevented myocardial fibrosis. ECG analysis showed PR interval normalization but heart rate and QRS duration were not corrected. Closed-chest hemodynamic assay in 22-m-old mice showed normal systolic function and ejection fraction. Heart specific mini-dystrophin therapy also improved the dobutamine stress response and survival. Surprisingly, only moderate improvement was seen in diastolic function, stroke volume and cardiac output. Heart-restricted mini-dystrophin expression also enhanced treadmill performance. Interestingly, over-expressing the mini-dystrophin gene in the normal heart profoundly displaced endogenous dystrophin yet heart function was not compromised. We demonstrate that the skeletal muscle proven mini-dystrophin gene can correct cardiac histopathology but cannot normalize heart function. These results suggest that the requirements for heart therapy could be different from those for skeletal muscle therapy. Furthermore, these findings highlight the potential importance of skeletal muscle disease in modulating heart function. Novel therapeutic strategies have to be developed to treat DMD cardiomyopathy.
Univ of Missouri, Columbia, MO
Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by mutations in the dystrophin gene. Lack of dystrophin causes muscle wasting that eventually leads to premature death from respiratory and/or heart failure. Heart disease can be detected as early as six years of age in DMD patients. As heart disease progresses, patients begin to show overt signs of severe dilated cardiomyopathy and up to 40% of patients die from heart disease. Adeno-associated virus (AAV) mediated micro-dystrophin gene therapy brings the hope of ameliorating DMD. Dystrophin-deficient mdx mice exhibit a cardiac phenotype similar to human DMD patients albeit at a delayed progression (Bostick et al 2008 Cir Res102:121–130). Delineating the potential of gene therapy at different stages of dystrophic cardiomyopathy is critical to developing a successful gene therapy strategy. Investigators, including ourselves, have previously shown that AAV-9 can efficiently transduce the adult mouse heart. In this study, we delivered an AAV-9 micro-dystrophin vector to mdx mice at both the pre-clinical and symptomatic stages of disease (1–5 x 1012 viral particles per mouse). Micro-dystrophin expression and cardiac function were analyzed six weeks to four months post gene transfer. Immunofluorescence staining revealed widespread micro-dystrophin expression in the heart. ECG analysis of mice treated during the pre-clinical stage showed normalization of heart rate, PR interval and QT interval. The cardiomyopathy index was also improved. Mice treated during the later stages of disease showed an incomplete improvement. Notably, ECG analysis showed an improvement in the QRS duration in treated mice (AAV treated, 7.10 ± 0.25 ms; untreated control, 9.36 ± 0.14 ms; p < 0.05). Mice in the later stages of disease also showed a significant increase in the maximal rate of pressure development (dP/dt max) during left ventricular catheterization assay (AAV treated, 11,810 ± 1,225 mmHg/sec; untreated control, 4,864 ± 1,099 mmHg/sec; p < 0.05). In summary, our results suggest that AAV-9 mediated micro-dystrophin gene therapy is a promising approach to treat DMD heart disease. However, the extent of dystrophic cardiomyopathy when gene therapy is initiated may be a critical modifier of disease rescue.
Johns Hopkins Univ, Baltimore, MD
Despite the success of cardiac magnetic resonance (CMR) for identification of cohesive scar, its utility for quantification of diffuse myocardial fibrosis is unknown. We hypothesized that delayed enhancement intensity variance (DEIV) predicts conduction system disease due to diffuse myocardial fibrosis in myotonic muscular dystrophy (MMD). Thirty seven patients with MMD underwent CMR. The DEIV of the entire left ventricle was calculated by obtaining the variance of the mean intensity of each of 20 sectors per inversion recovery prepared gradient echo image plane (120 –200 sectors/patient, Medis software). Standard (ECG) and signal averaged electrocardiography (SAECG) with frank orthogonal leads at a sampling rate of 1 kHz/channel and enough QRS complexes to reduce the noise level to <1 microvolt were performed (Norav software). After correcting for potential confounders of DEIV and conduction disease (body mass index, age and left ventricular ejection fraction), DEIV was predictive of QRS duration on ECG (0.261 ms increased QRS duration / unit increase in DEIV, 95% CI 0.153– 0.369). DEIV was also predictive of low amplitude (<40 microvolt) signal duration on SAECG (0.338 ms increased signal duration / unit increase in DEIV, 95% CI 0.049 – 0.627). Ten fold cross validation yielded a receiver operating characteristic area of 0.742 for the predictive value of DEIV on QRS > 120 ms (figure). The DEIV of the entire left ventricle predicts physiologic QRS prolongation due to late depolarization of tissue within islands of patchy fibrosis and may improve the quantification of diffuse myocardial fibrosis in MMD and other cardiomyopathies.
8 -
(Hum. Mol. Genet., Nov 2008)
Shifts in macrophage phenotypes and macrophage competition for arginine
metabolism affect the severity of muscle pathology in muscular dystrophy
Duchenne muscular dystrophy (DMD) is the most common, lethal, muscle-wasting disease of childhood. Previous investigations have shown that muscle macrophages may play an important role in promoting the pathology in the mdx mouse model of DMD. In the present study, we investigate the mechanism through which macrophages promote mdx dystrophy and assess whether the phenotype of the macrophages changes between the stage of peak muscle necrosis (4 weeks of age) and muscle regeneration (12 weeks). We find that 4-week-old mdx muscles contain a population of pro-inflammatory, classically-activated M1 macrophages that lyse muscle in vitro by NO-mediated mechanisms. Genetic ablation of the iNOS gene in mdx mice also significantly reduces muscle membrane lysis in 4-week-old mdx mice in vivo. However, 4-week mdx muscles also contain a population of alternatively-activated, M2a macrophages that express arginase. in vitro assays show that M2a macrophages reduce lysis of muscle cells by M1 macrophages through the competition of arginase in M2a cells with iNOS in M1 cells for their common, enzymatic substrate, arginine. During the transition from the acute peak of mdx pathology to the regenerative stage, expression of IL-4 and IL-10 increases, either of which can deactivate the M1 phenotype and promote activation of a CD163+, M2c phenotype that can increase tissue repair. Our findings further show that IL-10 stimulation of macrophages activates their ability to promote satellite cell proliferation. Deactivation of the M1 phenotype is also associated with a reduced expression of iNOS, IL-6, MCP-1 and IP-10. Thus, these results show that distinct subpopulations of macrophages can promote muscle injury or repair in muscular dystrophy, and that therapeutic interventions that affect the balance between M1 and M2 macrophage populations may influence the course of muscular dystrophy.
8 - Heart rate variability and hypercapnia in Duchenne muscular dystrophy
8 - Sarcospan reduces dystrophic pathology: stabilization of the utrophin-glycoprotein complex
8 - Serum levels of vascular endothelial growth factor elevated in patients with muscular dystrophy
OCTOBER
26 - (Nature, 2008) Sarcolemma-localized nNOS is required to maintain activity after mild exercise
Yvonne M. Kobayashi
, Erik P. Rader, Robert W. Crawford, Nikhil K. Iyengar, Daniel R. Thedens, John A. Faulkner, Swapnesh V. Parikh, Robert M. Weiss, Jeffrey S. Chamberlain, Steven A. Moore & Kevin P. Campbell - USAMany neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response that is disproportionate to activity level. This fatigue is not necessarily correlated with greater central or peripheral fatigue in patients, and some patients experience severe fatigue without any demonstrable somatic disease. Except in myopathies that are due to specific metabolic defects, the mechanism underlying this type of fatigue remains unknown. With no treatment available, this form of inactivity is a major determinant of disability3. Here we show, using mouse models, that this exaggerated fatigue response is distinct from a loss in specific force production by muscle, and that sarcolemmalocalized signalling by neuronal nitric oxide synthase (nNOS) in skeletal muscle is required to maintain activity after mild exercise. We show that nNOS-null mice do not have muscle pathology and have no loss of muscle-specific force after exercise but do display this exaggerated fatigue response to mild exercise. In mouse models of nNOS mislocalization from the sarcolemma, prolonged inactivity was only relieved by pharmacologically enhancing the cGMP signal that results from muscle nNOS activation during the nitric oxide signalling response to mild exercise. Our findings suggest that the mechanism underlying the exaggerated fatigue response to mild exercise is a lack of contraction-induced signalling from sarcolemma-localized nNOS, which decreases cGMP-mediated vasomodulation in the vessels that supply active muscle after mild exercise. Sarcolemmal nNOS staining was decreased in patient biopsies from a large number of distinct myopathies, suggesting a common mechanism of fatigue. Our results suggest that patients with an exaggerated fatigue response to mild exercise would show clinical improvement in response to treatment strategies aimed at improving exercise-induced signalling.
Study may explain exercise-induced fatigue in muscular dystrophies
25 - (CHEST, 2008) HOME MECHANICAL IN-EXSUFFLATION: SAFETY AND EFFICACY IN NEUROMUSCULAR DISORDERS (NMD)
PURPOSE: NMD patients frequently have impaired cough. Inefficient bronchial clearance may cause ventilatory failure. Mechanical in-exsufflation (MI-E) is effective to improve airway clearance, however limited data exist on its home long-term use. Our purpose was to determine safety and effectiveness of home MI-E treatment.
METHODS: Retrospective analysis of 17 NMD patients (12 males) with median age of 54.5 yrs-old. All patients were on continuous home mechanical ventilation (3 under tracheostomy) and MI-E treatment with oximetry feed-back. Data were recorded as function of diagnosis, spirometry, respiratory muscle strength, unassisted peak cough flow and level of ventilatory support. Safety and efficacy were assessed based on number of complications and hospitalizations related to secretion encumbrance. Side effects were also assessed.
RESULTS: Patients fell into 5 different NMD: Amyotrophic Lateral Sclerosis (ALS) (10), Duchenne Muscular Dystrophy (DMD) (3), other Muscular Dystrophy (MD) (2), Multiple Sclerosis (MS) (1) and Transverse Myelitis (TM) (1). Pulmonary function previous to initiation of MI-E revealed: median FVC=0.78L (29%), median FEV1=0.68L (33%), median MIP=28cmH2O, median MEP=22cmH2O and median PCF=90L/min. Median follow-up under home MI-E was 9 months (1–35 months). Seven patients (6 with ALS, 1 with DMD) used MI-E daily, while 10 patients used it intermittently, during exacerbations. All patients with tracheostomy used MI-E every day and more times a day than patients under NIV, but none of those patients needed antibiotherapy. MI-E was well-tolerated. There were no complications related to the treatment. In general, MI-E was considered effective by caregivers. Timely initiation of MI-E (guided by oximetry feed-back) avoided hospitalization in 6 patients. Only 2 patients (in 3 occasions) had to be hospitalized due to difficulty in handling secretions.Four patients have died (all ALS), 3 of them related to disease progression and 1 from unknown reason.
CONCLUSION: Home MI-E was well tolerated and effective. We consider it safe if used adequately by well prepared caregivers.
CLINICAL IMPLICATIONS: Increased MI-E use during respiratory infections avoided hospitalization in non-tracheostomized patients. Daily home MI-E use decreased frequency of tracheobronchial infections in tracheostomized patients.
PURPOSE:Duchenne patients eventually require 24 hour ventilatory support. Many centers recommend tracheostomy rather than noninvasive ventilation (NIV). We report the outcome of 19/23 adult DMD patients on nocturnal NIV plus daytime mouthpiece ventilation (MPV).
METHODS:16/23 patients used nocturnal bilevel NIV, avg. pressure 17/6 cmH2O. 3/23 used volume-targeted ventilators, Vtavg 900 ml, RRavg 13/min. Outpatient-initiated 24 hour NIV patients (12/23), used MPV, a chair-mounted ventilator and mouthpiece. Oximetry, downloads and clinical assessment assured adequate ventilation. All patients used lung volume recruitment and 16/23 had CoughAssistTM devices.
RESULTS:23 DMD patients were followed at the Ottawa Hospital Rehabilitation Centre. Nocturnal NIV was used in 19/23 patients. Average values at initiation of nocturnal NIV were; FVC 1.08L (25%pred.), PaCO2 52mmHg, age 17.5 years. Duration on nocturnal NIV was 1 to 11.3 years with one death after 2 years on NIV. 12 patients subsequently required 24 hour support, indications; dyspnea, tachypnea and hypercapnea despite adequate nocturnal NIV. Average values at initiation of 24 hour support were; FVC 0.57L (13.2%pred.), PaCO2 53mmHg, age 19.3 years. PaCO2 was reduced to 48mmHg with Nocturnal NIV and to 43mmHg with 24 hour NIV. Duration on 24 hour NIV/MPV is up to 12 years with two deaths to date (after 3.75 and 4 years on NIV). No patient required a permanent tracheostomy. In 81 patient-years on NIV there have been only 4 respiratory-related hospitalizations.
CONCLUSION:To date all of our patients with DMD have been managed long beyond nocturnal NIV with MPV. No patient required a tracheostomy for respiratory failure.
CLINICAL IMPLICATIONS:MPV should be considered as an alternative to tracheostomy for 24 hour support in DMD.
25 - (Archives of Physical Medicine and Rehabilitation, Volume 89, Issue 10, October 2008, Page e8) Quantification of Longitudinal Changes in Muscle Pathology in Boys With Duchenne’s Muscular Dystrophy Using Magnetic Resonance Imaging and Spectroscopy
Sunita Mathur (University of Florida, Gainesville, FL), D. Lott, C. Senesac, S. Germain, G. Walter, K. Vandenborne.
Objectives: To examine longitudinal changes in muscle size, damage, lipid content, and function in boys with Duchenne’s muscular dystrophy (DMD) and to compare this with controls. Design: Cohort study, with 6- to 12-month follow-up. Setting: Research lab. Participants: Volunteer sample, boys with DMD (n6; age range, 6-13y); age- and sex-matched controls (n6). Interventions: Not applicable. Main Outcome Measures: Magnetic resonance imaging (MRI) and spectroscopy (MRS) (at 1.5T or 3.0T) were used to quantify muscle cross-sectional area (CSA), T2 relaxation time (muscle damage), and lipid content of the lower leg. Functional tests included timed 30-foot walk and isometric muscle strength. Results: Increases of 2% to 32% in lipid and 2% to 11% in T2 of the soleus were observed in boys with DMD. These values were higher than in controls (P<.05). Time to walk 30ft increased an average of 30% over time in boys with DMD and was longer than for controls (P<.01). CSA of the plantar- and dorsiflexors was 15% to 143% higher, whereas strength was 49% to 74% lower in boys with DMD compared with controls (P<.01). Conclusions: MRI and MRS provide unique, noninvasive quantification of progressive muscle pathology in boys with DMD and may be applicable in clinical trials examining new therapies for DMD.
25 - (Archives of Physical Medicine and Rehabilitation, Volume 89, Issue 10, October 2008, Page e48) The Effects of Ankle-Foot Orthoses in the Treatment on the Evolution of Plantarflexion Contracture in Muscular Dystrophy.
Denise Troise (Muscular Dystrophy Association of Brazil, San Paulo, Brazil), Maria Fornari, Michele Hukuda, Maria Hayashi.
Objective: To evaluate the effect of ankle-foot orthoses (AFOs) on the evolution of plantarflexion contractures in patients with muscular dystrophy. Design: Prospective study. Setting: Patients from Muscular Dystrophy Association of Brazil. Participants: 29 patients diagnosed with muscular dystrophy, both male and female, between 6 and 42 years of age. They were divided in 2 groups: the adherent group (n=25) and no adherent group (n=14). Interventions: Use of an AFO for 4 hours a day. Main Outcome Measures: Passive range of motion (ROM) of ankle dorsiflexion and plantarflexion measured with a goniometer. Patients were followed for 12 months and evaluated initially and at 6-month intervals. Results: Significant improvement in ankle dorsiflexion and plantarflexion was related in the adherent group after 6 months as compared with the no adherent group. After 12 months, the adherent group maintained improvements in ankle dorsiflexion. Conclusions: AFOs improved the ROM of ankle in patients with muscular dystrophy.
23 - (Journal of Cardiology, 2008) Beneficial effects of beta-blockers and angiotensin-converting enzyme inhibitors in Duchenne muscular dystrophy
Hitoko Ogata (MD, FJCC), Yuka Ishikawa (MD), Yukitoshi Ishikawa (MD), Ryoji Minami (MD) - Japan
Background: Patients with Duchenne muscular dystrophy (DMD) often have severe heart failure with a high mortality rate. Most DMD patients with cardiomyopathy became symptomatic in their early to middle teens and usually die of congestive heart failure within 2—3 years from the onset of symptoms. It has been reported that the combination of an angiotensin-converting enzyme (ACE) inhibitor and a betablocker has additive benefits in patients with heart failure. The aim of this study was to assess whether the combination of an ACE inhibitor and a beta-blocker is associated with long-term survival of DMD patients with left ventricular (LV) dysfunction. Methods: We retrospectively analyzed the outcomes of 52 DMD patients who had begun treatment for heart failure with an ACE inhibitor and a beta-blocker at National Yakumo Hospital during the period from 1992 to 2005. All patients used wheelchairs in their daily lives. Patients were classified as symptomatic or asymptomatic at the initiation of treatment with these two drugs. Twelve patients who had already had apparent symptoms due to heart failure were enrolled in a treatment group. Forty patients who had no symptoms with reduced LV ejection fraction (≤45% in echocardiography) were enrolled in a prevention group. Results: Five-year and 7-year survival rates of all patients were 93 and 84%, respectively. In the treatment group, 5-year and 7-year survival rate were 81 and 71%, respectively. Survival rate became zero at 10.9 years. In the prevention group, 5-year and 7-year survival rates were 97 and 84%, respectively, and 10-year survival rate was 72%. Nine patients in the prevention group remained event-free over 10 years.
18 - (Neurobiology of Disease, Volume 32, Issue 2, November 2008, Pages 243-253) Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle
Annamaria De Luca, Beatrice Nico, Jean-François Rolland, Anna Cozzoli, Rosa Burdi , Domenica Mangieri, Viviana Giannuzzi, Antonella Liantonio, Valentina Cippone, Michela De Bellis, Grazia Paola Nicchia, Giulia Maria Camerino, Antonio Frigeri, Maria Svelto and Diana Conte Camerino - Italy
Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8–12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6–8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.
11 - Calcium antagonists for Duchenne muscular dystrophy
4 - Duchenne muscular dystrophy; a cardiomyopathy that can be prevented?
SEPTEMBER
20 - (Archives of Oral Biology, 2008) Orofacial dysfunction in Duchenne muscular dystrophy
Sebastien Botteron , Catherine Morel Verdebout, Pierre-Yves Jeannet , Stavros Kiliaridis - Switzerland
Duchenne muscular dystrophy (DMD) affects orofacial function. Our aim was to evaluate certain characteristics of orofacial function in DMD and relate possible deteriorations to the age of the patients and to the diminished internal structure quality of the masseter muscle. Bite force and finger force were measured in 16 DMD patients (6–20 years old) and 16 age matched controls. The thickness and internal structure quality of the masseter muscle were evaluated ultrasonographically. We found reduced mouth opening but no signs of masticatory muscle tenderness. Bite force values were lower for DMD patients. Masseter thickness showed no significant differences between the two groups, but poorer internal muscle structure quality characterised the elder, non-walking DMD patients explaining their low bite force values. In conclusion, the masseter muscle follows the general progress of the disease. Orofacial function in DMD patients is becoming ever more important as their life expectancy increases.
19 - (PNAS,2008) Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer
Bo Wu, Hong M. Moulton, Patrick L. Iversen, Jiangang Jiang, Juan Li, Jianbin Li, Christopher F. Spurney, Arpana Sali, Alfredo D. Guerron, Kanneboyina Nagaraju, Timothy Doran, Peijuan Lu, Xiao Xiao, and Qi Long Lu -USA
Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cell-penetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients
14 - Exon Skipping, a therapy for Duchenne muscular dystrophy
Reading through premature stop codons with PTC124. Project Catalyst to find more Duchenne drugs
10 - (Hum.Mol.Genet., 2008) Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function
Antisense oligonucleotides (AOs) have the potential to induce functional dystrophin protein expression via exon skipping by restoring in-frame transcripts in the majority of patients suffering from Duchenne muscular dystrophy (DMD). AOs of morpholino phosphoroamidate (PMO) and 2'-O-methyl phosphorothioate RNA (2'Ome RNA) chemistry have been shown to restore dystrophin expression in skeletal muscle but not in heart, following high dose systemic delivery in murine models of muscular dystrophy (mdx). Exploiting the cell transduction properties of two basic arginine-rich cell penetrating peptides, we demonstrate widespread systemic correction of dystrophin expression in body-wide muscles and cardiac tissue in adult dystrophic mdx mice, with a single low dose injection of peptide-conjugated PMO AO. This approach was sufficient to restore uniform, high level dystrophin protein expression in peripheral muscle and cardiac tissue, with robust sarcolemmal relocalization of the dystrophin-associated protein complex and functional improvement in muscle. Peptide-conjugated AOs therefore have significant potential for systemic correction of the DMD phenotype.
10 - (European Heart Journal, 2008) Long-term blinded placebo-controlled study of SNT-MC17/idebenone in the dystrophin deficient mdx mouse: cardiac protection and improved exercise performance
Aims: Duchenne muscular dystrophy (DMD) is a severe and still incurable disease, with heart failure as a leading cause of death. The identification of a disease-modifying therapy may require early-initiated and long-term administration, but such type of therapeutic trial is not evident in humans. We have performed such a trial of SNT-MC17/idebenone in the mdx mouse model of DMD, based on the drug’s potential to improve mitochondrial respiratory chain function and reduce oxidative stress. Methods and results: In this study, 200 mg/kg body weight of either SNT-MC17/idebenone or placebo was given from age 4 weeks until 10 months in mdx and wild-type mice. All evaluators were blinded to mouse type and treatment groups. Idebenone treatment significantly corrected cardiac diastolic dysfunction and prevented mortality from cardiac pump failure induced by dobutamine stress testing in vivo, significantly reduced cardiac inflammation and fibrosis, and significantly improved voluntary running performance in mdx mice. Conclusion: We have identified a novel potential therapeutic strategy for human DMD, as SNT-MC17/idebenone was cardioprotective and improved exercise performance in the dystrophin-deficient mdx mouse. Our data also illustrate that the mdx mouse provides unique opportunities for long-term controlled prehuman therapeutic studies.
10 - Endurance training improves fitness and strength in patients with Becker muscular dystrophy
4 - Cognitive and adaptive deficits in young children with Duchenne muscular dystrophy (DMD)
3 - (International Journal of Biochemistry and Cell Biology (2007)) Regenerative pharmacology in the treatment of genetic diseases: the paradigm of muscular dystrophy
Chiara, M., Giulia, M., & Lorenzo, P. P. - Italy
Current evidence supports the therapeutic potential of pharmacological interventions that counter the progression of genetic disorders by promoting regeneration of the affected organs or tissues. The rationale behind this concept lies on the evidence that targeting key events downstream of the genetic defect can compensate, at least partially, the pathological consequence of the related disease. In this regard, the beneficial effect exerted on animal models of muscular dystrophy by pharmacological strategies that enhance muscle regeneration provides an interesting paradigm. In this review, we describe and discuss the potential targets of pharmacological strategies that promote regeneration of dystrophic muscles and alleviate the consequence of the primary genetic defect. Regenerative pharmacology provides an immediate and suitable therapeutic opportunity to slow down the decline of muscles in the present generation of dystrophic patients, with the perspective to hold them in conditions such that they could benefit of future, more definitive, therapies.
3 - (European Meeting of Cardiology, 2008) Differential effects on IGF-1 expression in myocardium and skeletal muscle of wild type and mdx -/- mice after voluntary exercise
N. Mangner1 , S. Gielen1, M. Sandri1, R. Hoellriegel1, V. Adams1, R. Hambrecht2 , G. Schuler1. 1University Leipzig - Heart Center, Department for Cardiology, Leipzig, Germany; 2Klinik Links der Weser - Heart Center, Bremen, Germany
Both clinical and experimental data show conflicting results with regard to exercise recommendations in hereditary muscle dystrophy: On the one hand overloading of diseased muscle fibres may accelerate disease progression, on the other hand myocardial affection by dystrophy may limit the cardiac training response. Aimof this study was to examine cardiac and skeletal muscle adaptations to voluntary wheel running in mdx -/- mice (MDX), an animal model of Duchenne muscular dystrophy vs. C57/Bl6 wild type (WT) with regard to local IGF-1 expression. Methods: Six months old WT and MDX were assigned to 4 weeks of voluntary wheel running (VR) or to nonexercised control group (C). Running distance was recorded daily. After 4 weeks body and heart weight were measured and myocardium, M. extensor digitorum longus (EDL) and M. soleus (SOL) were collected. IGF-1 was quantified as ratio of IGF-1 to 18S-RNA by quantitative rt-PCR. Results: WT ran a longer distance than MDX (5.71±0.36 vs. 4.27±0.18 km/d, p<0.01). Relative heart weight increased both in WT by 16.1% and in MDX by 10.2% after VR, but was significantly higher in the WT training group (p<0.01 vs. C and WT vs. MDX). Sedentary MDX and WT showed comparable expression levels of IGF-1 in myocardium. VR resulted in an increase of myocardial IGF-1 expression from 0.25±0.04 to 0.69±0.19 in WT (p<0.05) whereas it remained unaffected in MDX. Sedentary MDX showed higher expression levels of IGF-1 in EDL and SOL than WT (0.79±0.19 vs. 0.23±0.04 and 1.66±0.49 vs. 0.53±0.08, p<0.01). VR resulted in an increase of IGF-1 expression from 0.23±0.04 to 0.53±0.12 in EDL of WT (p<0.05) whereas remaining unchanged in SOL. No changes of IGF-1 expression in EDL or SOL of MDX were detectable after VR. Conclusion: Both WT and MDX developed a cardiac hypertrophy in response to voluntary wheel running which was more distinct in WT. MDX exhibited a compensatory higher baseline anabolic activation (by local IGF-1 levels) in EDL and SOL as compared to WT. Contrary to WT animals MDX were not capable to further increase IGF-1 expression in response to voluntary running. These data indicate a significantly limited capacity of MDX.
AUGUST
20 - (American Journal of Physical Medicine & Rehabilitation. 87(9):726-730, September 2008) Cough Augmentation in Duchenne Muscular Dystrophy
Objective: The purpose of this work was to compare
the relative importance of deep lung insufflation with the abdominal thrust and
their combination in augmenting cough peak flows (CPF). Design:
Unassisted CPF and CPF assisted by air stacking to deep lung volumes (CPFair),
assisted by abdominal thrusts (CPFthrust), and assisted by both air stacking and
abdominal thrusts (aCPF) were measured for 61 patients with Duchenne
muscular dystrophy (DMD). Results:
Overall, mean unassisted CPF were 138 +/- 70 liters/min, CPFthrust were 204 +/-
75 liters/min, CPFair were 236 +/- 68 liters/min, and aCPF were 302 +/- 78
liters/min. The differences between each were statistically significant (P <
0.0001). Conclusions: Thus, air stacking was significantly more effective
than abdominal thrust in increasing CPF, but the combination was the most
effective. The CPF of the quartile of patients with the lowest unassisted CPF
were also significantly (P <= 0.04) more augmented by air stacking and thrusting
than for the milder quartiles of patients. Thus, the greatest improvements in
CPF were for patients with the weakest coughs.
15 - 2008 Annual CONNECT Conference Presentations
15 - (International Journal of Cardiology, 2008) Cardiac involvement in a female carrier of Duchenne muscular dystrophy
Thomas Walcher, Markus Kunze, Peter Steinbach, Anne-Dorte Sperfeld, Christof Burgstahler d, Vinzenz Hombach a, Jan Torzewski - Germany
A 42 year-old female carrier of Duchenne muscular dystrophy (DMD) was referred with suspected subacute myocarditis and nonsustained ventricular tachycardia. Echochardiography and cardiac catheterization revealed severely reduced left ventricular function (LVF). Coronary artery disease was excluded. Cardiac magnetic resonance imaging showed transmural, intramural and subepicardial late gadolinium enhancement. Myocardial biopsy excluded viral infection and showed severe myopathic changes with abnormal expression of dystrophin and utrophin. Moleculargenetic analysis of the DMD gene revealed frameshift duplication of exon 2. The patient received conventional heart failure therapy, implantable cardioverter/defibrillator-implantation and prednisolone to attenuate cardiac degradation. 6 months later she had improved clinically though LVF was still severely reduced.
15 - (Hum. Mol. Genet., Sep 2008; 17: 2622 - 2632) RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology. The peak of the pathology attributed to dystrophin deficiency happens between 3 and 8 weeks of age in mdx mice, the animal model of DMD. Accordingly, we hypothesized that the pathology observed with dystrophin deficiency may be developmentally regulated. Initially, we demonstrated that profound small interfering RNA-mediated dystrophin knockdown could be achieved in mouse primary muscle cultures. The use of adeno-associated virus vectors to express short-hairpin RNAs targeting dystrophin in skeletal muscle in vivo yielded a potent and specific dystrophin knockdown, but only after 5 months, indicating the very long half-life of dystrophin. Interestingly, and in contrast to what is observed in congenital dystrophin deficiency, long-term (1 year) dystrophin knockdown in adult mice did not result, per se, in overt dystrophic pathology or upregulation of utrophin. This supports our hypothesis and suggests new pathophysiology of the disease. Furthermore, taking into account the rather long half-life of dystrophin, and the notion that the development of pathology is age-dependent, it indicates that a single gene therapy approach before the onset of pathology might convey a long-term cure for DMD.
9 - (Acta Biomaterialia, Volume 4, Issue 5, September 2008, Pages 1411-1420) Matrix metalloproteinase-1 treatment of muscle fibrosis
Joel L. Kaar, Yong Li, Harry C. Blair, Gemma Asche, Richard R. Koepsel, Johnny Huard and Alan J. Russell - USA
The onset of scarring after injury may impede the regeneration and functional recovery of skeletal muscle. Matrix metalloproteinase-1 (MMP-1) hydrolyzes type I collagen and thus may improve muscle regeneration by resolving fibrotic tissue. We examined the effect of recombinant human MMP-1 on fibrosis in the lacerated gastrocnemius muscle of NOD/scid mice, allowing treatment potential to be ascertained in isolation from immune response. The efficacy of proMMP-1 and active MMP-1 were compared with or without poly(ethylene glycol) (PEG) modification, which was intended to increase the enzyme’s stability. Active MMP-1 was most effective in reducing fibrosis, although treatment with proMMP-1 was also beneficial relative to controls. PEG-modified MMP-1 had minimal activity in vivo, despite retaining activity towards a thioester substrate. Moreover, the modified enzyme was inactivated by trypsin and subtilisin at rates comparable to that of native MMP-1. These results and those of computational structural studies suggest that modification occurs at the C-terminal hemopexin domain of MMP-1, which plays a critical role in collagen turnover. Site-specific modifications that spares catalytic and substrate binding sites while protecting susceptible proteolytic digestion sites may be beneficial. We conclude that active MMP-1 can effectively reduce muscle scarring and that its activity is related to the ability of the enzyme to digest collagen, thereby facilitating remodeling of the injured muscle
JULY
31 - (Arch. Dis. Child., Jul 2008) Update on the management of Duchenne muscular dystrophy
Adnan Y Manzur, Maria Kinali, Francesco Muntoni - UK
Duchenne Muscular Dystrophy (DMD) is familiar to paediatricians as the most common childhood muscular dystrophy, leading to severe disability and early death in late teenage years, if untreated. Improvement in general care, glucocorticoid corticosteroid treatment, non-invasive ventilatory support, cardiomyopathy and scoliosis management have significantly changed the course of the DMD in treated individuals, so that survival into adulthood is now a realistic possibility for most DMD patients. This has important implications for the medical and social sectors to provide transition to adult medical services, suitable employment, and social care. Multidisciplinary team working for optimal management of the DMD-specific multisystem complications is essential, and collaboration in disease specific national clinical networks is recommended. Several curative therapeutic strategies including cell and gene therapy in DMD are being pursued, but these are still at an experimental stage.
31 - (The American Journal of Pathology) Muscle CD31(-) CD45(-) Side Population Cells Promote Muscle Regeneration by Stimulating Proliferation and Migration of Myoblasts
CD31(-) CD45(-) side population (SP) cells are a minor SP subfraction that have mesenchymal stem cell-like properties in uninjured skeletal muscle but that can expand on muscle injury. To clarify the role of these SP cells in muscle regeneration, we injected green fluorescent protein (GFP)-positive myoblasts with or without CD31(-) CD45(-) SP cells into the tibialis anterior muscles of immunodeficient NOD/scid mice or dystrophin-deficient mdx mice. More GFP-positive fibers were formed after co-transplantation than after transplantation of GFP-positive myoblasts alone in both mdx and NOD/scid muscles. Moreover, grafted myoblasts were more widely distributed after co-transplantation than after transplantation of myoblasts alone. Immunohistochemistry with anti-phosphorylated histone H3 antibody revealed that CD31(-) CD45(-) SP cells stimulated cell division of co-grafted myoblasts. Genome-wide gene expression analyses showed that these SP cells specifically express a variety of extracellular matrix proteins, membrane proteins, and cytokines. We also found that they express high levels of matrix metalloproteinase-2 mRNA and gelatinase activity. Furthermore, matrix metalloproteinase-2 derived from CD31(-) CD45(-) SP cells promoted migration of myoblasts in vivo. Our results suggest that CD31(-) CD45(-) SP cells support muscle regeneration by promoting proliferation and migration of myoblasts. Future studies to further define the molecular and cellular mechanisms of muscle regeneration will aid in the development of cell therapies for muscular dystrophy.
30 - (Neuromuscular Disorders, 2008) Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone
Tidhar Turgeman , Yosey Hagai , Kyla Huebner , Davinder S. Jassal , Judy E. Anderson , Olga Genin , Arnon Nagler , Orna Halevy , Mark Pines - Israel
Fibrosis is a known feature of dystrophic muscles, particularly the diaphragm, in the mdx mouse. In this study we evaluated the effect of halofuginone, a collagen synthesis inhibitor, on collagen synthesis in various muscles of young wild-type (C57/BL/6J) and mdx mice. Halofuginone prevented the age-dependent increase in collagen synthesis in the diaphragms of mdx with no effect on wild-type mice (n = 5 for each time point). This was associated with a decrease in the degenerated areas and number of central nuclei. Halofuginone also inhibited collagen synthesis in cardiac muscle. Moreover, enhanced motor coordination, balance and improved cardiac muscle function were observed implying reduced muscle injury. Halofuginone inhibited Smad3 phosphorylation downstream of TGFb in the diaphragm and cardiac muscles, in C2 cell line and in primary mouse myoblast cultures representing various muscular dystrophies. We suggest that via its effect on Smad3 phosphorylation, halofuginone inhibits muscle fibrosis and improves cardiac and skeletal muscle functions in mdx mice.
28 - (Nature Medicine) PlGF–MMP-9–expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle
Cesare Gargioli
, Marcello Coletta, Fabrizio De Grandis, Stefano M Cannata & Giulio Cossu - ItalySclerosis and reduced microvessel density characterize advanced stages of muscular dystrophy and hamper cell or gene delivery, precluding treatment of most individuals with Duchenne muscular dystrophy. Modified tendon fibroblasts expressing an angiogenic factor (placenta growth factor, PlGF) and a metalloproteinase (matrix metalloproteinase-9, MMP-9) are able to restore a vascular network and reduce collagen deposition, allowing efficient cell therapy in aged dystrophic mice. These data open the possibility of extending new therapies to currently untreatable individuals.
Fresh hope for muscular dystrophy
Terapia Cellulare: nuovi sviluppi per la DMD (Italian)
26 - Clinical use of creatine in neuromuscular and neurometabolic disorders
18 - Family sues co. for muscular dystrophy drug
Advocating a Treatment, but Denied Access to It
13 - (Cell, 2008) Highly Efficient, Functional Engraftment of Skeletal Muscle Stem Cells in Dystrophic Muscles
Massimiliano Cerletti, Sara Jurga, Carol A. Witczak, Michael F. Hirshman, Jennifer L. Shadrach, Laurie J. Goodyear and Amy J. Wagers - USA
Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.
Public release: Purified stem cells restore muscle in mice with muscular dystrophy
13 - Combining stem cells and exon skipping strategy to treat muscular dystrophy
13 - Pneumothorax Associated with Mechanical Insufflation-Exsufflation and Related Factors
1 - (GENES & DEVELOPMENT 22:1747-1752, 2008) Fibrinogen drives dystrophic muscle fibrosis via a TGFβ/alternative macrophage activation pathway
In the fatal degenerative Duchenne muscular dystrophy (DMD),
skeletal muscle is progressively replaced by fibrotic tissue.
Here, we show that fibrinogen accumulates in dystrophic muscles of
DMD patients and mdx mice. Genetic loss or pharmacological
depletion of fibrinogen in these mice reduced fibrosis and dystrophy
progression. Our results demonstrate that fibrinogen–Mac-1 receptor
binding, through induction of IL-1β, drives the synthesis of
transforming growth factor-β (TGFβ) by mdx macrophages, which
in turn induces collagen production in mdx fibroblasts.
Fibrinogen-produced TGFβ further amplifies collagen accumulation
through activation of profibrotic alternatively activated macrophages.
Fibrinogen, by engaging its
vβ3
receptor on fibroblasts, also directly promotes collagen synthesis.
These data unveil a profibrotic role of fibrinogen deposition
in muscle dystrophy.
JUNE
27 - (J Appl Physiol, Jun 2008) Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract
Duchenne muscular dystrophy is characterized by the absence of dystrophin from muscle cells. Dystrophic muscle cells are susceptible to oxidative stress. We tested the hypothesis that 3 weeks of endurance exercise starting at age 21-d in young male mdx mice would blunt oxidative stress and improve dystrophic skeletal muscle function, and these effects would be enhanced by the antioxidant, green tea extract (GTE). In mice fed normal diet, average daily running distance increased 300% from week 1 to week 3, and total distance over 3 weeks was improved by 128% in mice fed GTE. Running, independent of diet increased serum antioxidant capacity, EDL tetanic stress and total contractile protein content, heart citrate synthase and heart and quadriceps -hydroxyacyl-CoA dehydrogenase activities. GTE, independent of running decreased serum creatine kinase, heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate synthase activity. These data suggest that both endurance exercise and GTE may be beneficial therapeutic strategies to improve muscle function in mdx mice.
24 - (Developmental Medicine & Child Neurology. 50(7):546-552, July 2008) Pain and activity limitations in children with Duchenne or Becker muscular dystrophy.
Zebracki, Kathy ; Drotar, Dennis - USA
The purpose of this study was to examine the prevalence and characteristics of pain in children with Duchenne (DMD) or Becker (BMD) muscular dystrophy, including the nature of disagreements concerning pain symptoms among children, parents, and physicians, and limitations in daily activities. Male children (age 8-18y, n = 53) and parents (n = 53) completed questionnaires assessing pain intensity (visual analogue scale), pain frequency (Likert scale [LS]), pain duration (LS), emotional distress due to pain (LS), and pain location (body outline markings). The Child Activity Limitations Interview was also completed by both raters to assess daily activities that are limited by recurrent pain. Physicians completed a form indicating medical history and pain symptoms. The majority of children with DMD (mean age 13y 11mo [SD 3.38]; range 8-18y) or BMD (mean 14y 10mo [SD 1.48]; range 12-17y) were non-ambulatory (79 and 50% respectively) and experienced pain according to self (54-80%) and parent reports (70-90%). Pain typically occurred at least once per week and was of mild to moderate intensity. Most children experienced pain for less than a few hours and little to moderate levels of emotional distress due to pain. Pain occurred in the lower back, spine, and legs, and was described as 'aching'. Children and parents indicated significantly more intense pain than the physician. Actual agreement between parent and child report on pain symptoms was poor to fair. Pain is a common occurrence in children with DMD or BMD, yet may be under-recognized. Pain assessment needs to be a standard part of care and may identify difficulties faced by these children to be targeted by interventions.
21 - (Experimental Physiology, 2008) IGF-I analogue protects muscles of dystrophic mdx mice from contraction-mediated damage
Contraction-mediated injury is a major contributing factor to the pathophysiology of muscular dystrophy and so therapies that can attenuate this type of injury have clinical relevance. Systemic administration of insulin-like growth factor-I (IGF-I) has been shown to improve muscle function in dystrophic mdx mice, an effect associated with a shift to a more oxidative muscle phenotype and a reduced susceptibility to contraction-mediated damage. The actions of IGF-I in vivo are modulated by IGF binding proteins (IGFBPs), which generally act to inhibit IGF-I signaling. We tested the hypothesis that an analogue of IGF-I (LR IGF-I) with significantly reduced binding affinity for IGFBPs, would improve the dystrophic pathology by reducing muscle susceptibility to injury. Dystrophic mdx and wild type (C57BL/10) mice were administered LR IGF-I continuously (~1.5 mg/kg/day) via osmotic mini-pump for 4 weeks. LR IGF-I administration reduced the susceptibility of EDL, soleus and diaphragm muscles to contraction damage as evident from lower force deficits after a protocol of lengthening contractions. In contrast to the mechanism of protection conferred by administration of IGF-I, that conferred by LR IGF-I was independent of changes in muscle fatigue and oxidative metabolism. The findings support the contention that modulating IGF-I signalling has therapeutic potential for muscle diseases.
21 - AAV-9 micro-dystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice
Since its introduction several decades ago, solid organ transplantation has had a wide impact as successful therapy for end-stage organ failure. Refinements in surgical techniques have allowed for the expansion of the spectrum of transplantation to replacement of “nontraditional” organs and tissue such as extremity limbs, facial structures, and laryngeal allografts. Duchenne muscular dystrophy (DMD) is the most common and severe of the human muscular dystrophies, affecting 1 in 3500 live male births. Currently no efficacious therapy is available for this disease, and death generally occurs in the late teens to early twenties from respiratory complications resulting from diaphragmatic dysfunction. Diaphragm transplantation could thus represent a novel therapy for this disease. As the ultimate success of transplantation hinges on adequate revascularization, the goal of this study was to develop surgical techniques for orthotopic diaphragmatic transplantation.
To our knowledge, this is the first demonstration of technical feasibility of diaphragmatic transplantation. We are in the process of establishing a long-term transplantation model that will allow us to study physiology, immunosuppression, and long-term survival and metabolic requirements of a transplanted diaphragm. Methods for reinnervation or direct diaphragmatic pacing will be explored. Inasmuch as a homolog of DMD has been identified in a muscular dystrophic Golden Retriever, the canine model may provide an opportunity for future preclinical large animal trials.
MAY
29 - (Hum. Mol. Genet., May 2008) Cell-lineage Regulated Myogenesis for Dystrophin Replacement: a Novel Therapeutic Approach for Treatment of Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles of myofiber necrosis and regeneration leading to loss of muscle fibers and replacement with fibrotic connective and adipose tissue. The ongoing activation and recruitment of muscle satellite cells for myofiber regeneration results in loss of regenerative capacity in part due to proliferative senescence. We explored a method whereby new myoblasts could be generated in dystrophic muscles by transplantation of primary fibroblasts engineered to express a micro-dystrophin/eGFP (µDys/eGFP) fusion gene together with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)]. Fibroblasts isolated from mdx4cv mice, a mouse model for DMD, were efficiently transduced with lentiviral vectors expressing µDys/eGFP and MyoD-ER(T) and underwent myogenic conversion when exposed to tamoxifen. These cells could also be induced to differentiate into µDys/eGFP-expressing myocytes and myotubes. Transplantation of transduced mdx4cv fibroblasts into mdx4cv muscles enabled tamoxifen-dependent regeneration of myofibers that express micro-dystrophin. This lineage control method therefore allows replenishment of myogenic stem cells using autologous fibroblasts carrying an exogenous dystrophin gene. This strategy carries several potential advantages over conventional myoblast transplantation methods including: 1) the relative simplicity of culturing fibroblasts compared with myoblasts, 2) a readily available cell source and ease of expansion, and 3) the ability to induce MyoD gene expression in vivo via administration of a medication. Our study provides a proof of concept for a novel gene/stem cell therapy technique and opens another potential therapeutic approach for degenerative muscle disorders.
29 - (Hum. Mol. Genet., May 2008) RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology. The peak of the pathology attributed to dystrophin deficiency happens between 3 and 8 weeks of age in mdx mice, the animal model of DMD. Accordingly, we hypothesized that the pathology observed with dystrophin deficiency may be developmentally regulated. Initially, we demonstrated that profound siRNA-mediated dystrophin knockdown could be achieved in mouse primary muscle cultures. The use of adeno-associated virus (AAV) vectors to express shRNAs targeting dystrophin in skeletal muscle in vivo yielded a potent and specific dystrophin knockdown, but only after 5 months, indicating the very long half-life of dystrophin. Interestingly, and in contrast to what is observed in congenital dystrophin deficiency, long-term (1 year) dystrophin knockdown in adult mice did not result, per se, in overt dystrophic pathology or up-regulation of utrophin. This supports our hypothesis and suggests new pathophysiology for the disease. Furthermore, taking into account the rather long half-life of dystrophin, and the notion that the development of pathology is age-dependent, it indicates that a single gene therapy approach before the onset of pathology might convey a long term cure for DMD.
27 - Long-Term Benefit of Adeno-Associated Virus/Antisense-Mediated Exon Skipping in Dystrophic Mice
27 - (Journal of Speech, Language, and Hearing Research Vol.51 586-595 June 2008) Laryngeal Muscles Are Spared in the Dystrophin Deficient mdx Mouse
Purpose: Duchenne muscular dystrophy (DMD) is caused by the loss of the cytoskeletal protein, dystrophin. The disease leads to severe and progressive skeletal muscle wasting. Interestingly, the disease spares some muscles. The purpose of the study was to determine the effects of dystrophin deficiency on 2 intrinsic laryngeal muscles, the posterior cricoarytenoid and the thyroarytenoid, in the mouse model. Method: Larynges from dystrophin-deficient mdx and normal mice were examined histologically. Results: Results demonstrate that despite the absence of dystrophin in the mdx laryngeal muscles, membrane damage, inflammation, necrosis, and regeneration were not detected in the assays performed. Conclusions: The authors concluded that these muscles are 1 of only a few muscle groups spared in this model of dystrophin deficiency. The muscles may count on intrinsic and adaptive protective mechanisms to cope with the absence of dystrophin. Identifying these protective mechanisms may improve DMD management. The study also highlights the unique aspects of the selected laryngeal skeletal muscles and their dissimilarity to limb skeletal muscle.
A) C57 sham injected mouse. KI= 3.86 B) mdx sham injected. KI=3.05 C) mdx AO treated. KI=3.65
21 - (Am J Physiol Cell Physiol, May 2008) Poloxamer 188 reduces the contraction-induced force decline in lumbrical muscles from mdx mice
Duchenne Muscular Dystrophy is a genetic disease caused by the lack of the protein dystrophin. Dystrophic muscles are highly susceptible to contraction-induced injury, and following contractile activity, have disrupted plasma membranes that allow leakage of calcium ions into muscle fibers. Because of the direct relationship between increased intracellular calcium concentration and muscle dysfunction, therapeutic outcomes may be achieved through the identification and restriction of calcium influx pathways. Our purpose was to determine the contribution of sarcolemmal lesions to the force deficits caused by contraction-induced injury in dystrophic skeletal muscles. Using isolated lumbrical muscles from dystrophic (mdx) mice, we demonstrate for the first time that P188, a membrane sealing poloxamer, is effective in reducing the force deficit in a whole mdx skeletal muscle. A reduction in force deficit was also observed in mdx muscles that were exposed to a calcium-free environment. These results, coupled with previous observations of calcium entry into mdx muscle fibers during a similar contraction protocol, support the interpretation that extracellular calcium enters through sarcolemmal lesions and contributes to the force deficit observed in mdx muscles. The results provide a basis for potential therapeutic strategies directed at membrane stabilization of dystrophin-deficient skeletal muscle fibers.
12 - (PNAS, 105 (19): 7028-33) Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency
M. Khairallah, R. J. Khairallah, M. E. Young, B. G. Allen, M. A. Gillis, G. Danialou, C. F. Deschepper, B. J. Petrof, and C. Des Rosiers - Canada
We recently demonstrated early metabolic alterations in the dystrophin- deficient mdx heart that precede overt cardiomyopathy and may represent an early ‘‘subclinical’’ signature of a defective nitric oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC+/0). When perfused ex vivo in the working mode, 12- and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC+/0. At the metabolic level, mdx/GC+/0 displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.
5- (The American Journal of Pathology, May 2008) L-Arginine Decreases Inflammation and Modulates the Nuclear Factor-kB/Matrix Metalloproteinase Cascade in Mdx Muscle Fibers
Duchenne muscular dystrophy (DMD) is a lethal,
X-linked disorder associated with dystrophin deficiency that results
in chronic inflammation, sarcolemma damage, and severe skeletal
muscle degeneration. Recently, the use of L-arginine, the substrate
of nitric oxide synthase (nNOS), has been proposed as a pharmacological
treatment to attenuate the dystrophic pattern of DMD. However,
little is known about signaling events that occur in dystrophic
muscle with L-arginine treatment. Considering the implication of
inflammation in dystrophic processes, we asked whether L-arginine
inhibits inflammatory signaling cascades. We demonstrate that
L-arginine decreases inflammation and enhances muscle regeneration in
the mdx mouse model. Classic stimulatory signals, such as
proinflammatory cytokines interleukin-1,
interleukin-6, and tumor necrosis factor-
,
are significantly decreased in mdx mouse muscle, resulting in lower
nuclear factor (NF)-
B
levels and activity. NF-B
serves as a pivotal transcription factor with multiple levels of
regulation; previous studies have shown perturbation of NF-B
signaling in both mdx and DMD muscle. Moreover, L-arginine decreases
the activity of metalloproteinase (MMP)-2 and MMP-9, which are
transcriptionally activated by NF-B.
We show that the inhibitory effect of L-arginine on the NF-B/MMP
cascade reduces
-dystroglycan
cleavage and translocates utrophin and nNOS throughout the sarcolemma.
Collectively, our results clarify the molecular events by which
L-arginine promotes muscle membrane integrity in dystrophic muscle
and suggest that NF-B-related
signaling cascades could be potential therapeutic targets for
DMD management.
APRIL
29 - SUMMIT REPORTS PROGRESS OF DUCHENNE MUSCULAR DYSTROPHY PROGRAMME AT INTERNATIONAL CONFERENCE
29 - (Thorax, May 2008; 63: 430 - 434) Effect of non-invasive ventilation on respiratory muscle loading and endurance in patients with Duchenne muscular dystrophy
M Toussaint1,
P Soudon1,
W Kinnear2 1
Centre for Home Mechanical Ventilation, Ziekenhuis Inkendaal, Vlezenbeek,
Belgium
2 Department of Respiratory Medicine, University Hospital, Nottingham,
UK
Background: Respiratory muscle weakness in patients with Duchenne muscular dystrophy (DMD) leads to respiratory failure for which non-invasive positive pressure ventilation (NIPPV) is an effective treatment. This is used initially at night (n-NIPPV) but, as the disease progresses, diurnal use (d-NIPPV) is often necessary. The connection between NIPPV and relief of respiratory muscle fatigue remains unclear. A study was undertaken to determine the extent to which n-NIPPV and d-NIPPV unload the respiratory muscles and improve respiratory endurance in patients with DMD.
Methods: Fifty patients with DMD were assessed at 20.00 and 08.00 h. More severely affected patients with nocturnal hypoventilation received n-NIPPV; those with daytime dyspnoea also received d-NIPPV via a mouthpiece (14.00–16.00 h). Lung function, modified Borg dyspnoea score, spontaneous breathing pattern, tension-time index (TT0.1 = occlusion pressure (P0.1)/maximum inspiratory pressure (MIP) x duty cycle (Ti/Ttot)) and respiratory muscle endurance time (Tlim) against a threshold load of 35% MIP were measured.
Results: More severe respiratory muscle weakness was associated with a higher TT0.1 and lower Tlim. In contrast to non-dyspnoeic patients, patients with dyspnoea (Borg score >2.5/10) showed an increase in Tlim and decrease in TT0.1 after n-NIPPV. At 16.00 h, immediately after d-NIPPV, patients with dyspnoea had lower TT0.1 and Borg scores with unchanged Tlim. Compared with the control day without d-NIPPV, TT0.1, Borg scores and Tlim were all improved at 20.00 h.
Conclusions: In patients with dyspnoea with DMD, the load on respiratory muscles increases and endurance capacity decreases with increasing breathlessness during the day, and this is reversed by n-NIPPV. An additional 2 h of d-NIPPV unloads respiratory muscles and reverses breathlessness more effectively than n-NIPPV alone.
26 - A canine minidystrophin is functional and therapeutic in mdx mice
25 - (Neuromuscular Disorders, 2008) Dystrophin-deficient cardiomyopathy in mouse: Expression of Nox4 and Lox are associated with fibrosis and altered functional parameters in the heart
Christopher F. Spurney Susan Knoblach , Emidio E. Pistilli ,Kanneboyina Nagaraju , Gerard R. Martin , Eric P. Hoffman - USA
Duchenne muscular dystrophy (DMD; dystrophin-deficiency) causes dilated cardiomyopathy in the second decade of life in affected males. We studied the dystrophin-deficient mouse heart (mdx) using high-frequency echocardiography, histomorphometry, and gene expression profiling. Heart dysfunction was prominent at 9–10 months of age and showed significantly increased LV internal diameter (end systole) and decreased posterior wall thickness. This cardiomyopathy was associated with a 30% decrease in shortening fraction. Histologically, there was a 10-fold increase in connective tissue volume (fibrosis). mRNA profiling with RT-PCR validation showed activation of key pro-fibrotic genes, including Nox4 and Lox. The Nox gene family expression differed in mdx heart and skeletal muscle, where Nox2 was specifically induced in skeletal muscle while Nox4 was specifically induced in heart. This is the first report of an altered profibrotic gene expression profile in cardiac tissue of dystrophic mice showing echocardiographic evidence of cardiomyopathy.
25 - (American Thoracic Society
Meeting, Toronto, May - 2008) Clinical Profiles and Survival in Duchenne
Muscular Dystrophy
M. Kohler, M.D., C.F. Clarenbach, M.D., C. Bahler, E.W. Russi, M.D.,
K.E. Bloch, M.D., Zurich, Switzerland
Rationale: Duchenne muscular dystrophy (DMD) leads to progressive
impairment of muscle function, respiratory failure and premature death.
Longitudinal data on the course of physical disability and respiratory function
are sparse.
Objectives: To prospectively assess physical impairment and disability,
respiratory function and survival in DMD patients over several years in order to
accurately describe the course of the disease.
Methods: In 43 patients with DMD, aged 5-35 years, physical disability
was assessed yearly by the Duchenne muscular dystrophy physical Impairment and
Dependence on care (DID) score ranging from 9 (no disability) to 80 (complete
dependence). Forced vital capacity (FVC) and survival were also recorded. The
mean
SD
observation period was 5.4
2.1
years. Inter-observer-agreement of the DID score was tested in 40 patients.
Measurements and main results: DID scores were correlated with age
according to a hyperbolic function (f=85.33*age/(10.05+age), R=0.62, P<0.0001).
FVC revealed an exponential decline with age (f=145.30*e(-0.08*age)),
R=0.55, P<0.0001. Mean age
SD
at the beginning of assisted positive-pressure ventilation was 19.8
3.9
(range 14 to 31) years. Three patients deceased during the observation period.
Kaplan-Meier analysis revealed a median survival of 35 years.
Inter-observer-agreement of the DID score was excellent (bias
2SD=2
5
points).
Conclusions: In DMD, physical disability and respiratory function are
strongly related to age, which allows the expected clinical course to be
predicted. Compared to historical data, survival has considerably improved,
probably due to treatment with assisted positive pressure ventilation.
23 - (Neuromuscular Disorders,
2008) Corticosteroid treatment retards development of ventricular
dysfunction in Duchenne muscular dystrophy
Larry W. Markham, Kathi Kinnett, Brenda L. Wong, D. Woodrow Benson, Linda H. Cripe - USA
Duchenne muscular dystrophy (DMD) is characterized by a predictable decline in cardiac function with age that contributes to early death. Although corticosteroids are a clinically effective pharmacologic therapy for skeletal muscle function, there is limited published work documenting the impact on cardiac function. The primary objective of this work is to determine benefit from steroid treatment on the development of ventricular dysfunction in DMD. We performed a historical cohort study of DMD cases undergoing serial cardiac evaluations from 1998–2006. In addition to the history of steroid use, basic medical characteristics and serial echocardiographic measures were obtained for each identified case meeting inclusion criteria. Data from initial (7.5 ± 0.8 years) and follow-up (12 ± 0.7 years) evaluation was collected from untreated (n = 23) and steroid treated (n = 14) DMD cases. Kaplan–Meier freedom from ventricular dysfunction was 93% for steroid treated cases versus 53% for untreated cases at 1500 days. Treatment with steroids was protective against ventricular dysfunction (Hazard ratio 0.16 95% CI 0.04, 0.70). We demonstrate here that steroid treatment, begun prior to ventricular dysfunction retards the anticipated development of ventricular dysfunction.
22 - (Human Molecular Genetics, 2008) Major basic protein-1 promotes fibrosis of dystrophic muscle and attenuates the cellular immune response in muscular dystrophy
The immune response to dystrophin-deficient muscle promotes the pathology of Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD. In this investigation, we find that the release of major basic protein (MBP) by eosinophils is a prominent feature of DMD and mdx dystrophy and that eosinophils lyse muscle cells in vitro by the release of MBP-1. We also show that eosinophil depletions of mdx mice by injections of anti-chemokine receptor-3 reduce muscle cell lysis, although lysis of mdx muscle membranes is not reduced by null mutation of MBP-1 in vivo. However, ablation of MBP-1 expression in mdx mice produces other effects on muscular dystrophy. First, fibrosis of muscle and hearts, a major cause of mortality in DMD, is greatly reduced by null mutation of MBP-1 in mdx mice. Furthermore, either ablation of MBP-1 or eosinophil depletion causes large increases in cytotoxic T-lymphocytes (CTLs) in mdx muscles. The increase in CTLs in MBP-1-null mice does not reflect a general shift toward a Th1 inflammatory response, because the mutation had no significant effect on the expression of interferon-gamma, inducible nitric oxide synthase or tumor necrosis factor. Rather, MBP-1 reduces the activation and proliferation of splenocytes in vitro, indicating that MBP-1 acts in a more specific immunomodulatory role to affect the inflammatory response in muscular dystrophy. Together, these findings show that eosinophil-derived MBP-1 plays a significant role in regulating muscular dystrophy by attenuating the cellular immune response and promoting tissue fibrosis that can eventually contribute to increased mortality.
20 - Emergent Dilated Cardiomyopathy Caused by Targeted Repair of Dystrophic Skeletal Muscle
19 - ABSTRACTS PRESENTED IN ANNUAL MEETING OF AMERICAN ACADEMY OF NEUROLOGY, CHICAGO, 2008/APRIL
16 - (Abstracts of the P2T Congres de Physiologie,de Pharmacologie et de Therapeutique, 2008 - Fundamental & Clinical Pharmacology, 22:1-102, 2008) Effect of L-arginine, a NOS substrate, on mdx mouse diaphragm
K Hnia, J Gayraud, A Lacampagne, G Hugon, M Ramonatxo, D Mornet, S Matecki - France
Introduction: L-arginine was proposed as a pharmacological tool in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease due to a mutation in the dystrophin gene. Administration of L-arginine (the substrate of nitric oxide synthase) promotes a better muscle membrane integrity in mdx mice, the animal model of the muscle dystrophin-deficiency. Despite the beneficial effect on L-arginine on muscle weakness and force, mechanisms by each this molecule acts on muscle remain poorly investigated. Methods: Five-week-old Male dystrophin-deficient Animals (mdx control; n = 7 and mdx treated; n = 7) were treated for 2 weeks with intraperitoneal injections. Control group was injected with (Ss) physiological saline solution (mdx-Ss). Treated group was injected with L-arginine, (Sigma), 20 ll/vol/g at a Cc of 200 mg/kg (mdx-L-arg). After beheading, the diaphragm was dissected into 2 parts. One part was immediately frozen in liquid nitrogen-cooled isopentane and stored at -80C and the other part was used freshly to determine enzymatic activities, Ca2+-spark’s properties and resistance to eccentric contractions. Results: Here, we showed that L-arginine improve RyR gating mechanisms with a decreased inflammatory secreted cytokine IL-1alpha, IL-6 and TNFalpha targeted to dystrophic muscle fibres. This leads to decrease level and activity of NF-kB and its targeted proteins such as the muscle specific metalloproteinase MMP-2 and MMP-9 which are responsible of the betadystroglycan cleavage into a ~30 kDa form in mdx muscles. The betadystroglycan is a key transmembrane glycoprotein of the dystrophin glycoprotein complex which anchors utrophin to sarcolemma. Conclusion: Our results demonstrate that L-arginine administration promotes a better membrane stability of beta-dystroglycan/utrophin couple and re-localizes nNOS in subsarcolemmal compartment of the dystrophic fibres which leads to improve dystrophic pattern in mdx diaphragm with increase resistance to contraction-induced mechanical stress. Our data strengthen the usefulness of L-arginine as a powerful pharmacological tool in Duchenne muscular dystrophies and also enlarge its use in other muscle-inflammation mediated myopathies.
12 - (European Journal of Paediatric Neurology, 2008) Effect of deflazacort on cardiac and sternocleidomastoid muscles in Duchenne muscular dystrophy: A magnetic resonance imaging study
Sophie Mavrogeni, Antigoni Papavasiliou, Marouso Douskou, Genovefa Kolovou, Evangelia Papadopoulou, Dennis V. Cokkinos - Greece.
Objective: To evaluate the involvement of cardiac and sternocleidomastoid muscles by magnetic resonance imaging (MRI) measurement of T2 relaxation time and the left ventricular systolic function in patients with Duchenne muscular dystrophy (DMD) on treatment with deflazacort and compare them with DMD patients without treatment. Subjects: Seventeen patients with DMD (aged 17–22 years) on treatment with deflazacort for at least 7 years and 17 boys with DMD of younger age (12–15 years) without steroid treatment. All patients were free of cardiac or respiratory symptoms and had normal ECG and Holter monitor examination. Methods: T2 relaxation time of the myocardium (H), left (SCM-L) and right sternocleidomastoid (SCM-R) muscles and left ventricular systolic function were evaluated by magnetic resonance imaging (MRI) in two groups of DMD patients. Myocardial and sternocleidomastoid muscles T2 relaxation time was calculated using 16 TEs (10–85 msec) and TR at least 2000ms and T2 maps were created. Results: DMD on deflazacort had higher T2 relaxation time values of the heart and of both sternocleidomastoid muscles (T2H median (range): 47 (41–48) vs. 33 (31–37) ms, po0.001, T2 SCM-L median (range): 35 (30–37) vs. 23 (20–26) ms, po0.001, T2 SCM-R median (range): 35 (32–37) vs. 23 (20–27) ms, po0.001) and left ventricular systolic function (LVEDV median (range): 95 (75–120) vs. 90 (80–105) ml, p ¼ 0.03, LVESV median (range): 45 (38–55) vs. 47 (41–51) ml, p ¼ 0.81(NS), LVEF median (range): 53% (51–57) vs. 48% (42–51), po0.001) compared to DMD without treatment. Conclusions: DMD patients on deflazacort are characterized by better preservation of the T2 relaxation time of myocardium and sternocleidomastoid muscles and better LV systolic function. The duration of this beneficial effect needs to be studied prospectively.
12 - (Journal of the American Society of Echocardiography, 2008) Early Regional Myocardial Dysfunction in Young Patients With Duchenne Muscular Dystrophy
Luc Mertens, Javier Ganame, Piet Claus, Nathalie Goemans, Daisy Thijs, Bénédicte Eyskens, David Van Laere, Bart Bijnens, Jan D'hooge, George R. Sutherland, Gunnar Buyse
5 - The therapeutic potential of antisense-mediated exon skipping
5 - Special Report on DMD-BMD Research
MARCH
30 - (Neuromuscular Disorders, 2008) Reduced muscle necrosis and long-term benefits in dystrophic mdx mice after cV1q (blockade of TNF) treatment
Hannah G. Radley, Marilyn J. Davies, Miranda D. Grounds - Australia
Tumour necrosis factor (TNF) is a potent inflammatory cytokine that appears to exacerbate damage of dystrophic muscle in vivo. The monoclonal murine specific antibody cV1q that specifically neutralises murine TNF demonstrated significant anti-inflammatory effects in dystrophic mdx mice. cV1q administration protected dystrophic skeletal myofibres against necrosis in both young and adult mdx mice and in adult mdx mice subjected to 48 h voluntary wheel exercise. Long-term studies (up to 90 days) in voluntarily exercised mdx mice showed beneficial effects of cV1q treatment with reduced histological evidence of myofibre damage and a striking decrease in serum creatine kinase levels. However, in the absence of exercise long-term cV1q treatment did not reduce necrosis or background pathology in mdx mice. An additional measure of well-being in the cV1q treated mice was that they ran significantly more than control mdx mice.
30 - (Developmental Medicine & Child Neurology, Volume 50, Issue s113, 49-59, 2008) PEDIATRIC PATIENTS WITH DUCHENNE MUSCULAR DYSTROPHY
Fiona Adams, Susan Bostock, Angela Potter - Australia
Objective: This review aims to give recommendations for best respiratory management (and associated costs) for pediatric patients with Duchenne Muscular Dystrophy, in accordance with evidence in the literature and usual practice in Australia and overseas.
Design: Narrative review of the literature, and a survey of usual practice.
Method: Database search using Medline, CINAHL, PubMed, Cochrane and PEDro from 2003–2007. A survey was conducted amongst paediatric physiotherapists (and 2 physiotherapists working with adults), seeking information (including protocols) on usual management. Liaison with professionals regarding cost and training for recommended interventions also took place.
Results: The interventions supported by the strongest current evidence include mechanical in-exsufflation (MI-E), intrapulmonary percussive ventilation (IPV), nocturnal noninvasive ventilation (NIV), manually assisted cough (MAC), breath- stacking, glossopharyngeal breathing (GPB) and mechanical insufflation. The use of PEP treatment, chest physiotherapy, autogenic drainage and IPPB are supported by low-level evidence, while suction has little and conflicting evidence. Survey findings and protocols indicate majority of practitioners use postural drainage, percussion, MAC, manual insufflation, MI-E, suction and NIV, yet very few include IPV in management of clients with DMD. Technique application is variable depending on patient and physiotherapist. Implementation of MI-E is costly but time efficient. Conclusion: There remains a lack of strong evidence for certain techniques commonly used for children with DMD, such as postural drainage, percussion and suction. Due to strong evidence showing benefit, MI-E , MAC and nocturnal NIV should be continued in practice, while IPV, breath stacking, GPB and mechanical insufflation should be re-considered for implementation (if not already used).
30 - (Neuropathology, Volume 28, Issue 2, Page 177-226, 2008) Progress on Molecular Therapy toward Muscular Dystrophy
S Takeda - Japan
Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder of striated muscle caused by mutations in the DMD gene. A recombinant adeno-associated virus (AAV)-mediated gene transfer is one of attractive approaches to the treatment of DMD. To examine therapeutic effects and the safety issue of the approach in larger animal models, we recently established a Beagle-based dystrophic dog colony in Japan, CXMDJ. First, we injected a recombinant AAV2 encoding the LacZ gene (rAAV2- CMVLacZ) into skeletal muscles of normal Beagles. b-galactosidase (b-gal) was expressed only in a few fibers, together with marked cellular infiltration. Immunosuppressants improved b-gal expression, though the effect was not complete (Gene Ther. 14:1249–1260, 2007). We, then, generated a type 8 recombinant AAV (rAAV8)-CMVLacZ, and injected it into skeletal muscle of normal Beagles. Importanly, we found more b-galpositive fibers in AAV8-injected muscles than in AAV2-injected muscles. We, therefore, injected recombinant AAV8 encoding a canine microdystrophin (cDCS1) gene, a truncated version of the dystrophin gene into skeletal muscle of CXMDJ and confirmed widespread expression of micro-dystrophin at the sarcolemma. We also demonstrate that the administration of recombinant AAV8 encoding DCS1 through limb perfusion is a feasible approach to DMD.
21 - Fidelity of Gamma-Glutamyl Transferase (GGT) in Differentiating Skeletal Muscle From Liver Damage
19 - (Am Heart J 2008;0:1-7.) Characteristics and outcomes of cardiomyopathy in children with Duchenne or Becker muscular dystrophy: A comparative study from the Pediatric Cardiomyopathy Registry
David M. Connuck, Lynn A. Sleeper, Steven D. Colan, Gerald F. Cox MD, , Jeffrey A. Towbin , April M. Lowe , James D. Wilkinson , E. John Orav , Leigha Cuniberti , Bonnie A. Salbert , Steven E. Lipshultz and for the Pediatric Cardiomyopathy Registry Study Group - USA
Objective:
The aim of this study was to determine in pediatric Duchenne (DMD) and Becker muscular dystrophy (BMD) or other dilated cardiomyopathies (ODCM) whether outcomes differ by diagnosis. Background: Children with dilated cardiomyopathy are treated as a single undifferentiated group. Methods: This cohort study of 128 children with DMD, 15 with BMD, and 312 with ODCM uses outcome measures of left ventricular (LV) size and function, death, heart transplant, and death or transplant. Results: At cardiomyopathy diagnosis, the DMD and BMD groups had similar mean ages (14.4 and 14.6 years), prevalence of congestive heart failure (CHF) (30% and 33%), and LV fractional shortening (FS) Z-scores (median, −5.2 for DMD and −6.7 for BMD). The BMD group had more severe mitral regurgitation (P = .05) and a higher mean LV enddiastolic dimension Z-score than the DMD group (2.9 ± 1.5 vs 1.2 ± 1.9, P = .002). Duchenne muscular dystrophy group survival was lower than in BMD or ODCM groups (P = .06) at 5 years (57%, 100%, and 71%, respectively). In BMD, 25% received cardiac transplants within 0.4 years of cardiomyopathy diagnosis. The combined DMD and BMD group had less LV dilation and a closer-to-normal LV FS at cardiomyopathy diagnosis than the ODCM group. After 2 years, LV dilation increased, and LV FS did not change in the combined DMD and BMD group; for ODCM patients, LV dilation did not progress, and LV FS improved. Conclusions: Children with DMD and cardiomyopathy have a higher mortality. Becker muscular dystrophy has a high heart transplantation rate in the 5 years after diagnosis of cardiomyopathy. Serial echocardiography demonstrates a different disease course for DMD and BMD patients compared with ODCM patients.16 - (Nature Medicine, March, 2008) Genetic and pharmacologic inhibition of mitochondrial dependent necrosis attenuates muscular dystrophy
Douglas P Millay, Michelle A Sargent, Hanna Osinska, Christopher P Baines, Elisabeth R Barton, Gre´goire Vuagniaux, H Lee Sweeney, Jeffrey Robbins
& Jeffery D Molkentin - USAMuscular dystrophies comprise a diverse group of genetic
disorders that lead to muscle wasting and, in many instances, premature death.
Many mutations that cause muscular dystrophy compromise the support network that
connects myofilament proteins within the cell to the basal lamina outside the
cell, rendering the sarcolemma more permeable or leaky. Here we show that
deletion of the gene encoding cyclophilin D (Ppif) rendered mitochondria
largely insensitive to the calcium overload–induced swelling associated with a
defective sarcolemma, thus reducing myofiber necrosis in two distinct models of
muscular dystrophy. Mice lacking
-sarcoglycan
(Scgd-/- mice) showed markedly less dystrophic disease in both
skeletal muscle and heart in the absence of Ppif. Moreover, the premature
lethality associated with deletion of Lama2, encoding the
-2
chain of laminin-2, was rescued, as were other indices of dystrophic disease.
Treatment with the cyclophilin inhibitor Debio-025 similarly reduced
mitochondrial swelling and necrotic disease manifestations in mdx mice, a
model of Duchenne muscular dystrophy, and in Scgd-/- mice.
Thus, mitochondrial-dependent necrosis represents a prominent disease mechanism
in muscular dystrophy, suggesting that inhibition of cyclophilin D could provide
a new pharmacologic treatment strategy for these diseases.
New Treatment Strategy Possible For Muscular Dystrophy, Mouse Studies Show
15 - (J. Neurol. Neurosurg. Psychiatry 2008;79;366-367) Creatine treatment in muscle disorders: a meta-analysis of randomised controlled trials
R A Kley, M A Tarnopolsky, M Vorgerd - Canada
Creatine is a naturally occurring amino acid derivative that is obtained by endogenous synthesis and dietary intake. Creatine phosphate has a high phosphoryl group transfer potential and serves as an ATP buffer during muscle contraction. Creatine supplementation increases muscle strength in healthy persons.
CONCLUSIONS
There is evidence from randomised controlled trials that short and medium term creatine treatment improves muscle strength, increases lean body mass and is well tolerated in patients with muscular dystrophies. The available evidence does not support.the use of creatine in metabolic myopathies. It is noteworthy that high dose creatine supplementation increased muscle pain in glycogenosis type V. There is a need for long term creatine studies in muscular dystrophies and creatine trials in hereditary myopathies that have not been targeted for evaluation so far such as myofibrillar myopathies and fatty acid oxidation defects. Furthermore, the effect of creatine supplementation on activities of daily living has to be evaluated.
15 - Standards of care for Duchenne muscular dystrophy - Brief TREAT-NMD recommendations
15 - A phase I/IItrial of MYO-029 in adult subjects with muscular dystrophy
11 - (PNAS, 2008) Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors
Increasing the size and strength of muscles represents a promising therapeutic strategy for musculoskeletal disorders, and interest has focused on myostatin, a negative regulator of muscle growth. Various myostatin inhibitor approaches have been identified and tested in models of muscle disease with varying efficacies, depending on the age at which myostatin inhibition occurs. Here, we describe a one-time gene administration of myostatin-inhibitor-proteins to enhance muscle mass and strength in normal and dystrophic mouse models for >2 years, even when delivered in aged animals. These results demonstrate a promising therapeutic strategy that warrants consideration for clinical trials in human muscle diseases.
11 - Two-tiered hypotheses for Duchenne muscular dystrophy
FEBRUARY
26 - Disodium cromoglycate protects dystrophin-deficient muscle fibers from leakiness
14 - (Pediatric Neurology, Volume 38, Issue 3, March 2008, Pages 200-206) Deflazacort Use in Duchenne Muscular Dystrophy: An 8-Year Follow-Up
Sylvie Houde, Michèle Filiatrault, Anne Fournier, Julie Dubé, Sylvie D’Arcy, Denis Bérubé, Yves Brousseau, Guy Lapierre and Michel Vanasse - Canada
Data reported here were collected over an 8-year period for 79 Duchenne muscular dystrophy patients, 37 of whom were treated with deflazacort. Mean length of treatment was 66 months. Treated boys stopped walking at 11.5 + 1.9 years, compared with 9.6 + 1.4 years for untreated boys. Cardiac function was better preserved with the use of deflazacort, as shown by a normal shortening fraction in treated (30.8 + 4.5%) vs untreated boys (26.6 + 5.7%, P < 0.05), a higher ejection fraction (52.9 + 6.3% treated vs 46 + 10% untreated), and lower frequency of dilated cardiomyopathy (32% treated vs 58% untreated). Scoliosis was much less severe in treated (14 + 2.5°) than in untreated boys (46 + 24°). No spinal surgery was necessary in treated boys. Limb fractures were similarly frequent in treated (24%) and untreated (26%) boys, but vertebral fractures occurred only in the treated group (7/37) (compared with zero for the untreated group). In both groups, body weight excess tripled between the ages of 8 and 12 years. All untreated patients grew normally (>4 cm/year), as opposed to only 15% of treated boys. Deflazacort improves cardiac function, prolongs walking, and seems to eliminate the need for spinal surgery, although vertebral fractures and stunted growth occur. The overall impact on quality of life appears positive.
8 - (Am J Physiol Heart Circ Physiol, Feb 2008) Functional resolution of fibrosis in mdx mouse dystrophic heart and skeletal muscle by halofuginone
The effect of halofuginone (Halo) on established fibrosis in older mdx dystrophic muscle was investigated. Mice (8-9-months) treated with Halo (or saline in controls) for 5, 10 or 12 weeks were assessed weekly for grip strength and voluntary running. Echocardiography was performed at 0, 5, and 10 weeks. Respiratory function and exercise-induced muscle damage were tested. Heart, quadriceps, diaphragm and tibialis anterior muscles were collected to study fibrosis, collagen I and III expression, collagen content using a novel collagenase-digestion method, and cell proliferation. Hepatocyte growth factor (HGF) and alpha-smooth muscle actin (SMA) proteins were assayed in quadriceps. Halo decreased fibrosis, collagen I and III expression, collagen protein and SMA content after 10 weeks treatment. Muscle-cell proliferation increased at 5 weeks, and HGF increased by 10 weeks treatment. Halo markedly improved cardiac function and respiratory function, and reduced damage and improved recovery from exercise. The overall impact of already-established dystrophy in cardiac and skeletal muscles was reduced. Marked improvements in vital-organ functions implicate Halo as a strong candidate drug to reduce morbidity and mortality in DMD.
7 - (J. Physiol., Feb 2008) N-acetylcysteine ameliorates skeletal muscle pathophysiology in mdx mice
Nicholas Paul Whitehead, Catherine Pham, Othon L Gervasio, and David G. Allen - Australia
Duchenne muscular dystrophy (DMD) is a severe degenerative muscle disease caused by a mutation in the gene encoding dystrophin, a protein linking the cytoskeleton to the extracellular matrix. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) provided protection against dystrophic muscle damage in the mdx mouse, an animal model of DMD. In isolated mdx muscles, NAC prevented the increased membrane permeability and reduced the force deficit associated with stretch-induced muscle damage. Three week old mdx mice were treated with NAC in the drinking water for 6 weeks. Dihydroethidium staining showed that NAC treatment reduced the concentration of reactive oxygen species (ROS) in mdx muscles. This was accompanied by a significant decrease in centrally nucleated fibres in muscles from NAC-treated mdx mice. Immunoblotting showed that NAC treatment decreased the nuclear protein expression of NF-B, a transcription factor involved in pro-inflammatory cytokine expression. Finally, we show that NAC treatment reduced caveolin-3 protein levels and increased the sarcolemmal expression of -dystroglycan and the dystrophin homologue, utrophin. Taken together, our findings suggest that ROS play an important role in the dystrophic pathogenesis, both in terms of activating damage pathways and in regulating the expression of some dystrophin-associated membrane proteins. These results offer the prospect that antioxidants such as NAC could have therapeutic potential for DMD patients.
7 - Glucocorticoid corticosteroids for Duchenne muscular dystrophy
2 - (Clinica Chimica Acta, 2008) Wide ranges of serum myostatin concentrations in Duchenne muscular dystrophy patients - Letter to the editor
Hiroyuki Awano, Yasuhiro Takeshima,
Yo Okizuka, Kayoko Saiki, Mariko Yagi, Masafumi Matsuo - JapanDuchenne muscular dystrophy (DMD), a common inherited myopathy that affects approximately 1 in 3,500 males, is characterized by progressive muscle wasting due to a deficiency in muscle dystrophin. DMD progresses with a rather uniform pattern of muscle weakness; i.e., DMD causes affected individuals to lose their ability to walk by the age of 12 y, and patients succumb during their twenties due to either respiratory or cardiac failure. The deficiency in dystrophin is caused by translational reading frame shift or nonsense mutations in the dystrophin gene [1]. However, the existence of a modifying gene has been suggested by the identification of unusually mild DMD phenotypes [2-4]. Although some phenotypic variability may arise due to environmental factors, such as diet or exercise, genetic components are likely to contribute to this variability. Myostatin, also known as growth and differentiation factor 8 (GDF8), is a muscle-specific secreted peptide that limits muscle growth [5,6]. However, genotyping of the myostatin gene failed to disclose any nucleotide changes that behaved as a phenotypic modifier of DMD [7]. Remarkably, blocking endogenous myostatin has been shown to result in anatomic, biochemical, and physiologic improvements in the dystrophic phenotype of mdx mice, a mouse model for DMD, including particularly prominent enlarged fiber diameters and greatly reduced fatty fibrosis [8-10]. These results suggest that blocking endogenous myostatin is a potential strategy to treat DMD [11]. We examined the hypothesis that serum myostatin is increased in DMD, thereby enabling treatment by myostatin blockage. Forty-two DMD patients followed at Kobe University Hospital were enrolled in this study. All but 1 of the mutations in the dystrophin gene were found to introduce premature stop codons in the dystrophin mRNA: 24 cases with mutations that induced a translational reading frame-shift due to exon deletion or duplication; 5 cases with nonsense mutations; 7 cases with exon mutations involving one or a few nucleotides deleted or inserted; 3 cases with intron mutations that induced splicing errors; and one case with an abnormal chromosome (Table 1). The subjects’ ages ranged from 1 to 22 y (average: 8.3 y). Regular clinical checkups, including determination of serum creatine kinase (CK) concentrations, were performed at the outpatient clinic. All protocols were approved by the ethics committee of the Kobe University School of Medicine. Blood samples were taken after written informed consent was obtained from all patients, and serum was separated using a clinical centrifuge. Serum myostatin was measured using the Human Myostatin ELISA (Prodomain Specific) kit purchased from BioVendor Laboratory Medicine, Inc. (Bmo, Czech Republic). The upper limit of determination was 50 ng/ml, and normal adults have serum concentrations of 0.19 to 9.02 ng/ml (BioVendor Laboratory Medicine, Inc.). The Pearson product-moment coefficient was calculated to quantify the relationship. Serum myostatin concentrations in DMD patients ranged from 1.1 to >50 ng/dl (Table 1). Remarkably, 13 samples were >50 ng/ml, and the lowest concentration was 1.1 ng/ml (Table 1). Though age differences were examined in 2 conditions either including or deleting 13 samples with >50 ng/ml, no significant correlationship between age and serum myostatin concentration was found (Fig. 1). We next examined whether concentrations of myostatin were related to the type or location of mutations in the dystrophin gene. Though serum myostatin concentrations were compared based on their mutation types (exon deletion/duplication or others), no clear difference between two groups was revealed (Fig. 1). There found no significant difference in serum myostatin concentration between patients with mutation in the 5’ and 3’ regions of the dystrophin gene (Table 1). Considering that myostatin is an inhibitor of muscle growth, cases with high serum myostatin concentrations were predicted to present rather severe phenotypes. Transgenic overexpression of myostatin in mice was shown to result in cachexia [12]. However, the ages when DMD patients with high myostatin concentrations became wheelchair-bound were not different from those of patients with low myostatin concentrations, and signs of muscle weakness appeared mostly between ages 4 and 5. Furthermore, serum CK concentrations were not significantly lower in DMD cases with high myostatin concentrations than in those with low concentrations (Table 1). This indicates that serum myostatin did not modify the DMD phenotype even though blocking endogenous myostatin has been shown to result in improvements in the dystrophic phenotype of mdx mice [8-10]. In this study, we measured myostatin that reacted with a monoclonal antibody recognizing the prodomain of myostatin. Considering that myostatin is secreted as an inactive propeptide and is cleaved to produce the active form, further studies would be required to measure active or latent myostatin individually. Expression of the myostatin gene has been examined previously in skeletal muscle by measuring mRNA and protein concentrations [13]. Serum myostatin concentration has been determined by the Western blot analysis in a patient with a mutation in the myostatin gene, disclosing an absence of myostatin [14]. Furthermore the Western blot analysis disclosed that serum myostatin concentration was lower than that of rat [14]. But no further study has been conducted on serum myostatin concentrations. Our results disclosed a wide range in serum myostatin concentrations in DMD patients. Though myostatin blockage is attracting attention as a novel target for increasing muscle growth in cases of DMD [11], our results suggest that myostatin blockage therapy would only be effective in DMD cases involving high serum myostatin concentrations. Therefore, myostatin blockage therapy should be applied carefully as a treatment for DMD.
JANUARY
30 - (Nature Medicine, February 2008) Functional skeletal muscle regeneration from differentiating embryonic stem cells
Radbod Darabi, Kimberly Gehlbach, Robert M Bachoo, Shwetha Kamath, Mitsujiro Osawa, Kristine E Kamm, Michael Kyba
& Rita C R Perlingeiro - USALittle progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-a receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy.
17 -
Safety and Efficacy Study of PTC124 in Duchenne and
Becker Muscular Dystrophy
16 - (Paediatrics
and Child Health, Volume 18, Issue 1, January 2008, Pages 22-26)
Recent developments in the management of Duchenne
muscular dystrophy
Maria Kinali, Adnan
Y. Manzur and Francesco Muntoni - UK
Duchenne muscular dystrophy (DMD) is the most common and severe childhood muscular dystrophy, resulting in progressive muscle weakness and wasting, disability and decreased survival. Although the molecular defect in DMD is known, no curative treatment is available. The treatment options of glucocorticoid corticosteroids and supportive measures, such as ventilation and management of cardiac insufficiency, have become accepted clinical practice in the last decade, and these are of major interest to the paediatrician as they have significantly changed the course of the disease in treated individuals. This has implications not only for the affected individual and his family, but also for the medical and social sectors to provide transition to adult medical services and for provision of suitable employment, and social care.
Several experimental therapeutic strategies, including cell and gene therapy, are promising, with encouraging results in relevant animal models and in cultured human cells. As a number of approaches are in early clinical trials, expectations are raised of their impact as a cure for DMD; nevertheless, it is not realistic to expect that these approaches will have a substantial impact on disease course in the short term. While waiting for a curative therapy to become available, symptomatic and palliative treatment is essential to enhance the patient’s quality of life. This review addresses the advances in these therapies aimed at improving function and quality of life in patients with DMD, and the current status of research into the DMD experimental therapies.
16 -
(Am J Physiol Regul Integr Comp Physiol , January 16,
2008) Stimulation of
calcineurin-A
activity attenuates muscle pathophysiology in mdx dystrophic mice
Calcineurin activation ameliorates the dystrophic
pathology of hindlimb muscles in mdx mice and decreases their
susceptibility to contraction damage. In mdx mice, the
pathology of the diaphragm is more severe than in hindlimb muscles,
and more representative of Duchenne muscular dystrophy.
Constitutively active calcineurin-A
was over-expressed in skeletal muscles of mdx dystrophic mice
(mdx CnA*)
to test whether muscle morphology and function would be improved.
Contractile function of diaphragm strips and EDL and soleus muscles
from adult mdx CnA
* and mdx mice were examined in vitro. Hindlimb muscles from
mdx CnA
* mice had a prolonged twitch time course and were more resistant to
fatigue. Due a slower phenotype and a decrease in fiber
cross-sectional area, normalized force (sPo) was lower in
fast and slow hindlimb muscles of mdx CnA*
than in mdx mice. In diaphragm, despite a slower phenotype and
an ~35% reduction in fiber size, sPo was preserved. This
was likely mediated by the reduction in the area of diaphragm
undergoing degeneration (i.e. mononuclear cell, connective, and
adipose tissue infiltration). The proportion of centrally nucleated
fibers was reduced in mdx CnA*
compared with mdx mice, indicative of improved myofiber
viability. In hindlimb muscles of mdx mice, calcineurin
activation increased expression of markers of regeneration,
particularly developmental myosin heavy chain (MyHC) isoform and
MEF2a. Thus, activation of the calcineurin signal transduction
pathway has potential to ameliorate the mdx pathophysiology
especially in the diaphragm through its effects on muscle
degeneration and regeneration and endurance capacity.
7 - FDA Issues Alert on Bone Drugs
5 - (Brain and Development, 2008) Isoprostanes in dystrophinopathy: Evidence of increased oxidative stress
Salvatore Grosso, Serafina Perrone, Mariangela Longini, Carlo Bruno, Claudio Minetti, Diego Gazzolo, Paolo Balestri and Giuseppe Buonocore - Italy
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are degenerative disorders of muscle. Although the mechanisms underlying muscle degeneration are still uncertain, oxidative-damage has been proposed to play a key role. Isoprostanes are markers of free radical-catalyzed lipid peroxidation; the aim of our study was to evaluate plasma isoprostane levels in group of patients affected by Duchenne and Becker muscular dystrophies. PF2-isoprostane levels were measured by colorimetric enzyme immunoassay in the plasma of 17 patients with DMD and 24 with BMD. When compared to a group of healthy controls, affected patients showed significantly higher plasma levels of isoprostanes (p = 0.001). When patients were stratified according to the clinical diagnosis, isoprostane levels were not statistically different between DMD and BMD patients. In conclusion whether the condition of oxidative stress found in plasma depends on the degenerative process occurring in muscles or on different mechanisms, such as the release of myoglobin in the blood, should be ascertained. However, our study confirms that oxidative stress findings in DMD/BMD patients are effectively present at the plasma levels. The condition of oxidative stress might act as an adjunctive cause of extra-muscular cell damage to which these patients are exposed for their entire life.
5 - Local dystrophin restoration with antisense oligonucleotide PRO051
5 - (Pediatric Research, December 2007) L-glutamine administration reduces oxidized glutathione and MAP Kinase signaling in dystrophic muscle of mdx mice
Mok, Elise; Constantin, Bruno; Favreau, Frederic; Neveux,
Nathalie; Magaud, Christophe; Delwail, Adriana; Hankard, Regis - France
To determine whether glutamine (Gln) reduces the ratio of
oxidized to total glutathione (GSSG/GSH) and extracellular signal-regulated
kinase (ERK1/2) activation in dystrophic muscle. 4-week old mdx mice, an animal
model for Duchenne muscular
dystrophy (DMD) and control (C57BL/10)
received daily intraperitoneal injections of L-Gln (500mg/kg/d) or 0.9% NaCl for
3 days. GSH and GSSG concentrations in gastrocnemius were measured using a
standard enzymatic recycling procedure. Free amino acid concentrations in
gastrocnemius were determined by ion exchange chromatography. Phosphorylated
protein levels of ERK1/2 in quadriceps were examined using Western Blot. L-Gln
decreased GSSG and GSSG/GSH (an indicator of oxidative stress). This was
associated with decreased ERK1/2 phosphorylation. Muscle free Gln, glutamate (Glu),
and the sum (Gln+Glu) were higher in mdx versus C57BL/10, at the basal level.
Exogenous Gln decreased muscle free Glu and Gln+Glu in mdx only, whereas Gln was
not affected. In conclusion, exogenous Gln reduces GSSG/GSH and ERK1/2
activation in dystrophic skeletal muscle of young mdx mice, which is associated
with decreased muscle free Glu and Gln+Glu. This antioxidant protective
mechanism provides a molecular basis for Gln's antiproteolytic effect in DMD
children.
5 - (Developmental Medicine & Child Neurology, January 2008) Abstracts that will be presented in 2008 British Paediatric Neurology Association Conference
A) Establishing the parameters for clinical trials of antisense oligonucleotide therapy in Duchenne muscular dystrophy
M KINALI MD MRCPCHA, V ARECHAVALA-GOMEZA MPHARM MSC PHDA, L FENG PHDA, A GLOVERB, M GUGLIERI MDC, H JUNGBLUTH MD PHD MRCP MRCPCHD, H ROPER FRCPCHE, RM QUINLIVAN FRCP FRCPCHF, D HUNT FRCSG, AY MANZUR FRCPCHA, A HENDERSON PHDC, J GOSALAKKALH, K HOLLINGSWORTH PHDI, J ALLSOP DCR(R)J, E MERCURI PHDA,K, J MORGAN PHDA, D J WELLS PHD MRCVSL, C SEWRY PHD FRCPATHA,F, V STRAUB MD PHDC, K BUSHBY MB CHB MSC MD FRCPC, M RUTHERFORD MD FRCR FRCPCHJ, F MUNTONI FRCPCH FMEDSCA - UK
Background: The progressive course of the muscle weakness in Duchenne muscular dystrophy (DMD) has been documented by several, exhaustive natural history studies; however, the progression of muscle pathology with time in individual muscle groups is poorly defined. There are currently no effective treatments to halt the muscle breakdown in DMD, although there are planned clinical trials using gene-based techniques. Most of these techniques, such as antisense oligonucleotides (AON), will require a sufficiently well preserved muscle to be effective. The choice of the muscles to be studied and the role of non-invasive methods to assess muscle preservation, therefore, require further evaluation. Method: Our principal objective was to study the degree of muscle involvement in the lower-leg muscles of 22 patients with DMD aged >8 years, using non-invasive imaging techniques (muscle MRI). In a subgroup of eight patients we also correlated the muscle MRI findings with the histology of muscle biopsies obtained during surgery from a foot muscle, the extensor digitorum brevis (EDB). Muscle MRI involvement was assigned using a 1 to 4 scale (normal–severe involvement). All patients had a gradient of involvement of their lower-leg muscles: posterior compartment (gastrocnemius>soleus) was most severely affected. Anterior compartment (tibialis anterior/posterior, popliteus, extensor digitorum longus) was least affected. Results: Muscle MRI showed a progressive involvement of the EDB but this did not directly correlate with age or time since loss of independent ambulation. There was a good correlation between the MRI and the EDB histopathology, with MRI grades 3 to 4 associated with a more fibro-adipose tissue replacement. Conclusion: Our results indicate that the EDB was sufficiently preserved even in non-ambulant patients and that MRI grading 2 to 3 is associated with sufficiently preserved structure. This information will allow patient recruitment in future clinical trials using AON based on the severity of the involvement of muscle on MRI without necessarily having to verify the muscle preservation invasively.
B) Long-term use of intermittent low-dosage prednisolone therapy in Duchenne muscular dystrophy: tolerance and effect on functional outcome
C POULTON MRCPCHA, M KINALI MD MRCPCHA, S A ROBB MD FRCP FRCPCHA, M MAIN MA MCSPB, AY MANZUR FRCPCHA, F MUNTONI FRCPCH FMEDSCA - UK
We reviewed functional outcome and steroid tolerance in 51 males with Duchenne muscular dystrophy (DMD) treated from 2000 onwards with intermittent prednisolone (0.75mg/kg/ day 10 days on/20 days off, or 10 days on/10 days off) for 18 months or more and compared this with natural history motor ability scores (MAS) previously published from this centre. Twentyseven out of 51 patients remained ambulant (Group 1) and 24 of 51 lost ambulation (Group 2). Regimes and ages at commencement were similar: Group 1 mean age 7 years (range 3y 10mo–11y 8mo); Group 2: mean age 7y 6mo (range 4–11y). Mean prednisolone duration was 2 years 7 months (Group 1) and 3 years 4 months (Group 2). Group 1 had better MAS than the natural history agematched patients (p<0.001) at the outset with a mean MAS 30. ANOVA tests showed stabilization of MAS for 24 months. Group 2 was similar to the natural history patients at outset with mean MAS <30, dropping to 23 after 24 months, a significant variance (p=0.069). Mean age of loss of independent ambulation was 10 years 5 months. Mean age of the still ambulant population was 10 years. Six males remained ambulant beyond 12 years. Age of loss of ambulation did not correlate with the MAS at onset. Forced vital capacity was >70% at 24 months after commencing therapy (n=23). Both steroid regimes were tolerated well. Six patients discontinued therapy, due to weight gain, epigastric pain (n=4), and parental choice (n=1). Behavioural changes data were available for 36 of 51 patients. Six had moderate changes, two required therapy modification. One child, with hydrocephalus, sustained a vertebral fracture. Eleven of 51 patients developed mild cardiomyopathy and two of 51 developed mild scoliosis. This open study demonstrates that in DMD, an intermittent prednisolone regime has a positive, though limited functional benefit in prolonging ambulation and preserving forced vital capacity, without major side effects. Use of the intermittent regime may be helpful to assess the likelihood of side effects, before transition to a daily regime, or may be continued with some benefit, especially if concerned about side effects.
C) Feeding difficulties in Ullrich congenital muscular dystrophy
N MCSWEENEY, A ALOYSIUS, T DAVIS, F MUNTONI - UK
Background: Ullrich congenital muscular dystrophy (UCMD) is an inherited progressive disorder due to a deficiency in Collagen VI, characterized by early onset hypotonia, contractures, distal joint laxity, spinal rigidity, and respiratory insufficiency. Feeding difficulties are frequently observed and patients might require dietary intervention or insertion of a gastrostomy tube. Objectives: To determine: (1) the incidence, (2) the severity of feeding difficulties, and (3) the outcome of intervention in UCMD. Methods: Medical notes of patients followed up at the Dubowitz Neuromuscular Centre with an established diagnosis of UCMD were reviewed. Results:We reviewed 38 case notes of patients with a clinical diagnosis of UCMD (28 males, 10 females, age range 3y 6mo– 32y), established by immunocytochemistry and/or genetic studies. Twenty-four had feeding difficulties and 14 did not. Feeding difficulties were defined as difficulty chewing, dysphagia with liquids or solids, prolonged mealtimes (>30 min), or having a poor appetite. Mean age at onset of feeding difficulties was age 13 years 6 months (median 14y, range birth–30y). Four patients had gastroesophageal reflux, which was treated medically. Faltering growth (weight <0.4th centile) was documented in 16 patients who had feeding difficulties. Fourteen patients received nutritional supplements, which only resulted in weight gain in three. The two patients who had nasogastric feeding had improved weight gain and three of the five who had gastrostomy had improved weight gain. Clinical course was generally more severe in those with feeding difficulties, with increased incidence of lower respiratory tract infections, non-invasive ventilation (mean age 15y), and scoliosis. Conclusion: Feeding difficulties are common in UCMD and broadly correlate with the overall disease severity. Dietary intervention with oral nutritional supplements seems to have little impact on faltering growth. Intervention using enteral feeding was more successful and suggests that intervention with gastrostomy should be considered early in the course of this progressive disorder.
D) Sibling adjustment in Duchenne muscular dystrophy
J READ BSC RGN, M KINALI PHD, F MUNTONI FMEDSCI FRCPCH, ME GARRALDA MD FRCPSYCH FRCPCH - UK
Objective: To document psychosocial adjustment in siblings of males with Duchenne muscular dystrophy (DMD). Methods: Participants were siblings of patients with DMD attending a specialist centre, aged 11 to 18 years, with no physical problems. We used quantitative measures: parent rated sociodemographic and illness questionnaires and interviews, and sibling self-rated questionnaires on physical and mental health (SF-36, Strengths and Difficulties Questionnaire, Hospital Anxiety and Depression Scale). We used qualitative methods (semi-structured interviews and qualitative data analysis) to describe siblings’ views on illness impact on their lives. Results: Out of 71 eligible families, 38 took part in the study; 29/38 of patients with DMD were wheelchair-dependent. Non-participant siblings were significantly more likely to be females and tended to be younger than the affected child; some families gave current family stress as a reason for not taking part. Participating siblings (n=45) were a mean age of 14 years 6 months (SD 2y 4mo), 22 were female and 23 male; 36 were Caucasian, 23 came from intact homes, and all but six had made adequate or good educational progress. Their mean scores (and rates above cut-offs for problems) on the physical and mental health questionnaires were within expected general population rates. Qualitative interviews identified considerable personal involvement with the affected sibling. Nevertheless, siblings reported little in the way of impact/burden on their lives, whilst 18/38 parents said that the condition had a severe impact on their own lives. Conclusions: Participating siblings of young people with DMD were well adjusted physically and mentally; most impact was reported on main carers. Some non-participating families may be overwhelmed by the condition and/or associated psychosocial problems. Further consideration may be given to ways of accessing and assisting them.
E) National audit of personal practice and experience relating to prevention and treatment of steroid-induced osteoporosis in Duchenne muscular dystrophy
S R CHANDRATRE MRCPCH DNB, R QUINLIVAN FRCPCH FRCP -UK
Objective:To audit personal practice and experience relating to prevention and treatment of steroid-induced osteoporosis in Duchenne muscular dystrophy (DMD). Methods: A questionnaire based on the Consensus Bone Protection Guideline of 2004 was e-mailed to 15 centres managing children with DMD on steroids. Completed questionnaires were returned by e-mail. Data were analyzed anonymously. Results: Eleven of 15 centres treating 315 patients returned the completed questionnaire. Investigations performed before commencing steroid therapy included calcium levels and varicella immune status in 9/11 centres, vitamin D levels in 7/11, and dual energy X-ray absorptiometry (DXA) scans in 6/11 centres, while 9/11 centres had access to DXA scan and facilities for interpretation. Frequency of DXA scans to monitor steroid therapy ranged from 1 to 2 years. Other investigations included spinal X-ray, annual bone profile and vitamin D levels, and vertebral morphometry. Losses of vertebral height or vertebral fractures were recorded in 5.6% of patients with lower limb fractures in 4.7%. Vitamin D or calcium were not routinely supplemented. Dieticians were involved in 8/11 centres. Bisphosphonates could be used in most centres (10/11). Overt vertebral fracture with or without back pain was the consensus indication for treatment with bisphosphonates. Opinion was divided over treatment of reduced vertebral height or back pain, but not overt fracture and long bone fractures. None of the centres recommended prophylactic treatment. Bisphosphonates were used in 28 (8.9%) patients. Pamidronate was used in 6/11 centres compared with risedronate in 2/11 centres. Adverse events reported were mild with good compliance. Some noted dramatically reduced bone fracture pain after infusion. Steroids were continued despite vertebral fractures in other patients. Conclusions: Adherence to the Consensus Bone Protection Guideline 2004 is good but can be improved. Bisphosphonate treatment is safe and well tolerated and needs a consensus guideline on indications for its use.
F) Correlation of hand-held myometry with various methods of assessment of muscle strength and function in patients with Duchenne muscular dystrophy
C MARCHESI MDA,B, M KINALI MD MRCPCHA, M MAIN MA MCSPC, F MUNTONI FRCPCH FMEDSCA - UK
Background: Quantitative muscle testing (QMT) has been used as a surrogate measure of muscle strength in Duchenne muscular dystrophy (DMD). However, its correlation with other measures of muscle strength or function is not well
known. Further studies are needed to assess the feasibility and reliability of QMT in different DMD age groups, so that QMT results could be used as surrogate markers for future clinical trials. Methods: We used hand-held myometry (HHM) to quantify muscle strength in 49 patients with DMD (age range 4y 11 mo–19y 8mo). Twenty-four patients (mean age 8y 4mo [SD 1 11mo) were ambulant (group 1); 25 (14y 1mo [SD 2y]) were non-ambulant (group 2). We correlated myometry values with patient’s age, manual muscle strength (%MRC), forced vital capacity (%FVC), and function: Motor Ability Score (MAS), 10-metre walk, and timed rise from the floor, where applicable. Results: In group 1, %MRC and MAS declined with age, timed 10-metre walk and TRF increased with age. Contrary to healthy age- and sex-matched controls where myometry values increase over time, myometry remained stable overall in group 1. Total myometry did not correlate with %MRC or MAS, while knee extensor myometry correlated positively with the MAS and negatively with the timed tests. Myometry for hip flexor, grip, and total distal arm strength correlated positively with patients’ age. In group 2, myometry of grip, pinch, key grip, and total distal arm correlated with %FVC. Grip myometry correlated positively with age at loss of ambulation. In both groups, distal arm muscle myometry peaked at age 10 to 11 years (mean age at loss of ambulation 11y 4mo), then gradually declined. Conclusions: Knee extensor myometry reliably correlates with MAS in the ambulant whereas %FVC correlates with grip myometry in non-ambulant patients. HHM is feasible in this latter group of patients and could thus form part of an assessment tool for evaluating efficacy of therapeutic interventions in DMD.G) Dystrophin positive revertant fibres do not increase with age in Duchenne muscular dystrophy
V ARECHAVALA-GOMEZA MPHARM MSC PHDA, M KINALI MD MRCPCHA, L FENG PHDA, M GUGLIERIB, G EDGE MD PHDC, M MAIN MA MCSPD, D HUNT FRCSE, J LEHOVSKY FRCSF, V STRAUB MD PHDB, K BUSHBY MB CHB MSC MD FRCPB, C SEWRY PHDA, J MORGAN PHDA, F MUNTONI FRCPCH FMEDSCA - UK
Objective: Muscle biopsies from patients with Duchenne muscular dystrophy (DMD) typically show absence of dystrophin.However, dystrophin traces or isolated positive (revertant) fibres are still found in up to 50% of DMD diagnostic biopsies. This phenomenon has been studied in the mdx mouse model for DMD, where revertants increase with advancing age. A number of experimental therapies currently under evaluation are aimed at restoring dystrophin expression in DMD. As dystrophin expression is an endpoint of these early studies, it is important to know if dystrophin expression also accumulates with age in males with DMD.Methods: We reviewed 63 DMD diagnostic muscle biopsy reports issued at the Dubowitz Neuromuscular Unit over the past 8 years to confirm the frequency of revertants and/or traces in the quadriceps muscle. Seven patients, in whom a second biopsy was obtained from the extensor digitorum brevis (EDB) muscle several years after the original diagnostic quadriceps biopsy, were studied to characterize the evolution of dystrophin expression with age. In these patients we performed a detailed comparison of dystrophin expression in the original and the recent biopsies. Results: Revertant fibres were present in 47% of the reports, traces in 33%; in 15% revertants and traces coexisted. In the seven patients who had second muscle biopsies, there was complete concordance between the original and the recent biopsies: presence or absence of revertants was maintained and did not increase with patient’s age. Conclusions: Revertant fibres do not increase with age in males with DMD in the EDB muscle, contrary to what has been reported on mdx mice, where regeneration continues over time. We devised a comparative assay to objectively measure dystrophin expression. Work is in progress to relate the presence of revertants or traces with functional abilities and dystrophin transcription in these patients. Our findings may have important implications for the planning of future dystrophin restoration studies