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QUESTIONARY ABOUT THE DRUGS USED IN DUCHENNE MUSCULAR DYSTROPHY
2009 NEWS
DECEMBER
22 - (Unpublished data, 2009) Effects of growth hormone (GH) in the evolution of Dystrophy disease in model B6.Wk-Lama2dy-2J/J
Maria Denise Fernandes Carvalho - research conducted to get the Master of Science in Bioscience Institute of Sao Paulo University under orientation of Mayana Zatz.
Muscular dystrophies (DM) are characterized by progressive and irrevertible degeneration of the skeleton muscles. Among them, Duchenne Muscular Dystrophy (DMD) consitutes lethal disease linked to chromossome X, distinguished by the absence of dystrophyn in the muscular fibers membrane, affecting one in each 3,000 boys born alive, who, most of them, will be confined to wheel chairs since the age of 6 and with life expectance, without special care, of 25 years. Despite modern diagnosis techniques, there is not an efficient treatment for these diseases. Some works from the 1970´s and 1980´s suggested that the inhibition of the growth hormone (GH) could retard the dystrophy process evolution in DMD patients. Nevertheless since this observation has not been proven scientifically, in the present work, the GH excess and inhibition effects on a murino model of muscular dystrophy, were studied: i) GH (10μg/mouse/day, i.p., for 10 days) was given to mice: a) dystrophic dy/dy (B6.Wk-Lama2dy-2J/J), with phenotype , resulting in the acceleration of the disease symptoms, confirmed by the performance worsening by 17% in the deambulation test (DT), 64% in the sustaining test (S), 28% in the mat test and 55% in the maximum strength test (MST), in relation to dystrophic mice which had not taken GH; only in the inclination resistance test (IRT) there was no significant difference; however, histological cuts of the gastrocnemius muscle of these mice did not show significant alterations, comparing to dystrophic mice which had not taken the drug: as to degeneration and regeneration markers (DRM) in these animals muscles, the GH did not alter Pax7, MyoD. Laminin and Desmin levels, although it increased by 90% the TGFβ-1 levels, an inflammatory cytokine, that induces fibrosis b) in normal mice, the GH, opposite to the results above, increased the performances by 13% in the DT and by 28% in the ST, and slightly, not significantly, in other two tests; histological cuts revealed that the GH increased by 16.5% the transversal section area of these animals muscles, what could explain the results described above; as to the MDR levels, including those of the TGFβ-1, they were similar to the control animals; ii) a new experimental model, resulting from the crossing of GH genetically deficient mice (Ghrthlit) with dystrophic dy/dy (B6.Wk-Lama2dy-2J/J), showing retardation in the disease evolution; the performances of the dystrophic dwarfs, compared to the normal dwarfs, were better than those of the dystrophic compared to normal animals, namely, 14% larger DT, 71% superior in the ST, 18% larger in the MT, 5.5% larger in the IRT and 102% superior in the MST; histological cuts showed that the interstice area in the dystrophic dwarfs muscles was 29% smaller than that of non-dwarfs dystrophic; the TGFβ-1 levels in these animals were the only MDR studied, which were found to be diminished by 36%, compared to those of the non-dwarfs dystrophic; the GH release inhibition by octreotide, somatostatin analogous, also showed a relative improvement or stabilization in the dystrophy evolution, demonstrated by the increase, ater 60 days treatment, of the performances in the muscle functional tests, compared to those of the dystrophic animals, without treatment: it was 12% larger in the DT, while the control dystrophic animals worsened by 32%; they maintained the same performance in the ST, and 43% in the seizing strength test, while the control dystrophic did not improve their performances in these tests. These results suggest a GH pathologic effect, possibly through the TGFβ-1 levels increase. The GH diminishing, consequently, could lead to the retardation in the disease evolution. These experimental studies open promissing perspectives to test the GH effects inhibition in human dystrophic diseases evolution.
22 - Tarantula-Venom-based Muscular Dystrophy Therapy to be Advanced by UB Scientists' Biotech Company
22 - (Circulation: Cardiovascular Genetics, 2009) Analysis of Dystrophin Deletion Mutations Predicts Age of Cardiomyopathy Onset in Becker Muscular Dystrophy
Rita Wen Kaspar, PhD, RN; Hugh D. Allen, MD; Will C. Ray, PhD; Carlos E. Alvarez, PhD; John T. Kissel, MD; Alan Pestronk, MD; Robert B. Weiss, PhD; Kevin M. Flanigan, MD; Jerry R. Mendell, MD and Federica Montanaro, PhD
From the Center for Gene Therapy (R.W.K., J.R.M., F.M.), The Research Institute at Nationwide Children’s Hospital; College of Nursing (R.W.K.), The Ohio State University; Division of Pediatric Cardiology (H.D.A.), The Ohio State University College of Medicine, Nationwide Children’s Hospital, Heart Center; Battelle Center for Mathematical Medicine (W.C.R.), The Research Institute at Nationwide Children’s Hospital; Biophysics Graduate Program (W.C.R.), The Ohio State University; Center for Molecular and Human Genetics (C.E.A.), The Research Institute at Nationwide Children’s Hospital; Departments of Pediatrics (C.E.A., J.R.M., F.M.) and Neurology (J.T.K., J.R.M.), The Ohio State University College of Medicine, Columbus, Ohio; Department of Neurology (A.P.), Washington University, St. Louis, Mo; and Departments of Genetics (R.B.W.) and Pediatrics (K.M.F.), University of Utah School of Medicine, Salt Lake City, Utah.
Background— Becker muscular dystrophy (BMD) and
X-linked dilated cardiomyopathy often result from deletion mutations
in the dystrophin gene that may lead to expression of an altered
dystrophin protein in cardiac muscle. Cardiac involvement is
present in
70%
of BMD and all X-linked dilated cardiomyopathy cases. To date, the
timing of cardiomyopathy development remains unpredictable. We
analyzed 78 BMD and X-linked dilated cardiomyopathy patients with
common deletion mutations predicted to alter the dystrophin protein
and correlated their mutations to cardiomyopathy age of onset. This
approach was chosen to connect dystrophin structure with function in
the heart.
Methods and Results— Detailed cardiac information was collected for BMD and X-linked dilated cardiomyopathy patients with defined dystrophin gene deletion mutations. Patients were grouped based on the dystrophin protein domain affected by the deletion. Deletions affecting the amino-terminal domain are associated with early-onset dilated cardiomyopathy (DCM; mid-20s), whereas deletions removing part of the rod domain and hinge 3 have a later-onset DCM (mid-40s). Further, we modeled the effects of the most common mutations occurring in the rod domain on the overall structure of the dystrophin protein. By combining genetic and protein information, this analysis revealed a strong correlation between specific protein structural modifications and DCM age of onset.
Conclusions— We identified specific regions of the dystrophin gene that when mutated predispose BMD patients to early-onset DCM. In addition, we propose that some mutations lead to early-onset DCM by specific alterations in protein folding. These findings have potential implications for early intervention in the cardiac care of BMD patients and for therapeutic approaches that target the heart in dystrophinopathies.
5 - (International Journal of Experimental Pathology, 2009) Calcium-binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy
Adriana Pertille, Candida Luiza Tonizza de Carvalho, Cintia Yuri Matsumura, Humberto Santo Neto and Maria Julia Marques - Brazil
Duchenne muscular dystrophy is one of the most common
hereditary diseases. Abnormal ion handling renders dystrophic muscle fibers more
susceptible to necrosis and a rise in intracellular calcium is an important
initiating event in dystrophic muscle pathogenesis. In the mdx
mice, muscles are affected with different intensities and some muscles are
spared. We investigated the levels of the calcium-binding proteins calsequestrin
and calmodulin in the non-spared axial (sternomastoid and diaphragm), limb (tibialis
anterior and soleus), cardiac and in the spared extraocular muscles (EOM) of
control and mdx mice. Immunoblotting analysis showed a
significant increase of the proteins in the spared mdx
EOM and a significant decrease in the most affected diaphragm. Both proteins
were comparable to the cardiac muscle controls. In limb and sternomastoid
muscles, calmodulin and calsequestrin were affected differently. These results
suggest that differential levels of the calcium-handling proteins may be
involved in the pathogenesis of myonecrosis in mdx
muscles. Understanding the signaling mechanisms involving Ca
5 - (Hum. Mol. Genet., Dec 2009) The artificial gene Jazz, a transcriptional regulator of Utrophin, corrects the dystrophic pathology in mdx mice
The absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF "Jazz" was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.
5 - (Acta Biochim Biophys Sin, Dec 2009; 41: 1053 - 1060) A non-viral vector for potential DMD gene therapy study by targeting a minidystrophin-GFP fusion gene into the hrDNA locus
Gene therapy has emerged as a promising approach for the lethal disorder of Duchenne muscular dystrophy (DMD). Using a novel non-viral delivery system, the human ribosomal DNA (hrDNA) targeting vector, we targeted a minidystrophin-GFP fusion gene into the hrDNA locus of HT1080 cells with a high site-specific integrated efficiency of 10–5, in which the transgene could express efficiently and continuously. The minidystrophin-GFP fusion protein was easily found to localize on the plasma membrane of HT1080 cells, indicating its possible physiologic performance. Our findings showed that the hrDNA-targeting vector might be highly useful for DMD gene therapy study.
NOVEMBER
14 - (Science Translational Medicine, Nov 2009; 1: 6ra15) Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates
Janaiah Kota, Chalonda R. Handy, Amanda M. Haidet, Chrystal L. Montgomery, Amy Eagle, Louise R. Rodino-Klapac, Danielle Tucker, Christopher J. Shilling, Walter R. Therlfall, Christopher M. Walker, Steven E. Weisbrode, Paul M. L. Janssen, K. Reed Clark, Zarife Sahenk, Jerry R. Mendell, and Brian K. Kaspar
Antagonists of myostatin, a blood-borne negative regulator of muscle growth produced in muscle cells, have shown considerable promise for enhancing muscle mass and strength in rodent studies and could serve as potential therapeutic agents for human muscle diseases. One of the most potent of these agents, follistatin, is both safe and effective in mice, but similar tests have not been performed in nonhuman primates. To assess this important criterion for clinical translation, we tested an alternatively spliced form of human follistatin that affects skeletal muscle but that has only minimal effects on nonmuscle cells. When injected into the quadriceps of cynomolgus macaque monkeys, a follistatin isoform expressed from an adeno-associated virus serotype 1 vector, AAV1-FS344, induced pronounced and durable increases in muscle size and strength. Long-term expression of the transgene did not produce any abnormal changes in the morphology or function of key organs, indicating the safety of gene delivery by intramuscular injection of an AAV1 vector. Our results, together with the findings in mice, suggest that therapy with AAV1-FS344 may improve muscle mass and function in patients with certain degenerative muscle disorders.
13 - (Circulation, 2009, 120) Abstract 2962: Prevention of Cardiac Injury and Ventricular Remodeling in Dystrophic Dogs by Chronic Administration of Membrane Sealant P188
1 Univ Minnesota, Minneapolis, MN
2 Univ of North Carolina, Minneapolis, MN
3 Univ Minnesota, Minneapolis, MN
Background: Duchenne muscular dystrophy (DMD) is a fatal disease resulting from the loss of the cytoskeletal protein dystrophin and consequent damage to both skeletal and cardiac muscle cells. In the absence of dystrophin small tears in the cardiac sarcolemma arise causing loss of membrane integrity, muscle wasting, and eventual heart failure in DMD patients. There are no reports of long-term efficacious treatments for dystrophic cardiomyopathy.
Hypothesis: The long term application of the chemical-based membrane sealant Poloxamer 188 (P188) will be safe and effectively slow the development of dystrophic cardiomyopathy.
Methods/Results: Here we show chronic administration of P188 to dystrophin-deficient golden retriever muscular dystrophy (GRMD) dogs in vivo is both safe and effective in blocking the development of cardiac disease. Intravenous administration of 60 mg/kg/hour P188 for 8 weeks in adult GRMD dogs prevented the onset of heart disease observed in saline infused animals. Specifically, significant reductions in fibrotic lesions were observed in P188 infusion (5.8±0.9% of total myocardium) compared to saline infused controls (11.0±1.1%). In saline infused dogs elevations of serum cTnI and BNP were observed. These elevations were not present in P188 infused dogs, indicating a reduction in both myocardial necrosis and congestion in P188 infused dogs. Treatment with P188 also prevented left ventricular dilation that was evident in untreated GRMD control dogs (diastolic volumes: 39±4 vs. 24±3 ml for saline and P188 infused dogs respectively). Regardless of treatment, adult cardiac myocytes isolated from either P188 or saline infused GRMD dogs revealed significantly abnormal passive tension-extension properties. These functional deficits of the isolated myocyte were rapidly reversed upon addition of P188.
Conclusion: Given the clinical prominence of cardiomyopathy and heart failure in DMD, there is an urgent need for effective therapies for the dystrophic heart. This study demonstrates that P188 has the promise of an immediately available therapeutic approach for mitigating the progression of cardiac disease in DMD.
13 - (Circulation, 2009, 120)
Abstract 2156: Idebenone Improves Peak
Systolic Strain in Children With Duchenne Muscular Dystrophy With
Preserved Ejection Fraction: Results of a 12 Month Double-blind, Randomized,
Controlled Trial
1 Univ Hosps Leuven, Leuven, Belgium
2 UMC Radboud, Nijmegen, Netherlands
3 Univ Hosp Antwerp, Antwerp, Belgium
4 Santhera Pharmaceuticals, Basel, Switzerland
5 Hosp for Sick Children, Toronto, Canada
Objective - To evaluate effect of treatment with idebenone compared to placebo on myocardial radial and longitudinal deformation parameters in children and adolescents with Duchenne muscular dystrophy (DMD).
Background - Idebenone supports mitochondrial respiratory chain function and reduces oxidative stress. We have shown that early long-term treatment with idebenone is cardioprotective and improves exercise performance in the dystrophin-deficient mdx mouse. Peak systolic strain is reduced in young patients with DMD and precedes overt cardiac dysfunction. We hypothesized that early treatment with idebenone in presymptomatic children has a cardioprotective effect.
Design/Methods - 21 DMD patients (8 –16 yr) without overt cardiac dysfunction but reduced baseline peak radial systolic strain values were enrolled in a phase II double-blind, randomized, placebo-controlled trial. Comedication with glucocorticoids was allowed at stable dosage; use of ACE-inhibitors was excluded. Thirteen patients received idebenone (450 mg daily) and 8 patients placebo for 52 weeks. The primary endpoint was the change from baseline in peak systolic radial strain of the left ventricular (LV) inferolateral wall, the region of the heart which is most affected in DMD.
Results - All subjects completed the study, and idebenone showed good safety and tolerability. Compared to placebo, treated patients showed significant improvement in peak systolic radial strain of the LV inferolateral wall (percent change 28.9 ± 40.7 placebo and 104.4 ± 92.2 idebenone; p= 0.030). Peak systolic longitudinal strain of the LV lateral wall improved in patients on idebenone and deteriorated in patients on placebo (percent change 42.3 ± 61.8 idebenone –4.5 ± 23.0 placebo; p=0.039). Ejection fraction was normal at baseline and did not significantly change in both groups.
Conclusions - Idebenone improves cardiac radial and longitudinal deformation parameters in the inferolateral wall in children with DMD which have not yet developed overt cardiac dysfunction. The results provide the basis and guidance for phase III studies with SNT-MC17/idebenone in DMD. This is the first clinical trial where strain imaging was used as a primary endpoint in children.
7 - (Clinical Nutrition, 2009) Green tea extract decreases muscle pathology and NF-κB immunostaining in regenerating muscle fibers of mdx mice
Nicholas P. Evans, Jarrod A. Call, Josep Bassaganya-Riera, John L. Robertson, Robert W. Grange - USA
Mdx breeder mice and pups were fed diets containing 0.25% or 0.5% green tea extract and compared to untreated mdx and C57BL/6J mice. Serum creatine kinase was assessed as a systemic indicator of muscle damage. Quantitative histopathological and immunohistochemical techniques were used to determine muscle pathology, macrophage infiltration, and NF-κB localization.
Early treatment of mdx
mice with green tea extract significantly decreased serum creatine kinase by
85%
at age 42 days (P ≤ 0.05). In these
mice, the area of normal fiber morphology was increased by as much as
32%
(P ≤ 0.05). The primary
histopathological change was a
21%
decrease in the area of regenerating fibers (P ≤ 0.05).
NF-κB staining in regenerating muscle fibers was also significantly decreased in
green tea extract-treated mdx mice when
compared to untreated mdx mice (P ≤ 0.05).
Early treatment with green tea extract decreases dystrophic muscle pathology potentially by regulating NF-κB activity in regenerating muscle fibers.
OCTOBER
31 - Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism
25 - (Mol.Ther, 2009) Prevention of Dystrophic Pathology in Severely Affected Dystrophin/Utrophin-deficient Mice by Morpholino-oligomer-mediated Exon-skipping
Aurélie Goyenvalle1, Arran Babbs1, Dave Powell1, Ryszard
Kole2, Sue Fletcher3, Steve D Wilton3 and Kay E Davies1
1MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics,
University of Oxford, Oxford, UK;
2AVI Biopharma, Corvallis, Oregon, USA;
3Center for Neuromuscular and Neurological Disorders, University of Western
Australia, Perth, Western Australia,
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the effciency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These fndings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.
25 - (Neuromuscular Disorders 19 (2009) 754–758) Prevalence and psychosocial impact of lower urinary tract symptoms in patients with Duchenne muscular dystrophy
Evaline van Wijk, Bert J. Messelink, Lily Heijnen, Imelda J.M. de Groot
- The NetherlandsPatients with Duchenne muscular dystrophy (DMD) frequently report lower urinary tract symptoms at the outpatient rehabilitation clinic. The purpose of this study was to determine the prevalence of lower urinary tract symptoms in the Dutch male DMD population and their effect on quality of life. A postal questionnaire was sent to members of Dutch DMD patient organisations. 199 male patients with con-.rmed DMD and over the age of 3 years were included. 170/199 (85%) patients reported one or more lower urinary tract symptoms. Generally, post micturition dribble, straining and feeling of incomplete emptying were most frequently mentioned. 42% of patients (range 18–76%) experienced the symptoms as a problem. In 49/170 (29%) patients, it reduced quality of life. In conclusion, lower urinary tract symptoms in DMD patients are under reported and under diagnosed. However, the vast majority of male DMD patients with symptoms experience them as a problem, often reducing quality of life.
21 - (Neurobiology of Disease, 2009; 36 (2): 252-258) MALDI reveals membrane lipid profile reversion in MDX mice
Farida Benabdellah, Hua Yu, Alain Brunelle, Olivier Laprévote, Sabine De La Porte - France
Duchenne muscular dystrophy (DMD), the most common and severe X-linked myopathy, is characterized by the lack of dystrophin, a sub-sarcolemmal protein necessary for normal muscle functions. In a previous study of the lipid content of skeletal muscles of dystrophic (mdx) mice, the animal model for DMD, by in situ Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry (MALDI-MS), an inversion of the phosphatidylcholine PC34:2/PC34:1 ion peaks intensity ratio was observed between destructured (abnormal fiber morphology) and structured (normal fiber morphology). A possible treatment for this dramatic disease is to introduce an exogenous nitric oxide (NO) donor into the organism, leading to an increase of utrophin and a regression of the dystrophic phenotype. In the present work, after confirmation by tandem mass spectrometry of the structure of these two phospholipids, their intensity ratio inversion was used to evidence a restoration of membrane lipid composition very similar to those of wild-type mice after the treatment of mdx mice with MOLSIDOMINE, a NO donor. This was associated with the observation by immunohistology of an increase of the regeneration process in the mice.
20 - (Cellular Immunology, 2009) Mesenchymal Stem Cells as Anti-inflammatories: Implications for Treatment of Duchenne Muscular Dystrophy
1Thomas E. Ichim, 2Doru T. Alexandrescu, 3Fabio Solano, 3Fabian Lara, 4Rosalia De Necochea Campion, 5Eugenia Paris, 6Erik J Woods, 7Michael P Murphy, 8Constantin A Dasanu, 9Amit N Patel, 10Annette M Marleau, 1
*Neil H. Riordan1Medistem Inc, San Diego, USA; 2Georgetown Dermatology, Washington DC, USA;
3Institute for Cellular Medicine, Panama City, Panama; 4University of California, San Diego, California; 5Hospital CIMA, San Jose, Costa Rica; 6General Biotechnology, Indianapolis, Indiana; 7Division of Medicine, Indiana University School of Medicine, Indiana, USA; 8Department of Hematology and Medical Oncology, St Francis Hospital and Medical Center, Hartford, CT; 9Dept of Cardiothoracic Surgery, University of Utah, Salt Lake City, Utah, USA; 10Department of Surgery, University of Nebraska Medical Centre, Omaha NebraskaDuchenne muscular dystrophy (DMD) is a lethal X-linked musculodegenerative condition consisting of an underlying genetic defect whose manifestation is augmented by inflammatory mechanisms. Previous treatment approaches using gene replacement, exon-skipping or allogeneic cell therapy have been relatively unsuccessful. The only intervention to mediate improvement in survival, albeit minor, is glucocorticoid treatment. Given this modality appears to function via suppression of underlying inflammation; we focus this review on the inflammatory response as a target for mesenchymal stem cell (MSC) therapy. In contrast to other cell based therapies attempted in DMD, MSC have the advantages of: a) ability to fuse with and genetically complement dystrophic muscle; b) possess anti-inflammatory activities; and c) produce trophic factors that may augment activity of endogenous repair cells. We conclude by describing one practical scenario of stem cell therapy for DMD. ...................................................
...."Case Report of Stem Cell Therapy for DMD
We report a case study of a 23 year old male diagnosed with DMD at age 3, who
manifested a progressive decrease in muscular strength and became wheelchair
bound at age of 12. Supportive treatment with intermittent courses of prednisone,
pain management, along with physical therapy was provided. Frequent respiratory
infections secondary to a poor respiratory effort with decrease clearance of the
secretions were managed with standard antibiotic therapy. On August 5-14th,
2008, the patient was treated with a combination of ERC and CD34 umbilical cord
blood, mixed lymphocyte reaction-matched positive cells,subsequently on November
25-28, the patient received another course of therapy including placental matrix
derived mesenchymal stem cells (Table 1). Cells were prepared and administered
as previously described by us [148, 149]. No adverse events were associated with
the stem cell infusion. A significant increase in muscle strength occurred in
all the muscle groups, and was accompanied by an increase in the functional
capacity of the patient. Thus, a pre-transplantation strength of 2-2.5/5 in the
neck, shoulder, upper, and lower extremities began to improve after each of the
two stem cell administrations, and reached a final 4/5 level 1 month after
second transplantation treatment. The increments in muscle strength after the
two stem cell administration appeared to be additive, with most benefit recorded
after the second. Upper extremity improvement in strength evolved from the
incapacity to lift against
gravity before the transplantation towards the ability to lift 2 lbs weights
after the procedure. Trunk balance and strength were also markedly improved. The
patient gained 20 lbs, along with an increased general activity level. The
frequency of respiratory infections decreased from 3-4/year before stem cell
therapy to none. The inspiratory effort improved from -32 to -40 cm H20. A
muscle biopsy taken in January 2009 demonstrated normal (>50%, normal = 50-100%
expression of normal-molecular size) levels of muscular dystrophin. The
improvement in muscular strength, clinical respiratory function, and general
level of activity are maintained to date.
To our knowledge this is the first report of profound dystrophin expression
occurring in a non-ambulatory DMD patient after ERC treatment .......
10 - (Cardiovasc Res, Oct 2009) Long-Term Improvement in mdx Cardiomyopathy after Therapy with Peptide-conjugated Morpholino Oligomers
1 Department of Pharmacology and Lineberger
Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina, USA
2 Thalassemia Research Center and Institute of Molecular Biology and
Genetics, Mahidol University, Bangkok, Thailand
3 AVI BioPharma, Inc., Corvallis, Oregon, USA
4 McAllister Heart Institute, University of North Carolina, Chapel
Hill, NC
5 Department of Pathology and Laboratory Medicine, University of
North Carolina, Chapel Hill, NC
Aims: The cardiomyopathy found in Duchenne muscular dystrophy (DMD) is responsible for death due to heart failure in 30% of patients and additionally contributes to many DMD morbidities. Strategies to bypass DMD-causing mutations to allow an increase in body-wide dystrophin have proven promising, but increasing cardiac dystrophin continues to be challenging. The purpose of this study was to determine if therapeutic restoration of cardiac dystrophin improved the significant cardiac hypertrophy and diastolic dysfunction identified in X-linked muscular dystrophy (mdx) dystrophin-null mouse due to a truncation mutation over time after treatment.
Methods and Results: Mice lacking dystrophin due to a truncation mutation (mdx) were given an arginine-rich, cell-penetrating, peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that delivered a splice-switching oligonucleotide-mediated exon skipping therapy to restore dystrophin in mdx mice before the development of detectable cardiomyopathy. PPMO successfully restored cardiac dystrophin expression, preserved cardiac sarcolemma integrity, and prevented the development of cardiac pathology that develops in mdx-null mice over time. By echocardiography and Doppler analysis of the mitral valve, we identified that PPMO treatment of mdx mice prevented the cardiac hypertrophy and diastolic dysfunction identified in sham-treated, age-matched mdx mice, characteristic of DMD patients early in the disease process, in as little as 5-6 weeks after the initiation of treatment. Surprisingly, despite the short-term replacement of cardiac dystrophin (<1% present after 12 weeks by PCR), PPMO therapy also provided a durable cardiac improvement in cardiac hypertrophy and diastolic dysfunction for up to 7 months after the initiation of treatment.
Conclusions: These results demonstrate for the first time that PPMO-mediated exon skipping therapy early in the course of DMD may effectively prevent or slow down associated cardiac hypertrophy and diastolic dysfunction with significant long-term impact.
10 - Exon Skipping update - Günter Scheuerbrandt report
6 - (Hum. Mol. Genet., Nov 2009; 18:4089 - 4101) Evaluation of the therapeutic potential of carbonic anhydrase inhibitors in two animal models of dystrophin deficient muscular dystrophy
1 Centre de Génétique Moléculaire et Cellulaire, UMR 5534, Université Lyon 1, 69622 Villeurbanne Cedex, France 2 Laboratory of Molecular Myology, Department of Neurology, Friedrich Baur Institute, Ludwig Maximilians University of Munich, Munich, Germany 3 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada, V5A 1S6 4 Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, UK
Duchenne Muscular Dystrophy is an inherited muscle degeneration disease for which there is still no efficient treatment. However, compounds active on the disease may already exist among approved drugs but are difficult to identify in the absence of cellular models. We used the Caenorhabditis elegans animal model to screen a collection of 1000 already approved compounds. Two of the most active hits obtained were methazolamide and dichlorphenamide, carbonic anhydrase inhibitors widely used in human therapy. In C. elegans, these drugs were shown to interact with CAH-4, a putative carbonic anhydrase. The therapeutic efficacy of these compounds was further validated in long-term experiments on mdx mice, the mouse model of Duchenne Muscular Dystrophy. Mice were treated for 120 days with food containing methazolamide or dichlorphenamide at two doses each. Musculus tibialis anterior and diaphragm muscles were histologically analyzed and isometric muscle force was measured in M. extensor digitorum longus. Both substances increased the tetanic muscle force in the treated M. extensor digitorum longus muscle group, dichlorphenamide increased the force significantly by 30%, but both drugs failed to increase resistance of muscle fibres to eccentric contractions. Histological analysis revealed a reduction of centrally nucleated fibers in M. tibialis anterior and diaphragm in the treated groups. These studies further demonstrated that a C. elegans-based screen coupled with a mouse model validation strategy can lead to the identification of potential pharmacological agents for rare diseases.
5 - (Neuropediatrics, 2008) Treatment of Duchenne muscular dystrophy with cyclosporin A - A randomized, double-blind, placebo controlled trial
J Kirschner, J Schessl, G Ihorst, R Korinthenberg, Muskeldystrophie Netzwerk MD-NET - Germany
Treatment with steroids has shown to slow down progression of muscle weakness in patients with Duchenne muscular dystrophy (DMD) but its use is limited by side effects. In a randomized, multi-centre, double-blind placebo-controlled trial we investigated the effect of cyclosporin A (CSA) in DMD. 153 patients were randomized to receive either placebo or 4mg/kg CSA. After three months both groups received additional treatment with intermittent prednisone (0.75mg/kg, 10 days on/10 days off) for another 12 months. In each group 73 patients were available for intention to treat analysis. Baseline characteristics were comparable in both groups. There was no significant difference between the two groups concerning primary (manual muscle strength according to MRC) and secondary (myometry, loss of ambulation, side effects) outcome measures. Peak CSA values were measured blindly and ranged from 12-658ng/ml (mean 210ng/ml) in the verum group. In conclusion, CSA does not improve muscle strength as a monotherapy and does not improve the efficacy of intermittent prednisone in DMD. Networks of rare diseases such as the BMBF-funded MD-NET facilitate the conduction of meaningful multicentre trials even in rare diseases.
The study was supported by BMBF, aktion bennie und co e.V., Novartis Pharma, and Deutsche Gesellschaft für Muskelkranke e.V. Participating trial sites were Basel, Berlin, Essen, Freiburg, Göttingen, Kiel, Mainz, Munich, Neuss, and Vienna.
3 - Cardiac assessment of patients with late stage Duchenne muscular dystrophy
SEPTEMBER
26 - (Experimental Neurology, September 2009) Flavocoxid counteracts muscle necrosis and improves functional properties in mdx mice: a comparison study with methylprednisolone
Sonia Messina, Alessandra Bitto, M'hammed Aguennouz, Anna Mazzeo, Alba Migliorato, Francesca Polito, Natasha Irrera, Domenica Altavilla, Gian Luca Vita, Massimo Russo, Antonino Naro, Maria Grazia De Pasquale, Emanuele Rizzuto, Antonio Musarò, Francesco Squadrito and Giuseppe Vita
Muscle degeneration in dystrophic muscle is exacerbated by the endogenous inflammatory response and increased oxidative stress. A key role in is played by nuclear factor(NF)- κB. We showed that NF-κB inhibition through compounds with also antioxidant properties has beneficial effects in mdx mice, the murine model of Duchenne muscular dystrophy (DMD), but these drugs are not available for clinical studies. We evaluated whether flavocoxid, a mixed flavonoid extract with anti-inflammatory, antioxidant and NF-κB inhibiting properties, has beneficial effects in mdx mice in comparison with methylprednisolone, the gold standard treatment for DMD patients. Five-week old mdx were treated for 5 weeks with flavocoxid, methylprednisolone or vehicle. The evaluation of in vivo and ex vivo functional properties and morphological parameters was performed. Serum samples were assayed for oxidative stress markers, creatine-kinase (CK) and leukotriene B-4 determination. Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), tumor necrosis factor-α, p-38, JNK1 expression was evaluated in muscle by western blot analysis. NF-κB binding activity was investigated by electrophoresis mobility shift assay. The administration of flavocoxid: 1) ameliorated functional properties in vivo and ex vivo; 2) reduced CK; 3) reduced the expression of oxidative stress markers and of inflammatory mediators; 4) inhibited NF-кB and mitogen-activated protein kinases (MAPKs) signal pathways; 5) reduced muscle necrosis and enhanced regeneration. Our results highlight the detrimental effects of oxidative stress and NF-κB, MAPKs and COX/5-LOX pathways in the dystrophic process and show that flavocoxid is more effective in mdx mice than methylprednisolone.
15 - A review of nutrition in Duchenne muscular dystrophy
6 - (J Pediatr 2009;155:380-5) Delayed Diagnosis in Duchenne Muscular Dystrophy: Data from the Muscular Dystrophy Surveillance, Tracking, and Research Network (MD STARnet)
Emma Ciafaloni, Deborah J. Fox, Shree Pandya, Christina P. Westfield, RN, Soman Puzhankara, Paul A. Romitti, Katherine D. Mathews, Timothy M. Miller, Dennis J. Matthews, Lisa A. Miller, Christopher Cunniff, Charlotte M. Druschel, and Richard T. Moxley - USA
Objective: To identify key factors for the delay in
diagnosis of Duchennemuscular dystrophy (DMD) without known family history.
Study design: The cohort comes from the Muscular Dystrophy Surveillance,
Tracking, and Research Network (MD STARnet), a multistate, multiple-source,
population-based surveillance system that identifies and gathers in- formation on
all cases of Duchenne and Becker muscular dystrophy born since 1982.We analyzed
medical records of 453 Duchenne and Becker muscular dystrophy boys to document
the time course and steps taken to reach a de- finitive diagnosis.
Results: Among 156 boys without known family history of DMD prior
to birth, first signs or symptoms were noted at a mean age of 2.5 years. Concerns
resulted in primary care provider evaluation of the child at a mean age of 3.6
years. Mean age at time of initial creatine kinase was 4.7 years. Mean age at
definitive diagnosis of DMD was 4.9 years.
Conclusions: There is a delay of about 2.5 years between onset of DMD
symptoms and the time of definitive diagnosis, unchanged over the previous 2
decades. This delay results in lost opportunities for timely genetic counseling
and initiation of corticosteroid treatment. We recommend checking creatine
kinase early in the evaluation of boys with unexplained developmental delay.
EDITORIAL: Missed Opportunities for Duchenne Muscular Dystrophy
Petra Kaufmann, MD, MSc, Columbia University, Department
Duchenne Muscular Dystrophy (DMD) continues to challenge affected boys and their families because it causes progressive weakness in children who were seemingly normal as infants and toddlers. The discovery of mutations in the dystrophin gene more than 2 decades ago
1 raised hopes for improved diagnosis and treatment, some of which have been ful-filled. For almost all patients, the diagnosis can be confirmed by genetic testing, largely obviating the need for muscle biopsy. The survival rate has shown significant improvement, with now more than half of the patients surviving past age 25 years,2 largely as a result of proactive pulmonary and cardiac management. There is clear evidence for prednisone delaying the loss of motor function3 and reducing mortality rates.4 Animal models are available,5 and clinical trials are underway.6 Despite these advances, there has been no significant change over the past 20 years in the time from symptomonset to diagnosis. Ciafaloni et al7 report that 2.5 years go by on average until patients without known family history are given a definite diagnosis. It is not only surprising that it takes 2.5 years until adefinite diagnosis is reached, but it is even more surprising that the interval has not decreased over the past 2 decades. The report by Ciafaloni et al7 suggests that there is room for improvement in appropriate training and ongoingmedical education that would allow practitioners to make the diagnosis in a timelier manner. The initial screen, a blood test for creatine kinase, is widely available and inexpensive. In the past, one might have argued that little is gained by making an earlier diagnosis of an ‘‘incurable’’ disease. Today, effective treatments are available that can temporarily preserve motor function and prolong survival. There is therefore newfound urgency in making a correct diagnosis that will become more pressing as novel treatments enter clinical trials. It is reasonable that practitioners do not include screening for a relatively rare and serious diagnosis such as DMD in the very first work-up for developmental delay in young boys when there is no calf hypertrophy, toe-walking or weakness. However, persistent delay should prompt screening earlier in the work-up so that a diagnosis can be made in less than 2.5 years from symptom onset. The report by Ciafaloni et al7 suggests that neurologists are more likely to order a creatine kinase test than primary care clinicians. Therefore educational initiatives aimed at primary care clinicians are a first approach to shorten the time to a correct diagnosis. Ciafaloni et al7 also report that about 15 additional affected maternal relatives were born in the interval from symptom onset to a definite diagnosis. These families did not have the genetic counseling opportunities thatwould have permitted an informed decision. It is imperative that we improve translating scientific advances into medical practice for boys with DMD and their families by educating medical students, pediatricians, and other primary care practitioners that DMD is a readily diagnosable and treatable condition. Although thought of as a muscle disease, a dystrophinopathy often affects language development early on in the course. Therefore checking for subtle signs of muscle weakness or calf hypertrophy and sending off a blood test for creatine kinase should be considered early on in the evaluation of any boy with not only motor, but also language delay. This simple message should be clearly incorporated in medical training and practice guidelines. Most experts support the implementation of newborn screening. However, until newborn screening is universally implemented, families depend on well-trained clinicians to afford them early access to a correct diagnosis and thus genetics counseling and treatment.6 - Santhera Starts Late-stage Trial In Rare Muscle Disease
AUGUST
26 - (Muscle & Nerve. 40 (3) 466-68,2009) Long-term therapy with deflazacort decreases myocardial fibrosis in mdx mice
Maria Julia Marques, Daniella Silva Oggiam, Isabel Cristina Chagas Barbin, Renato Ferretti, Humberto Santo Neto - Brazil
We evaluated the effects of long-term administration of deflazacort (DFZ) on the progression of myocardial fibrosis in mdx mice. Mdx mice (6 months old) were treated with DFZ for 15 months. Myocardial fibrosis (MF) was evaluated by histomorphometric methods, and treated and untreated mdx mice of the same age (21 months) were compared. DFZ significantly decreased MF. We conclude that long-term therapy with DFZ is effective in slowing down the progression of fibrosis in the dystrophin-deficient heart.
26 - (Muscle & Nerve. 40 (3) 443-54,2009) Effects of prednisolone on skeletal muscle contractility in mdx mice
Kristen A. Baltgalvis, Jarrod A. Call, Jason B. Nikas, Dawn A. Lowe - USA
Current treatment for Duchenne muscular dystrophy (DMD) is chronic administration of the glucocorticoid prednisolone. Prednisolone improves muscle strength in boys with DMD, but the mechanism is unknown. The purpose of this study was to determine how prednisolone improves muscle strength by examining muscle contractility in dystrophic mice over time and in conjunction with eccentric injury. Mdx mice began receiving prednisolone (n = 23) or placebo (n = 16) at 5 weeks of age. Eight weeks of prednisolone increased specific force of the extensor digitorum longus muscle 26%, but other parameters of contractility were not affected. Prednisolone also improved the histological appearance of muscle by decreasing the number of centrally nucleated fibers. Prednisolone treatment did not affect force loss during eccentric contractions or recovery of force following injury. These data are of clinical relevance, because the increase in muscle strength in boys with DMD taking prednisolone does not appear to occur via the same mechanism in dystrophic mice.
26 - (Muscle & Nerve. 40 (3) 476-80,2009) Pericardial effusion with cardiac tamponade as a cardiac manifestation of duchenne muscular dystrophy
Jainn-Jim Lin, Mao-Sheng Hwang, Shao-Hsuan Hsia, Hung-Tao Chung, Yu-Sheng Chang, Kuang-Lin Lin - Taiwan
Duchenne muscular dystrophy (DMD) is the most common hereditary neuromuscular disease in children. It is an X-linked hereditary dystrophinopathy due to the absence of dystrophin. Its onset is often in early childhood and presents with proximal distribution of weakness and a progressive course. Cardiac involvement in DMD is common, and its onset is usually after the age of 10 years. The most common cardiac manifestations are a dilated cardiomyopathy and cardiac arrhythmia. However, pericardial effusion with cardiac tamponade is a very rare cardiac complication of DMD. We report a boy with DMD who initially presented with progressive dyspnea and an enlarged cardiac silhouette on chest radiography who subsequently developed a large pericardial effusion with cardiac tamponade. Early recognition of pericardial effusion with cardiac tamponade is important for institution appropriate therapy.
25 - (Lancet Neurology, 2009) Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study
- UK.
Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder.
Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44—79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26·4%), and the mean intensity was 17% (range 11—21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0·002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot.
Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD.
20 - (Human Molecular Genetics, 2009) A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice
1 Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK 2 Tianjin Research Centre of Basic Medical Science, Tianjin Medical University, Qixiangtai Road, Heping District, Tianjin, 300070, China 3 AVI Biopharma Inc., Corvallis, Oregon, 97333, USA
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the synthesis of dystrophin protein. Antisense oligonucleotides (AOs) targeted to trigger excision of an exon bearing a mutant premature stop codon in the DMD transcript have been shown to skip the mutated exon and partially restore functional dystrophin protein in dystrophin-deficient mdx mice. To fully exploit the therapeutic potential of this method requires highly efficient systemic AO delivery to multiple muscle groups, to modify the disease process and restore muscle function. While systemic delivery of naked AOs in DMD animal models requires high doses and is of relatively poor efficiency, we and others have recently shown that short arginine-rich peptide-AO conjugates can dramatically improve in vivo DMD splice correction. Here we report for the first time that a chimeric fusion peptide (B-MSP-PMO) consisting of a muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-peptide) and conjugated to a morpholino oligomer (PMO) AO directs highly efficient systemic dystrophin splice correction in mdx mice. With very low systemic doses, we demonstrate that B-MSP-PMO restores high-level, uniform dystrophin protein expression in multiple peripheral muscle groups, yielding functional correction and improvement of the mdx dystrophic phenotype. Our data demonstrate proof-of-concept for this chimeric peptide approach in DMD splice correction therapy and is likely to have broad application.
JULY
23 - Abstracts of Papers at The Sixty-Third Annual Meeting of The Society of General Physiologists: MUSCLE in Health and Disease
Stretch-induced Muscle Damage and Duchenne Muscular Dystrophy D.G. ALLEN,1 O.L. GERVASIO,1 E.W. YEUNG,2 and N.P. WHITEHEAD1, 1School of Medical Sciences and Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia; 2Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
Duchenne muscular dystrophy is a severe muscle wasting disease caused by the absence of the cytoskeletal protein dystrophin. Experiments on the mdx mouse, which also lacks dystrophin, have shown that mdx muscles are particularly susceptible to stretch-induced damage. We have shown that Ca2+ entry through a channel blocked by streptomycin, Gd3+, or the spider venom peptide, GsMTx-4, contributes to stretch-induced muscle damage (Yeung, E.W., N.P. Whitehead, T.M. Suchyna, P.A. Gottlieb, F. Sachs, and D.G. Allen. 2005 J. Physiol. 562:367–380). The pharmacological and functional properties of this Ca2+ entry channel resemble a stretch-activated channel permeable to many cations known as SACNSC, which is expressed in muscle and for which a candidate gene is TRPC1 (Maroto, R., A. Raso, T.G. Wood, A. Kurosky, B. Martinac, and O.P. Hamill. 2005. Nat. Cell Biol. 7:179–185). We have also shown that Ca2+ entry into mdx muscle fibers can also be stimulated by H2O2 and entry is also blocked by streptomycin, suggesting that the physiological activator of SACNSC may be reactive oxygen species (ROS). To gain further understanding of the properties of TRPC1, it was expressed in C2 myoblasts. When expressed alone, it did not appear in the surface membrane, but when coexpressed with caveolin-3, the proteins co-located in the membrane (Gervásio, O.L., N.P. Whitehead, E.W. Yeung, W.D. Phillips, and D.G. Allen. 2008. J. Cell Sci. 121:2246–2255). When TRPC1 was located in the membrane, it could be activated by H2O2, causing Ca2+ influx that was blocked by PP2, an inhibitor of src kinase. These data suggest that in muscular dystrophy, excessive production of ROS might open SACNSC channels by activating src kinase. These pathways offer several new targets for therapy of muscular dystrophy.
23 - Utility of cystatin C to monitor renal function in duchenne muscular dystrophy
23 - Scoliosis repair in a teenager with Duchenne's muscular dystrophy: who calls the shots?
23 - Efficacy and tolerance of gastrostomy feeding in Duchenne muscular dystrophy
23 - Effect of VEGF on the Regenerative Capacity of Muscle Stem Cells in Dystrophic Skeletal Muscle
23 - Cardiac consequences to skeletal muscle-centric therapeutics for Duchenne muscular dystrophy
2 - (Am J Med Genet Part A 149A:1499-1503, 2009) Somatic mosaicism for Duchenne dystrophy: Evidence for genetic normalization mitigating muscle symptoms
Kesari A, Neel R, Wagoner L, Harmon B, Spurney C, Hoffman EP
We describe a young adult male presenting with cardiac failure necessitating cardiac transplantation 7 months after presentation. Skeletal muscle biopsy showed mosaic immunostaining for dystrophin. DNA studies showed somatic mosaicism for a nonsense mutation in the dystrophin gene (Arg2905X). The frequency of normal versus mutant genes were determined in blood/DNA (50:50), muscle/DNA (80:20) and muscle/mRNA (90:10). These data are consistent with genetic normalization processes that may biochemically rescue skeletal muscle in male somatic mosaic patients mitigating muscle symptoms (gradual loss of dystrophin-negative skeletal muscle tissue replaced by dystrophin-positive stem cells). To our knowledge, this is only the second reported case of a clinically ascertained patient showing somatic mosaicism for Duchenne muscular dystrophy (DMD). We hypothesize that many somatic mosaic males for DMD exist, yet they are not detected clinically due to genetic normalization. Somatic mosaicism for DMD should be considered in acute heart failure with dilated cardiomyopathy, as genetic normalization in heart is unlikely to occur.
JUNE
30 - (Unpublished data, 2009) The effect of losartan in the skeletal muscle morphology of Golden Retriever Muscular Dystrophy: a promising drug for dystrophic muscle regeneration? *
Marina Brito da Silva - research conducted to get the Master of Science in Department of Surgery of the Sao Paulo University, Faculty of Veterinary Medicine and Animal Science, Sao Paulo, SP, Brazil under orientation of Maria Angelica Miglino.
Duchenne Muscular Dystrophy (DMD) has the substitution of the muscle by connective tissue as its most relevant characteristic. Once fibrotic proliferation is a major obstacle to the efficacy of therapy for muscular dystrophies, early interventions to prevent it will probably be necessary as part of an effective treatment. A significant correlation between fibrosis and the expression of TGF-beta 1, a multifunctional cytokine, in Duchenne muscular Dystrophy has been reported, emphasizing the role of cytokine in the development of muscle fibrosis, and suggesting it as target for fibrosis therapies. In this study we evaluated the effect of losartan over the development of connective tissue on the skeletal musculature of the canine model GRMD. One dystrophic dog was previously used in the pilot study to estipulate the dosage and side effects caused by losartan. Five dystrophic dogs, two male and two female and one control animal were used in the experiment. A dose of 50mg of losartan was orally given once a day. The clinical and laboratorial exams did not show any adverse effect through the experimental period. Therefore losartan utilization showed to be safe therapy. Muscle biopsy fragments have been removed before starting losartan (T0) and after (Tf) were used for histology and TGF beta-1 imunohistochemistry to compare this two times. The evaluations range of motion and limb circunference measures within imunohistochemistry and collagen quantification results helped to infer about losartan effect in the dystrophyc muscle fibrosis. Range of motion and limb circumference values did not show statistical difference. Although the percentage of connective tissue deposition area in the animals in Tf was statistically lower lower than T0. The decrease of TGF beta-1 signalization showed in imunohystochimestry pictures within the decrease of connective tissue deposition, after losartan, suggest an inhibitory effect of this medication through this cytokine in the studied GRMD muscle.
* NOTE: the dogs were treated for 2 months on average.
26 - (Bone, 2009) Duchenne and Becker muscular dystrophies: A 4-year longitudinal follow-up study of bone mineral density
A.C. Söderpalma, A.K. Kroksmarka, P. Magnussonb, A.C. Åhlandera, J. Karlssona, M. Tuliniusa, D. Swolin-Eidea - Sweden
Duchenne muscular dystrophy (DMD) is an inherited X-linked recessive disorder that leads to reduced bone turnover and an increased risk of osteoporosis due to progressive muscular impairment. Becker muscular dystrophy is caused by a deletion in the same gene, but shows a relatively milder clinical course. Muscle mass and bone mass are closely related and it is known that inactivity and immobilisation lead to loss of mineral from the skeleton. Six boys with Becker (10.8–18.9 years at baseline) and 18 boys with DMD (2.3–19.7 years at baseline) were followed 4 years with respect to areal bone mineral density (BMD) in the hip and axial skeleton measured by DXA and calcaneal BMD measured by DXL Calscan BMD at all sites; total body (TB) (P=0.0002), TB head excluded (TBHE) (P<0.0001), spine (P=0.0001), hip (P=0.002) and calcaneal (P<0.0001), were significantly lower in the DMD group compared with Becker at baseline and follow-up for all sites, P<0.0001. Bone mineral accretion was significantly less in the DMD group at all sites after the study period in comparison with Becker; TB (P=0.002), TBHE (P<0.0001), spine (P=0.0001), hip (P=0.002) and calcaneal (P<0.05). BMD increased at all sites in Becker during the follow-up period, but only significantly for TB and spine, P<0.05. A smaller increase was found in the DMD group for TB, TBHE and spine, P<0.05; but decreased BMD in the hip (P<0.05) after 4 years. Summary: A greater bone mineral accretion was observed in the Becker group in comparison with DMD and we even observed a decreased hip BMD in the DMD group after the study period. We suggest that these findings could, in part, be explained by the better preserved muscle strength in Becker and the earlier appearing weakness in DMD of the lower extremities compared with the upper body.
25 - (Bone, 2009) Mechanisms underlying low bone density in muscular dystrophy
A. Rufoa, A.Del Fattorea,M.L.Bianchib, L.Morandif, E. Bertinic, A.Musaròd, S. Ferrarie, D. Pierroze, M. Capullia, N. Ruccia, F. De Benedettic, A. Tetia - Italy
Muscular dystrophies are characterized by inflammation, osteoporosis and increased risk of fractures other than myofiber necrosis and reduced muscular strength. We observed muscular atrophy and bone loss in mice overexpressing the pro-inflammatory cytokine IL-6 and propose that IL-6 may link the muscular and the bone phenotype in muscular dystrophies. Duchenne Muscular Dystrophy (DMD) is an X-linked disease due to mutations of the dystrophin gene. In DMD patients, we observed increased IL-6 in muscle biopsies and in sera. Similar to osteoblasts from IL-6 overexpressing mice and to osteoblasts treated with IL-6, human osteoblasts exposed to DMD sera failed to mineralize the extracellular matrix and showed reduced Osterix and Osteocalcin mRNA expression, despite normal alkalinephosphatase activity and Runx2 mRNA. The circulating RANKL/OPG protein ratio was low in DMD patients and inversely correlated with bone density. Transcriptional analysis revealed a similar reduction in RANKL/OPG ratio and increased IL-6 in osteoblasts exposed to DMD sera, along with up-regulation of further 26 genes and downregulation of further 90 genes associated with osteoblast function and osteoblast–osteoclast cross-talk. Despite low RANKL/OPG ratio, peripheral blood monocytes from patients and those from healthy donors exposed to DMD sera exhibited increased osteoclastogenesis similar to that observed in IL-6 overexpressing mice. In addition, mature osteoclasts expressed dystrophin, co-localized with F-actin in podosomes and actin rings, suggesting a role in cytoskeletal remodelling and bone resorption. Dystrophin-deficient mice (MDX) showed reduced tibial trabecular and cortical bone compared to WT, due to decreased osteoblast and increased osteoclast activity. Similar increase of osteoclast activity was observed in MDX calvariae. These latter bones are not subjected to muscular traction, suggesting systemic induction of osteoclastogenesis. We therefore propose that, besides mechanical failure, additional factors induce low bone density in DMD, among which we hypothesize relevant roles for systemic IL-6 and osteoclast dystrophin.
25 - (Bone, 2009) Trends in bone mineral density in children with Duchenne Muscular Dystrophy before and after glucocorticoid therapy
A. Peacock, B. Oldroyd, A. Shaw, A.M. Childs, T Mushtaq - UK
Background: Duchenne Muscular Dystrophy (DMD) is characterised by progressive muscle wasting. Glucocorticoids (GCs) are used to slow the progression of the disease and prolong ambulation, but they also suppress growth and bone metabolism. Aim: A retrospective study that assessed serial changes in body composition and bone mineral density (BMD) as measured by dualenergy absorptiometry (DXA) in boys with DMD before and after GC treatment. Method: 11 boys were treated with prednisolone (0.75 mg/kg/ day: 10 days on/10 days off). Standard deviation scores (SDS) were calculated from local reference data for total body BMD (TBBMD), Total Body Less Head BMD (TBLH), Lumbar Spine (LS) BMD (LSBMD) and LS Bone Mineral Apparent Density (LSBMAD). Serial scans were repeated if symptomatic or initial low BMD. Results:
By scan 3 there was a trend for decreasing BMD at all sites. The TBLH is lower than the TBBMD indicating the influence of the skull in the growing child. The LSBMAD is lower than the TBBMD which could reflect the detrimental GC effects on trabecular bone. Due to the limited number of scans there were no significant differences in the DXA BMD results. As there is no control group it is not possible to compare the effects of GC to those with reducing mobilisation. Conclusion: Serial BMD measurements in boyswith DMD may show a progressive reduction in bone density with time. (⁎Pre-steroids.)
25 - (Bone, 2009) Bone density and bone metabolism in Duchenne Muscular Dystrophy
S. Vaia, L. Morandib, M.L. Bianchia - Italy
A frequent complication of Duchenne Muscular Dystrophy (DMD) is an increased susceptibility to fractures, causing immobilization and worsening of muscle hypotrophy and weakness. There are very few data on bone density (BMD) and bone metabolism in DMD patients and it is unknown if DMD per se – independently of glucocorticosteroid (GC) treatment – causes bone metabolism derangements and osteoporosis. In a group of 15 DMD children (aged 3–6 years), we evaluated BMD (with DXA) and bone metabolism before starting GCs, at baseline and after 6 and 12 months. In most children, BMD (spine and total body) was lower than normal for age, and in 8 (53.3%) it was significantly reduced (spine BMAD Z-score −2.4±0.6). Low BMD was present in 2 (28.5%) of the 7 children aged 3– 4 years, but in 6 of 8 (75%) of those aged 5–6 years. We also measured the pituitary-adrenal response with a 1 mg dexamethasone overnight suppression test, to evaluate whether the acute response to GCs could predict the long-term GCs impact on bone. The suppressed morning serum cortisol and the increased insulin levels were correlated with changes in BMD and in bone turnover markers during the 12-month follow-up. Upon multiple regression analysis, cortisol suppression and insulin increase were correlated with spine BMD Z-score (p=0.03), BMD loss rate (p=0.02), and bone marker changes (NTx: p=0.02; BSAP: p=0.03). In children with suppressed cortisol levels below 50th percentile, spine BMD Z-score decreased (−0.7±0.5) after 12 months. These findings indicate that: BMD is often decreased in DMD even before GCs; Reduced BMD might be more prevalent in older than in younger boys; Cortisol suppression test may be used to evaluate the sensitivity to exogenous GCs and their ability to induce bone side effects.
Research Grant UILDM - TELETHON (GUP 0300537).
25 - (Bone, 2009) Fracture history and bone mineral density (BMD) in children with Duchenne Muscular Dystrophy (DMD) and cerebral palsy (CP)
H.H. Kecskemethy, H.T. Harckea,, S.J. Bachracha, - USA
This study examines the history of fracture in relation to BMD and ambulation as measured by group mean Z-scores for lateral distal femur (LDF) and lumbar spine (LS) in three cohorts of children: non-ambulatory DMD and CP and ambulatory DMD. Fracture history of 49 children with DMD (23 ambulatory, 26 nonambulatory) and 47 non-ambulatory children with CP was obtained at time of first BMD assessment. Mean LS and LDF Zscores (3 regions) were calculated for positive or negative fracture history.
Positive fracture history in ambulatory DMD was 4/23 with half occurring in the lower extremity. Non-ambulatory DMD fracture history was positive in 13/26 (with 69% in the lower extremity). Fracture history was positive in 19/47 of the CP group (lower extremity 89%). Both non-ambulatory groups had mean LDF Z-scores well below normal regardless of fracture history, whereas ambulatory DMD subjects had LDF values in normal/low-normal range. Ambulatory status did not influence LS Z-scores of DMD patients compared to LDF Z-scores. Differences in LDF Z-scores were noted in fracture and nonfracture groups except for the non-ambulatory DMD group. LDF BMD is easily obtained on disabled children and provides another DXA parameter for use in evaluating fracture risk.
25 - (Bone, 2009) Bone strength in boys with Duchenne Muscular Dystrophy (DMD): A longitudinal study
N.J. Crabtreea,b, K.A. Wardc, H. Roperd, J.E. Adamsc, M.Z. Mughale, N.J. Shawb - UK
DMD is the most common childhood neuromuscular disorder causing loss of ambulation in early life. Steroids are currently used to improve muscle strength and prolong ambulation although the effect on bone health in this group of children is still unclear. The aim of this study was to compare the longitudinal changes in bone strength in healthy children with those observed in children with DMD, who either remained ambulant or who lost independent ambulation during the period of follow-up. Forty children were studied, 17 healthy boys (9.1±1.5 years) and 23 boys with DMD (8.6±2.1 years), taking intermittent steroids. PQCT was used to measure bone geometry, density and strength of the non-dominant tibia. Measurements were made at the distal metaphysis and mid-diaphysis sites. Data were adjusted for age, height and duration of steroids. After 15.0±3.1 months, 7 DMD boys lost independent ambulation. Longitudinal growth between the groups remained constant. In DMD boys, who remained ambulant, there was a slowing down in periosteal bone growth at the mid diaphysis (0.8 vs. 2.6 mm2/month; p<0.05). Whereas for DMD boys who lost ambulation, there was a significant reduction in the rate of bone growth at the mid-diaphysis (0.4 mm2/month; p<0.05) and at the distal metaphysis (2.8 vs. 6.2 mm2/month; p<0.05). In contrast, the rate of change in bone density at the distal metaphysis (−2.8 vs. 0.3 mg/cm3/month; p<0.001) and cortical bone mass (−0.2 vs. 1.3 mg/mm/month; p<0.001) and stress-strain index (2.0 vs. 9.9 mm3/month; p<0.05) at the mid diaphysis was only significantly different from the healthy boys in the 7 boys who lost independent ambulation. These data suggest that ambulation and hence muscle function and gravitational load have the greatest effect on bone strength and density in boys with DMD. Whilst they remain ambulant the effect of the relatively high dose steroids appears to be negligible. However, when they eventually lose independent ambulation significant losses in bone strength occur as a direct result of diminished periosteal bone growth and bone mineral accrual.
22 - (FEBS Journal. 276 Suppl. 1:349, July 2009) Chemotherapy of Duchenne’s muscular dystrophy
Y. Urade, M. Hayashi, T. Maruyama, S. Kamauchi, I. Mohri and K. Aritake - Japan
Duchenne muscular dystrophy (DMD) is an X-linked muscular abnormality caused by the loss of dystrophin and is one of the most gravely genetic disorders. We have recently found that hematopoietic prostaglandin (PG) D synthase (H-PGDS) was induced in grouped necrotic muscle fibers in DMD patients (Okinaga T. et al., Acta Neuropathol. 2002; 104: 377–384). We developed novel H-PGDS inhibitors based on the X-ray crystallographic analysis of human H-PGDS complexed with its prototype inhibitor, HQL-79 (Aritake K. et al., J. Biol. Chem. 2006: 281: 15277–15286). In this study, we developed a novel therapy for DMD by inhibition of H-PGDS. H-PGDS was localized in the necrotic muscle fibers and accumulated macrophages in mdx mice. Oral administration of H-PGDS inhibitors for 5 days prevented the expansion of muscular necrosis in an mdx mouse model, as measured by X-ray computed tomography (CT) imaging enhanced by non-ionic contrast media. The treatment with H-PGDS inhibitors also decreased the expression of mRNAs of pro-inflammatory cytokines. These results indicate that PGD2 produced by H-PGDS plays important pathological roles on the expansion of muscle necrosis. H-PGDS inhibitor also accelerated the accumulation and activation of macrophages in the necrotic area. These results indicate that PGD2 produced by H-PGDS is involved in the expansion of muscle necrosis in DMD and that inhibition of H-PGDS is a novel therapy for DMD.
9 - (Journal of Neuroimmunology) Imatinib mesylate ameliorates the dystrophic phenotype in exercised mdx mice
João Carlos da Silva Bizario, Daniel Giuliano Cerri , Lilian Cataldi Rodrigues, Gislane L.V. Oliveira, Auro Nomizo, Daniela Dover de Araujo, Paula Sakemi Fukuhara, Juliana Caldas Ribeiro, Fabíola Attié de Castro, Maria Cristina Ramos Costa - Brazil
Myofiber degeneration, inflammation, and fibrosis are remarkable features of Duchenne muscular dystrophy. We hypothesized that the administration of imatinib mesylate, an inhibitor of tyrosine kinase and TGF-β profibrogenic activity, could improve the muscular conditions in mdx mice. Four-week old mdx mice were treated and exercised for 6 weeks. Gastrocnemius and diaphragm histopathology, strength, creatine kinase, and cytokine levels were evaluated. The treated group presented increased muscular strength and decreased CK levels, injured myofibers, and inflammatory infiltrates. Pro-inflammatory cytokines and TGF-β were also reduced, while IL-10 was increased, suggesting an immunomodulatory effect of imatinib, which can ameliorate the dystrophic phenotype in mdx mice.
3 - Corticosteroid effects on blood gene expression in Duchenne muscular dystrophy
3 - Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts
MAY
26 - Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice
25 - Pulmonary Management of Pediatrics Patients with Neuromuscular Disorders - 30th annual Carrell-Krusen Neuromuscular Symposium - 2008
article 1 article 2 article 3 article 4 article 5 article 6 article 7 article 8 article 9
23 - (Bone 44 (2009) S234–S252 S237) Mechanisms inducing low bone density in Duchenne Muscular Dystrophy
A. Rufoa, A. Del Fattorea, M.L. Bianchib, L. Morandic, E. Bertinid, A. Musaròe, S. Ferrarif, D. Pierrozf, M. Capullia, N. Ruccia, F. De Benedettid, A. Tetia - Italy
Duchenne Muscular Dystrophy (DMD) is induced by mutations of the dystrophin gene that cause disruption of sarcolemmal integrity, myofiber necrosis and inflammation. Besides muscular damage, patients show osteoporosis and increased risk of fractures, that we hypothesize are due to determinants other than mechanical unloading. Consistently, in DMD children we observed increased levels of IL-6, an inflammatory cytokine known to reduce osteoblast and increase osteoclast activity in vitro and in pre-pubertal mice. When exposed to DMD sera, osteoblasts from healthy donors failed to mineralize matrix nodules and showed reduced osterix and osteocalcin mRNA expression, despite normal alkaline phosphatase activity and Runx2 mRNA. Surprisingly, the circulating RANKL/OPG protein ratio was low in DMD patients and inversely correlated with bone density. Similar reduced transcriptional RANKL/OPG ratio and increased IL-6 mRNA expression were observed in osteoblasts exposed to DMD sera, along with up-regulation of further 26 genes and down-regulation of further 90 genes associated with osteoblast function and osteoblast–osteoclast cross-talk. Despite low RANKL/OPG ratio, peripheral blood monocytes from patients and those from healthy donors exposed to DMD sera exhibited increased ability to differentiate into osteoclasts. Mature osteoclasts expressed dystrophin, co-localized with F-actin, first in filopodia, then in podosomes and finally in actin rings, suggesting a role of the dystrophin complex in the organization of the sealing zone cytoskeleton. Dystrophin-deficient mice (MDX) showed reduced cortical and trabecular bone compared to WT, as assessed in both genders by microCT and histomorphometry, which was due to decreased osteoblast and increased osteoclast activity. Interestingly, similar alterations, especially for the osteoclast lineage, were observed in calvariae from MDX mice, in which mechanical forces play negligible roles as they are not subjected to muscular traction. In conclusion, we suggest that osteoporosis in DMD patients could be explained by factors other than mechanical unloading due to muscular failure, among which we underscored a possible role for IL-6 and osteoclast dystrophin.
19 - (Am J Phys Med Rehabil 2009;88:502–522) Dysregulated Intracellular Signaling and Inflammatory Gene Expression During Initial Disease Onset in Duchenne Muscular Dystrophy
Evans NP, Misyak SA, Robertson JL, Bassaganya-Riera J, Grange RW - USA
Duchenne muscular dystrophy is a debilitating genetic disorder characterized by severe muscle wasting and early death in affected boys. The primary cause of this disease is mutations in the dystrophin gene that result in the absence of the protein dystrophin and the associated dystrophin-glycoprotein complex in the plasma membrane of muscle fibers. In normal muscle, this complex forms a link between the extracellular matrix and the cytoskeleton that is thought to protect muscle fibers from contraction-induced membrane lesions and to regulate cell signaling cascades. Although the primary defect is known, the mechanisms that initiate disease onset have not been characterized. Data collected during early maturation suggest that inflammatory and immune responses are key contributors to disease pathogenesis and may be initiated by aberrant signaling in dystrophic muscle. However, detailed time course studies of the inflammatory and immune processes are incomplete and need to be characterized further to understand the disease progression. The purposes of this review are to examine the possibility that initial disease onset in dystrophin-deficient muscle results from aberrant inflammatory signaling pathways and to highlight the potential clinical relevance of targeting these pathways to treat Duchenne muscular dystrophy.
19 - (Experimental Cell Research, 2009) Engraftment of Mesenchymal Stem Cells into Dystrophin-Deficient Mice is not Accompanied by Functional Recovery
Eun Ji Gang, Radbod Darabi, Darko Bosnakovski, Zhaohui Xu, Kristine E. Kamm, Michael Kyba, Rita C.R. Perlingeiro - USA
Mesenchymal stem cell preparations have been proposed for muscle regeneration in musculoskeletal disorders. Although MSCs have great in vitro expansion potential and possess the ability to differentiate into several mesenchymal lineages, myogenesis has proven to be much more difficult to induce. We have recently demonstrated that Pax3, the master regulator of the embryonic myogenic program, enables the in vitro differentiation of a murine mesenchymal stem cell line (MSCB9- Pax3) into myogenic progenitors. Here we show that injection of these cells into cardiotoxin-injured muscles of immunodeficient mice leads to the development of muscle tumors, resembling rhabdomyosarcomas. We then extended these studies to primary human mesenchymal stem cells (hMSCs) isolated from bone marrow. Upon genetic modification with a lentiviral vector encoding PAX3, hMSCs activated the myogenic program as demonstrated by expression of myogenic regulatory factors. Upon transplantation, the PAX3-modified MSCs did not generate rhabdomyosarcomas but rather, resulted in donor-derived myofibers. These were found at higher frequency in PAX3-transduced hMSCs than in mock-transduced MSCs. Nonetheless, neither engraftment of PAX3-modified or unmodified MSCs resulted in improved contractility. Thus these findings suggest that limitations remain to be overcome before MSC preparations result in effective treatment for muscular dystrophies.
16 - Identification of three distinguishable phenotypes in golden retriever muscular dystrophy
My comment: It is possible to conclude something with only four months of treatment in boys with DMD?
1 - Research approaches for a Therapy of Duchenne Muscular Dystrophy. Part 1: Exon Skipping
APRIL
29 - (Human Molecular Genetics, 2009) Matrix Metalloproteinase-9 Inhibition Ameliorates Pathogenesis and Improves Skeletal Muscle Regeneration in Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disorder of skeletal muscle caused by mutation in dystrophin gene. Although the degradation of skeletal muscle extracellular matrix, inflammation, and fibrosis are the common pathological features in DMD, the underlying mechanisms remain poorly understood. In this study, we have investigated the role and the mechanisms by which increased levels of matrix metalloproteinase-9 (MMP-9) protein causes myopathy in dystrophin-deficient mdx mice. The levels of MMP-9 but not tissue inhibitor of MMPs were drastically increased in skeletal muscle of mdx mice. Besides skeletal muscle, infiltrating macrophages were found to contribute significantly to the elevated levels of MMP-9 in dystrophic muscle. In vivo administration of a nuclear factor-kappa B (NF-B) inhibitory peptide, NBD, blocked the expression of MMP-9 in dystrophic muscle of mdx mice. Deletion of Mmp9 gene in mdx mice improved skeletal muscle structure and functions and reduced muscle injury, inflammation, and fiber necrosis. Inhibition of MMP-9 increased the levels of cytoskeletal protein β-dystroglycan and neural nitric oxide synthase and reduced the amounts of caveolin-3 and transforming growth factor-β in myofibers of mdx mice. Genetic ablation of MMP-9 significantly augmented the skeletal muscle regeneration in mdx mice. Finally, pharmacological inhibition of MMP-9 activity also ameliorated skeletal muscle pathogenesis and enhanced myofiber regeneration in mdx mice. Collectively, our study suggests that the increased production of MMP-9 exacerbates dystrophinopathy and MMP-9 represents as one of the most promising therapeutic targets for the prevention of disease progression in DMD.
23 - FASEB Meeting 2009:
1) Resveratrol feeding may be therapeutic for dystrophic skeletal muscle
Joshua T. Selsby1,2, Kevin Morine2, Klara Pendrak2, Z. Tian3, Erica Blanco3, Elisabeth R Barton3, H Lee Sweeney2. 1Animal Science, Iowa State University, Ames, IA, 2Physiology, 3Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA
Increased PGC-1α, either through a transgenic animal or gene transfer, provides therapeutic benefit to dystrophic skeletal muscle likely by increasing slow and oxidative protein expression. Resveratrol has been shown to activate SIRT-1, a known deacetylase capable of acting on and increasing the activity of PGC-1α. The purpose of this investigation was to determine if resveratrol supplementation could rescue dystrophic skeletal muscle. We hypothesized that daily feeding of resveratrol would improve muscle function in a similar fashion as PGC-1α overexpression. To test this hypothesis, 1-mo old mdx mice were either fed a control diet (Con) or a diet containing resveratrol at 100 mg/kg/day (Res) for 8 wks. Following intervention, absolute muscle mass was reduced 18-30% in the Res group compared to Con, however, body mass was also reduced in the Res group by 20% compared to Con. Relative muscle mass was similar between groups, except for the EDL, which was significantly smaller in the Res group. Solei in the Res group were more fatigue resistant than in the Con group, however, resistance to contraction induced injury was similar between groups. These data indicate that partial rescue of dystrophic pathology is possible through resveratrol. Perhaps, alternative sources or doses of resveratrol will provide a greater response.
2) Postnatal PGC-1α
over-expression improves muscle function in a mouse model of Duchenne muscular dystrophyJoshua T. Selsby1,2, Kevin Morine2, Klara Pendrak2, Z. Tian3, Erica Blanco3, Elisabeth R Barton3, H Lee Sweeney2. 1Animal Science, Iowa State University, Ames, IA, 2Physiology, 3Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA
PGC-1α has received a great deal of attention due to its potential to induce oxidative and slow proteins. The purpose of this investigation was to: 1) extend observations made in an initial study demonstrating transgenic over-expression of PGC-1α reduced dystrophic pathology 2) eliminate effects that could have taken place during development. Neonatal mdx mice were injected in the right hind limb with 1x1011 gc of AAV causing overexpression of PGC-1α and sacrificed at 4 (n=6) or 6 wks of age (n=5). Muscle mass was reduced in limbs over-expressing PGC- 1α at 4 and 6 wks, however, tetantic force and specific force in the soleus and EDL were either maintained or improved when compared to control limbs. PGC-1α over-expression caused EDLs to be more resistant to damage at 6 wks and the soleus to be more fatigue resistant at 4 and 6 wks compared to control limbs. PGC- 1α increased expression of slow proteins as utrophin was increased nearly 2-fold and type I myosin heavy chain nearly 3-fold as well as expression of oxidative proteins as cytochrome C and uncoupling protein-1 were increased approximately 2-fold, complex IV subunit IV (cytochrome C oxidase) was increased 1.5- fold and myoglobin was increased 3-fold in limbs over-expressing PGC-1α compared to control limbs. These data demonstrate the potential therapeutic role of the PGC-1α pathway for dystrophic skeletal muscle.
3) EUK-134, a synthetic superoxide dismutase/catalase mimetic, protects against loss of muscle mass/body mass in diaphragm and gastrocnemius in mdx mice
Jong-Hee Kim1, BR Macias2, C Canon2, S Courtney2, John M Lawler2. 1Health and Kinesiology, Texas A&M University, College Station, TX, 2Texas A&M University, College Station, TX Duchenne muscular dystrophy (DMD) is characterized by devastating muscle degeneration associated with oxidative stress, loss of contractile tissue, muscle atrophy, muscle weakness and increased fibrosis in respiratory and locomotor muscles. We tested the hypothesis that protection against oxidative stress via the catalase/superoxide dismutase mimetic EUK-134 will prevent reduction of diaphragm and skeletal muscle mass/body mass during the early inflammatory phase (20-28 days) in mdx mice. C57BL(wild type) and mdx mice were given EUK-134 (30mg/kg body weight/day, i.p., injection) for 8 days, beginning at 20 days of age. Body mass was significantly lower in mdx mice (-37.7%) than wild type but, EUK-134 increased body mass by 15.5% in mdx mice. Absolute muscle mass was lower in diaphragm (-44.3%), gastrocnemius (-53.6%), tibialis anterior (-58.1%) in mdx mice. Muscle mass/body mass was lower in diaphragm (-11.4%), gastrocnemius (-26.3%), tibialis anterior (-34.4%), but not heart, plantaris, soleus, extensor digitorum longus in mdx mice. EUK- 134 had a significant positive effect in protecting against reduced muscle mass/body mass in diaphragm (+38.7%) and gastrocnemius (+27.1%) in mdx mice. These data indicate that EUK-134 provide protection against reduced muscle mass/body mass in diaphragm and gastrocnemius in mdx mice during this early phase of muscular dystrophy.
4) Transgenic Overexpression of AlphaBeta1 Integrin Stimulates p70S6K Phosphorylation in Mice with a Severe Form of Muscular Dystrophy
Marni Della Boppart1, Stephen J Kaufman2. 1Kinesiology and Community Health, 2Cell and Developmental Biology, University of Illinois, Urbana, IL
MCK-driven transgenic expression of the alpha7 integrin can ameliorate pathology in a mouse model of Duchenne muscular dystrophy (mdx/utrn-/-) and thus compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the alpha7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remain to be established. PURPOSE: To determine a role for signaling in the amelioration of muscular dystrophy by alpha7 integrin. METHODS: Five wk wild type, mdx/utrn-/-, and alpha7BX2-mdx/utrn-/- gastrocnemius muscles (n=3-8/group) were dissected and extracted. Activation of PI3K, ILK, AKT, mTOR, p70S6K, GSK-3, and p38 was measured using in vitro activity assays or phosphospecific antibodies and western blotting. RESULTS: Significant increases in ILK activity (2.0-fold), AKT- (P) (2.3-fold), mTOR-(P) (57%), p70S6K-(P) (11.7-fold), and ERK-(P) (66%) were observed in dystrophic mdx/utrn-/- muscle compared to wild type. Significant decreases in GSK-3-(P) (57%) and p38-(P)(2.9-fold) were also observed. Most of these signaling events were similar in dystrophic mice over-expressing the alpha7 integrin. However, a further increase in p70S6K-(P) (18-fold) and decrease in GSK-3-(P)(3.7-fold) were detected in alpha7BX2- mdx/utrn-/- compared to wild type mice and these changes were significant compared to mdx/utrn-/- mice. CONCLUSION: The alpha7beta1 integrin confers a protective effect in dystrophic mice and increased p70S6K activity appears important to this.
5) Sildenafil Ameliorates Cardiomyopathy in mdx Mice
Candace M. Parchen1, Justin M. Percival2, Dao-Fu Dai3, Heidi N Gray1, Stanley C. Froehner2, Joseph A. Beavo1. 1Pharmacology, 2Physiology and Biophysics, 3Pathology, University of Washington, Seattle, WA
Duchenne muscular dystrophy (DMD) is the most prevalent type of muscular dystrophy and is the result of an X-linked mutation in the dystrophin gene. The progression of skeletal muscle damage is rapid in DMD patients and cardiomyopathy soon follows. We have investigated whether or not sildenafil citrate, a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction in dystrophin-null (mdx) mice, a mouse model of DMD. Using echocardiography, we show that chronic sildenafil treatment improves several functional deficits in the cardiac performance of aged mdx mice. Collagen VI levels are also lower in the hearts of sildenafil-treated mdx mice, suggesting a remodeling of the extracellular matrix. This is the first study to report a cardioprotective effect of PDE5 inhibition in aged mdx mice. Overall, the data suggest that PDE5 inhibitors could be a useful treatment for the cardiomyopathy suffered by DMD patients.
6) Blastocyst Injection of Wild Type Embryonic Stem Cells Induces Global Corrections in Mdx Mice
Joseph M Vitale, Elizabeth Stillwell, Farah Khadim, Genie Elson, Aneela Altaf, Joel Schneider, Ghassan Yehia, Diego Fraidenraich. Cell Biology & Molecular Medicine, University of Medicine & Dentistry of New Jersey, Newark, NJ
Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection also normalizes the amount of fat, a tissue that does not express dystrophin. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally.
7) Murine mammary tumor growth is blunted in dystrophin deficient mdx mice
Mary Pat Meaney, Robert W. Grange, Young H. Ju. Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA
Breast cancer (BC) is the second leading cause of cancer death in women in the US. Changes to the dystrophin glycoprotein complex (DGC), a multi-protein structure that likely plays mechanical and signaling roles, have been reported in BC cells. For example, expression of α- and β-dystroglycan appears to be inversely related to tumor stage. However, systemic absence of the DGC, as seen in the mdx mouse model of Duchenne muscular dystrophy, does not appear to induce spontaneous mammary tumor formation, indicating that presence of the DGC may be necessary for breast cancer initiation. We therefore hypothesized that breast tumor growth would be altered in mdx mice compared to wild type (C57BL/6) mice. We injected E0771 murine mammary adenocarcinoma cells into mdx and C57BL/6 mice. After 3 weeks of tumor growth, blood, skeletal muscles and tumors were collected and analyzed. Growth of E0771 tumors and serum content of migration and invasion chemokine markers, RANTES and MCP-1, was dramatically blunted in mdx mice. The dystrophin protein was not detectable in E0771 cells or tumors, suggesting that its expression, like that of other DGC components, may be suppressed or altered in this cancer cell line. The exact mechanism(s) of tumor inhibition in mdx mice is not presently known; however, our results suggest a mechanism for suppressing BC growth and progression may depend on the absence of one or more proteins in the DGC.
20 - Potential Muscular Dystrophy drug
Related article: Laminin-111 restores regenerative capacity in a mouse model for alpha7 integrin congenital myopathy
9 - (Am. J. Pathol., Apr 2009) Inhibition of prostaglandin D synthase suppresses muscular necrosis
Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D2. In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D2 signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy . We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor 1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D2 production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy .
MARCH
25 - Prednisone 10 days on/10 days off in patients with Duchenne muscular dystrophy
16 - (FASEB J. 23, 2009) Imatinib attenuates skeletal muscle dystrophy in mdx mice
Ping Huang, Xinyu S. Zhao, Matthew Fields, Richard M. Ransohoff, and Lan Zhou
-USADuchenne-Meryon muscular dystrophy (DMD) is the most common and lethal genetic muscle disease. Ameliorating muscle necrosis, inflammation, and fibrosis represents an important therapeutic approach for DMD. Imatinib, an antineoplastic agent, demonstrated antiinflammatory and antifibrotic effects in liver, kidney, lung, and skin of various animal models. This study tested antiinflammatory and antifibrotic effects of imatinib in mdx mice, a DMD mouse model. We treated mdx mice with intraperitoneal injections of imatinib at the peak of limb muscle inflammation and the onset of diaphragm fibrosis. Controls received PBS vehicle or were left untreated. Muscle necrosis, inflammation, fibrosis, and function were evaluated by measuring serum CK activity, endomysial CD45 immunoreactive inflammation area, endomysial collagen III deposition, and hind limb grip strength. Phosphorylation of the tyrosine kinase targets of imatinib was assessed by Western blot in diaphragm tissue and in primary cultures of peritoneal macrophages and skeletal muscle fibroblasts. Imatinib markedly reduced muscle necrosis, inflammation, and fibrosis, and significantly improved hind limb grip strength in mdx mice. Reduced clinical disease was accompanied by inhibition of c-abl and PDGFR phosphorylation and suppression of TNF-alpha and IL-1 expression. Imatinib therapy for DMD may hold promise for ameliorating muscle necrosis, inflammation, and fibrosis by inhibiting c-abl and PDGFR signaling pathways and downstream inflammatory cytokine and fibrotic gene expression.
14 - (Journal of Orthopaedic Research, 27(4): 421-6, 2009) Systemic human minidystrophin gene transfer improves functions and life span of dystrophin and dystrophin/utrophin-deficient mice
Bing Wang , Juan Li , Freddie H. Fu , Xiao Xiao - USA
Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disease, caused by mutations in the dystrophin gene. No efficacious treatment is currently available. Here we report AAV vector systemic delivery and therapeutic benefits of the functional human minidystrophin gene in a severe and more reliable DMD mouse model, the dystrophin/utrophin double deficiency mouse (dys-/-:utrn-/-, dKO). These mice show many pathologic and phenotypic signs typical of DMD in humans including kyphosis and shorter life span, all of which are not seen in the mdx mice due to their utrophin upregulation that partially compensates the loss of dystrophin functions and leads to mild phenotypes. The therapeutic value of this new approach was demonstrated in both mdx and dKO murine models, in which we observed highly efficient minidystrophin gene expression, ameliorated muscle pathologies, improvement in growth and motility, inhibition of spine and limb deformation, and prolongation of life span.
14 - The art of medicine - Complicated lessons: Lorenzo Odone and medical miracles
12 - Blastocyst injection of wild type embryonic stem cells induces global corrections in mdx mice
7 - (J Bone Joint Surg Br Proceedings, Mar 2009; 91-B: 70) THE USE OF APROTININ IN SCOLIOSIS CORRECTION SURGERY IN PATIENTS WITH DUCHENNE MUSCULAR DYSTROPHY
O.R. Richards; M. DeMatas; C. Bruce; J. Dorgan; and M. Cunliffe - Switzerland
Aprotinin has been shown to reduce blood loss in a number of surgical specialities. Patients with Duchenne Muscular Dytrophy(DMD) bleed more during surgical procedures than patients without this condition. The aim of this study was to evaluate the effect of aprotinin in reducing blood loss in scoliosis correction surgery in patients with DMD.
A retrospective analysis of case notes was performed. Thirty two patients diagnosed with DMD who underwent surgical correction for scoliosis over the last 25 years were included. All patients underwent posterior spinal fusion and instrumentation, between the levels T3 and L3. All procedures were carried out by the same lead surgeon. Patient age, body weight, length of procedure, and estimated blood loss were recorded. Blood loss as a percentage of total circulating volume was calculated and compared between patients who had not received aprotinin (seven patients), and those who did (25 patients). Blood loss as a percentage of total circulating volume in the group of patients with aprotinin (range 37% – 107% mean 67%) was significantly lower (P<0.05) than the group without aprotinin (range 67% – 157% mean 111%). There was found to be no statistically significant relationship between blood loss and length of procedure. There was no statistically significant difference in the duration of the procedure between the two groups of patients. Despite the small number of patients this study shows a beneficial effect for aprotinin in reducing blood loss during scoliosis correction surgery in patients with DMD.
7 - Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy
FEBRUARY
28 - Autonomic function in patients with Duchenne muscular dystrophy
28 - (Clinical Neurology and Neurosurgery, 2009) Diagnostic utility of skin biopsy in dystrophinopathies
Nadeem Tanveer, Mehar C. Sharma, Chitra Sarkar, Sheffali Gulati, Veena Kalra, Sumit Singh and Rohit Bhatia - India
20 - (Journal of the Neurological Sciences, 2009) Intramuscular renin–angiotensin system is activated in human muscular dystrophy
Guilian Sun, Kazuhiro Haginoya, Hongmei Dai, Yoko Chiba, Mitsugu Uematsu, Naomi Hino-Fukuyo, Akira Onuma, Kazuie Iinuma, Shigeru Tsuchiya - Japan
To investigate the role of the muscular renin–angiotensin system (RAS) in human muscular dystrophy, we used immunohistochemistry and Western blotting to examine the cellular localization of angiotensin-converting enzyme (ACE), the angiotensin II type 1 receptor (AT1) and the angiotensin II type 2 receptor (AT2) in muscle biopsies from patients with Duchenne muscular dystrophy (DMD), Beckermuscular dystrophy (BMD), and congenital muscular dystrophy (CMD). In normal muscle, ACE was expressed in vascular endothelial cells and neuromuscular junctions (NMJs), whereas AT1 was immunolocalized to the smooth muscle cells of blood vessels and intramuscular nerve twigs. AT2 was immunolocalized in the smooth muscle cells of blood vessels. These findings suggest that the RAS has a functional role in peripheral nerves and NMJs. ACE and AT1, but AT2 immunoreactivity were increased markedly in dystrophic muscle as compared to controls. ACE and the AT1 were strongly expressed in the cytoplasm and nuclei of regenerating muscle fibers, fibroblasts, and in macrophages infiltrating necrotic fibers. Double immunolabeling revealed that activated fibroblasts in the endomysium and perimysium of DMD and CMD muscle were positive for ACE and AT1. Triple immunolabeling demonstrated that transforming growth factor-β1 (TGF-β1) and ACE were colocalized on the cytoplasm of activated fibroblasts in dystrophic muscle. Furthermore, Western blotting showed increases in the expression of AT1 and TGF-β1 protein in dystrophic muscle, which coincided with our immunohistochemical results. The overexpression of ACE and AT1 in dystrophic muscle would likely result in the increased production of Ang II, which may act on these cells in an autocrine manner via AT1. The activation of AT1 may induce fibrous tissue formation through overexpression of TGF-β1, which potently activates fibrogenesis and suppresses regeneration. In conclusion, our results imply that the intramuscular RAS–TGF-β1 pathway is activated in human muscular dystrophy and plays a role at least partly in the pathophysiology of this disease.
8 - (Nature Medicine, 2009) Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle
Andrew M Bellinger, Steven Reiken, Christian Carlson, Marco Mongillo, Xiaoping Liu, Lisa Rothman, Stefan Matecki, Alain Lacampagne
& Andrew R Marks - USADuchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex, leading to pathological calcium (Ca2+)-dependent signals that damage muscle cells1–5. We have identified a structural and functional defect in the ryanodine receptor (RyR1), a sarcoplasmic reticulum Ca2+ release channel, in the mdx mouse model of muscular dystrophy that contributes to altered Ca2+ homeostasis in dystrophic muscles. RyR1 isolated from mdx skeletal muscle showed an age-dependent increase in S-nitrosylation coincident with dystrophic changes in the muscle. RyR1 S-nitrosylation depleted the channel complex of FKBP12 (also known as calstabin-1, for calcium channel stabilizing binding protein), resulting in ‘leaky’ channels. Preventing calstabin-1 depletion from RyR1 with S107, a compound that binds the RyR1 channel and enhances the binding affinity of calstabin-1 to the nitrosylated channel, inhibited sarcoplasmic reticulum Ca2+ leak, reduced biochemical and histological evidence of muscle damage, improved muscle function and increased exercise performance in mdx mice. On the basis of these findings, we propose that sarcoplasmic reticulum Ca2+ leak via RyR1 due to S-nitrosylation of the channel and calstabin-1 depletion contributes to muscle weakness in muscular dystrophy, and that preventing the RyR1-mediated sarcoplasmic reticulum Ca2+ leak may provide a new therapeutic approach.
While Focusing On Heart Disease, Researchers Discover New Tactic Against Fatal Muscular Dystrophy
JANUARY
7 -
β-hydroxyl-β-methylbutyrate (HMB) stimulates
myogenic cell proliferation, differentiation and survival via the MAPK/ERK and
PI3K/Akt pathways
NOTE: HMB is a supplement.