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2014 NEWS
DECEMBER
30 - (Lancet Neurology, 2014) Eplerenone for early cardiomyopathy in Duchenne muscular dystrophy: a randomised, double-blind, placebo-controlled trial
Subha V Raman, Kan N Hor, Wojciech Mazur, Nancy J Halnon, John T Kissel, Xin He, Tam Tran, Suzanne Smart, Beth McCarthy, Michae l D Taylor, ohn L Jeff eries, Jill A Rafael-Fortney, Jeovanna Lowe, Sharon L Roble, Linda H Cripe - USA
Cardiomyopathy is a leading cause of death in patients with Duchenne muscular dystrophy and myocardial damage precedes decline in left ventricular systolic function. We tested the efficacy of eplerenone on top of background therapy in patients with Duchenne muscular dystrophy with early myocardial disease.
In this randomised, double-blind, placebo-controlled trial, boys from three centres in the USA aged 7 years or older with Duchenne muscular dystrophy, myocardial damage by late gadolinium enhancement cardiac MRI and preserved ejection fraction received either eplerenone 25 mg or placebo orally, every other day for the first month and once daily thereafter, in addition to background clinician-directed therapy with either angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB). Computer-generated randomisation was done centrally using block sizes of four and six, and only the study statistician and the investigational pharmacy had the preset randomisation assignments. The primary outcome was change in left ventricular circumferential strain (Ecc) at 12 months, a measure of contractile dysfunction. Safety was established through serial serum potassium levels and measurement of cystatin C, a non-creatinine measure of kidney function. This trial is registered with ClinicalTrials.gov, number NCT01521546.
Between Jan 26, 2012, and July 3, 2013, 188 boys were screened and 42 were enrolled. 20 were randomly assigned to receive eplerenone and 22 to receive placebo, of whom 20 in the eplerenone group and 20 in the placebo group completed baseline, 6-month, and 12-month visits. After 12 months, decline in left ventricular circumferential strain was less in those who received eplerenone than in those who received placebo (median ΔEcc 1·0 [IQR 0·3–2·2] vs 2·2 [1·3–3·1]; p=0·020). Cystatin C concentrations remained normal in both groups, and all non-haemolysed blood samples showed normal potassium concentrations. One 23-year-old patient in the placebo group died of fat embolism, and another patient in the placebo group withdrew from the trial to address long-standing digestive issues. All other adverse events were mild: short-lived headaches coincident with seasonal allergies occurred in one patient given eplerenone, flushing occurred in one patient given placebo, and anxiety occurred in another patient given placebo.
In boys with Duchenne muscular dystrophy and preserved ejection fraction, addition of eplerenone to background ACEI or ARB therapy attenuates the progressive decline in left ventricular systolic function. Early use of available drugs warrants consideration in this population at high risk of cardiac death, but further studies are needed to determine the effect of combination cardioprotective therapy on event-free survival in Duchenne muscular dystrophy.
30 - (Clinical & Experimental Immunology, 2014) Human immunoglobulin G for experimental treatment of Duchenne muscular dystrophy
J. Zschüntzsch, P. Jouvenal, Y. Zhang, F. Klinker, M. Tiburcy, D. Malzahn, D. Liebetanz, H. Brinkmeier and J. Schmidt - Germany
Duchenne muscular dystrophy is the most common inherited disorder of the skeletal muscle. It is caused by a mutation in the dystrophin gene on the X chromosome. Subsequent lack of the dystrophin protein leads to impaired stability of the myocytoskeleton and reduced contraction functionality of skeletal muscle fibres [1, 2]. This devastating myopathy leads to an enormous burden of disease and often death before 30 years of age. Despite a tremendous effort with numerous clinical trials that aimed to correct the gene defect, so far no effective therapy is available [3]. Current standard treatment includes the use of glucocorticosteroids, which aims to reduce the profound bystander inflammation in the skeletal muscle [4]. However, treatment with glucocorticosteroids is hampered by severe long-term side effects.In search for a more effective and tolerable treatment we used the mdx mouse, an established model of the disease, which harbours a homologue mutation of the dystrophin gene. Mice received 2 g/kg human immunoglobulin (Ig)G once per month compared to sham treatment of equal volume, administered by intraperitoneal injection. Each mouse was housed in a separate plastic cage equipped with a computerized running wheel, which continuously recorded the running behaviour and provided parameters such as number of runs, daily and total distance and distance per run [5]. Clinical parameters included body weight, grip-strength assessment and running-wheel performance. The cardiac function was monitored by ultrasound and at the end of the experiment mice were killed for ex vivo contraction analysis. Muscle pathology and expression profile of inflammatory mediators was assessed by immunohistochemistry and quantitative polymerase chain reaction (qPCR). During the early phase of the disease, IgG led to an improved running-wheel performance and ex vivo contraction analysis displayed an elevated endurance. In line with this, myopathic changes in the skeletal muscle were ameliorated and cellular infiltration was reduced. At the same time, release of the muscle enzyme creatine kinase was diminished. In the late phase of the disease, running-wheel performance and grip strength were improved upon treatment with IgG. This was accompanied by a superior cardiac function, as evidenced by ultrasound. In tissue sections of skeletal muscle and diaphragm, myopathic alterations and infiltration by inflammatory cells were reduced. Collectively, the results demonstrate a beneficial effect of human IgG in the treatment of mdx mice. This suggests that IgG may be a promising option for the future treatment of Duchenne muscular dystrophy. Apart from a monotherapeutic approach, IgG could potentially be of value in combination with gene therapy. A clinical proof-of-concept trial is warranted to study the effect of IgG in Duchenne muscular dystrophy.
26 - (Neuromuscular Disorders,2015, 25(1):19-23) Implantation of a left ventricular assist device as a destination therapy in Duchenne muscular dystrophy patients with end stage cardiac failure:Management and lessons learned
Francesca Iodice, Giuseppina Testa, Marco Averardi, Gianluca Brancaccio, Antonio Amodeo, Paola Cogo - Italy
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, characterized by progressive skeletal muscle weakness, loss of ambulation, and death secondary to cardiac or respiratory failure. End-stage dilated cardiomyopathy (DCM) is a frequent finding in DMD patients, they are rarely candidates for cardiac transplantation. Recently, the use of ventricular assist devices as a destination therapy (DT) as an alternative to cardiac transplantation in DMD patients has been described. Preoperative planning and patient selection play a significant role in the successful postoperative course of these patients. We describe the preoperative, intraoperative and postoperative management of Jarvik 2000 implantation in 4 DMD pediatric (age range 12–17 years) patients. We also describe the complications that may occur. The most frequent were bleeding and difficulty in weaning from mechanical ventilation. Our standard protocol includes: 1) preoperative multidisciplinary evaluation and selection, 2) preoperative and postoperative non-invasive ventilation and cough machine cycles, 3) intraoperative use of near infrared spectroscopy (NIRS) and transesophageal echocardiography, 4) attention on surgical blood loss, use of tranexamic acid and prothrombin complexes, 5) early extubation and 6) avoiding the use of nasogastric feeding tubes and nasal temperature probes. Our case reports describe the use of Jarvik 2000 as a destination therapy in young patients emphasizing the use of ventricular assist devices as a new therapeutic option in DMD.
NOVEMBER
22 - (American Heart Association Meeting, 2014) Heart-derived Cell Therapy for Duchenne Cardiomyopathy: Cardiosphere-derived Cells and their Exosomes Improve Function, Restore Mitochondrial Integrity and Reverse Degenerative Changes in the Hearts of Mdx Mice
Mark A Aminzadeh, Rachel Tobin, Rachel Smith, Linda Marbán, Eduardo Marbán, Cedars-Sinai Heart Inst, Los Angeles, CA
Abstract
Introduction: Cardiosphere-derived cells (CDCs) promote cardiomyogenesis and angiogenesis, while inhibiting oxidative stress, inflammation and fibrosis, in both ischemic and nonischemic cardiomyopathy. The mdx mouse model of Duchenne muscular dystrophy develops cardiomyopathy due to dystrophin deficiency and the resultant intense oxidative stress, inflammation and apoptosis. Here we tested the hypothesis that transplantation of CDCs or of exosomes derived from CDCs (CDC-XO) may be beneficial in mdx mice.
Methods and Results: A total of 78 mice were studied at a point when global cardiac dysfunction was already evident by echocardiography. Wild-type syngeneic mouse CDCs (105 cells total), CDC-XO (70µg), or vehicle only were injected intramyocardially in 5 left ventricular (LV) sites in 10-month old mdx mice. LV ejection fraction markedly improved over 3 months after treatment either with CDC or CDC-XO compared to vehicle-treated mice (60.4±1.6 vs 48.1±2.2; p<0.005). The functional improvement was associated with enhanced Nrf2 activation, upregulation of Nrf2 downstream gene products, increased expression of mitochondrial transcription factor A and cellular mitochondrial content, restored expression of mitochondrial respiratory chain subunits, reduced collagen I and III deposition, and attenuated infiltration of inflammatory cells [CD68+ macrophages and CD3+ T cells] in the CDC or CDC-XO-treated mouse hearts. Mitochondrial swelling and disorganization of cristae, prominent in vehicle-treated hearts by electron microscopy, were reversed by CDC treatment. Concomitantly, CDC-treated mdx mice exhibited higher maximal exercise capacity compared to vehicle-treated mice over 3 months of follow up (p<0.05). Conclusions: Cardiac function and exercise capacity improved in mdx mice treated with either CDCs or CDC-XO, accompanied by enhanced activation of the antioxidative Nrf2 pathway, attenuation of inflammation, reduction in collagen content and fibrosis, and augmented cardiomyogenesis in CDC-treated mdx hearts. The findings raise the possibility that CDCs and/or CDC-derived exosomes may be useful therapeutically in patients with Duchenne cardiomyopathy.
15 - (American Heart Association Meeting, 2014) Role of FOXO Transcription Factors in the Protective Mechanism by Resveratrol Against Cardiomyopathy in the Dystrophin-deficient Mdx Mouse
Atsushi Kuno, Rio Sebori, Tetsuji Miura, and Yoshiyuki Horio = Japan
Purpose: FOXO transcription factors (FOXO) transcriptionally regulate anti-oxidant and autophagy-related genes and elicit cellular stress resistance. We previously reported that treatment with resveratrol (RSV), an activator of the deacetylase SIRT1, ameliorates cardiomyopathy in the dystrophin-deficient mdx mouse (MDX), a model of Duchenne muscular dystrophy. Since SIRT1 is known to activate FOXO under stress conditions, we hypothesized that FOXO play roles in protection by RSV in the mdx heart.
Methods and Results: In Experiment 1, we analyzed cardiac phenotypes in MDX. At 42-week-old, MDX showed cardiac hypertrophy evaluated by heart weight and echocardiography compared with age-matched C57BL10 mice. Left ventricular (LV) systolic function was preserved in MDX at this age. Dihydroethidium (DHE) staining for analysis of cardiac superoxide anion showed that fluorescence intensity was 4.5-fold higher in MDX than control C57BL10. Immunoblot showed that myocardial p62 and LC3-II levels were increased in MDX at 24-week-old, and this was associated with increased phosphor-S6 level, suggesting impaired autophagy due to mTORC1 activation. In Experiment 2, MDX were divided into untreated (RSV0) and RSV-treated (400 mg/kg chow, RSV400) groups. RSV administration was started at 8-week-old, and mice were sacrificed at 65-week-old. Echocardiography at 62-week-old demonstrated that LV fractional shortening was higher (38±2% vs. 34±1%, P<0.05), IVS thickness was thinner, and end-diastolic LV dimension was smaller in RSV400 than those in RSV0. Compared with untreated MDX, RSV treatment significantly up-regulated cardiac mRNA levels of genes known as FOXO’s targets including anti-oxidant catalase (+2.0-fold), SOD1 (+2.8-fold) and autophagy-related LC3b (+2.5-fold), Bnip3 (+4.1-fold). DHE fluorescence intensity was significantly decreased by 45% by resveratrol treatment. Total cardiac LC3 level was rather decreased in RSV400, suggesting promotion of autophagic flux by RSV. Nuclear FOXO3a level assessed by immunostaining was increased in RSV400 compared with RSV0.
Conclusions: RSV ameliorated cardiomyopathy in the mdx mouse probably by attenuating oxidative stress and restoring autophagy via FOXO3a activation.
15 - (American Heart Association Meeting, 2014) Sodium Nitrate Recapitulates Sarcolemmal nNOS and Alleviates Functional Muscle Ischemia in Patients With Becker Muscular Dystrophy
Michael D Nelson, Florian Rader, Xiu Tang, Thomas Stabler, Sarah Shidban, Ryan Rosenberry, Shomari Hogan, Robert Elashoff, Jason D Allen, and Ronald G Victor - USA
Becker muscular dystrophy (BMD) is a progressive X-linked muscle wasting disease for which there is currently no treatment. Most BMD mutations disrupt sarcolemmal targeting of neuronal nitric oxide synthase (nNOS), causing functional muscle ischemia. In the mouse model of dystrophinopathy, chronic treatment with a nitric oxide (NO)-donating non-steroidal anti-inflammatory drug (NSAID) alleviates this ischemia and improves many features of the dystrophic phenotype. As a first step toward clinical translation, we tested whether acute treatment with sodium nitrat--a simple inorganic NO donor without a NSIAD moiety-- will alleviate functional muscle ischemia in patients with BMD. In 11 men with BMD we first performed an open-label and then a placebo-controlled acute treatment trial. Reflex sympathetic vasoconstriction was measured as a decrease in muscle oxygenation by near infrared spectroscopy, evoked by lower-body negative pressure at rest and during rhythmic handgrip exercise. Without treatment, reflex vasoconstriction was not appropriately attenuated during exercise (ΔHbO2:-18.0±0.6 vs. -17.6±1.5 %; rest vs. exercise), producing functional muscle ischemia. With treatment, the ischemia was alleviated (ΔHbO2: -19.6±1.2 vs. -10.5±1.2; P <0.01, rest vs. exercise), and normal muscle blood flow regulation fully restored, by a single oral dose of sodium nitrate (8.4 mmol), which is converted by oral flora to nitrite (NO2-). These results were replicated in the placebo-controlled trial as well. Mechanistically, the data also show that circulating NO2- serves as an alternative NO donor when reduced by deoxyhemoglobin and deoxymyoglobin, whose intramuscular concentrations are low at rest and rise sharply when the muscle is exercised. Together, the data suggest that sodium nitrate recapitulates the normal vasomotor function of the missing sarcolemmal nNOS in patients with BMD, and constitutes a putative new therapy for patients with dystrophinopathy.
15 - (American Heart Association Meeting, 2014) Serum vs. Imaging Biomarkers of Myocardial Injury in Duchenne Muscular Dystrophy: Findings from the E-SCAR DMD Trial
Subha V Raman, Kan N Hor, Wojciech Mazur, Nancy Halnon, Tam Tran, Suzanne Smart, Beth McCarthy, John T Kissel, Michael D Taylor, John L Jefferies, Jill Rafael-Fortney, Sharon Roble, and Linda H Cripe - USA
Introduction: Cardiomyopathy has become a leading cause of death in Duchenne muscular dystrophy (DMD). We previously showed that early mineralocorticoid receptor antagonist therapy reduces myocardial damage in a preclinical model of DMD. The Eplerenone for Subclinical Cardiomyopathy in DMD (E-SCAR DMD, NCT01521546) is a multicenter randomized placebo-controlled clinical trial evaluating eplerenone in boys with preserved left ventricular ejection fraction (LVEF) and evident myocardial injury by late gadolinium enhancement cardiac magnetic resonance (LGE-CMR).
Hypothesis: To better define biomarkers of early disease, we hypothesized that LGE has greater sensitivity vs. serum biomarkers for myocardial injury in DMD cardiomyopathy.
Methods: Boys with DMD age ≥ 7 years were enrolled across 3 centers. LGE-CMR images were acquired using comparable techniques across 3T scanners, and core laboratory LGE quantification was performed blinded to laboratory findings as a percentage of LV mass using software based on the full-width half-maximum technique. Troponin-I, creatine kinase (CK) and CK isoenzymes were measured from blood samples obtained at the time of CMR examination using standardized clinical assays.
Results: 42 boys age 16 ± 7 years had preserved LVEF (57 ± 6%), and LGE-positive regions averaged 5.0 ± 2.6% of LV myocardium. While 100% had evident myocardial injury by LGE, 43% had measurable CK-MB and only 18% had detectable troponin-I in serum (Figure). %LGE was higher (5.4 ± 2.7 vs. 3.3 ± 1.8%, p<0.05) and LVEF was lower (55.4 ± 4.9 vs. 59.0 ± 7.1%, p < 0.01) in boys with detectable vs. those with undetectable troponin-I, whereas detectable CK-MB did not predict higher %LGE or lower LVEF.
Conclusion: DMD patients with abnormal myocardium by LGE-CMR may have no detectable abnormalities by serum biomarkers, underscoring the importance of myocardial injury imaging in identifying patients with subclinical cardiomyopathy who may benefit from early treatment.
15 - (American Heart Association Meeting, 2014) Cx43 Hemichannel Inhibition Prevents Arrhythmias in Duchenne Muscular Dystrophy Mice
J P Gonzalez, Jayalakshmi Ramachandran, Jorge Contreras, and Diego Fraidenraich - USA
Duchenne muscular dystrophy (DMD) is a fatal disease characterized by skeletal and cardiac muscle pathologies due to the absence of the membrane stabilizing protein dystrophin. Although most research has focused on skeletal muscle, 100% of patients over the age of 18 are diagnosed with cardiomyopathy, associated with the development of arrhythmias and eventual heart failure. The connexin proteins, normally responsible for the formation of gap junctions, are known to play a significant role in cardiac conduction. Remodeling of these proteins has been linked to the development of arrhythmias following myocardial infarction.
Through analysis of Cx43, the most abundant connexin in the heart, we found that both mild mdx and severe mdx:utr(-/-) models of DMD display significant protein upregulation and mislocalization to the lateral sides of cardiomyocytes. Interestingly, this pattern is more extreme in the severe model, consistent with the degree of cardiac phenotype. To assess the role of Cx43 in DMD arrhythmogenesis, anesthetized mice were subjected to isoproterenol challenge (5mg/kg IP) and ECGs were recorded. mdx mice developed non-sustained ventricular tachycardia (VT) while mdx:utr(-/-) mice developed sustained VT and eventually died within 30 minutes of challenge. To specifically inhibit the lateralized hemichannel function of Cx43, the protein mimetics Gap 26 and Gap 19 were administered to mice prior to challenge (1ug/kg IV, 10ug/kg IV). Amazingly, no arrhythmias developed in mdx mice, and mdx:utr(-/-) mice at even late stages of disease showed significantly improved scores with prevention of heart failure and death due to the challenge. In addition, RR and QRS intervals in all mice treated were closer to baseline than following isoproterenol challenge without treatment. These findings represent a novel mechanism to treat DMD cardiomyopathy and may have the potential to treat similar forms of heart disease that display lateralized Cx43.
15 - (American Heart Association Meeting, 2014) Myocardial Fibrosis Burden Predicts Left Ventricular Ejection Fraction and is Modified by Age and Steroid Treatment Duration in Duchenne Muscular Dystrophy
Animesh Tandon, Chet R Villa, Kan N Hor, John L Jefferies, Zhiqian Gao, Jeffrey A Towbin, Brenda L Wong, Wojciech Mazur, Robert J Fleck, Joshua J Sticka, Dudley W Benson, and Michael D Taylor - USA
Background: Patients with Duchenne muscular dystrophy (DMD) typically exhibit progressive cardiac and skeletal muscle dysfunction, and are commonly treated with corticosteroids to prolong ambulation. Myocardial fibrosis and steroid treatment may modulate the progression of cardiac dysfunction in DMD patients. We aimed to longitudinally characterize the impact of myocardial fibrosis and steroid treatment on the progression of cardiac dysfunction using cardiac magnetic resonance (CMR) in a large DMD cohort.
Methods: Serial CMR studies performed on DMD patients were reviewed for LVEF and late gadolinium enhancement (LGE) status, a marker for myocardial fibrosis. LVEF was modeled by examining effects of patient age, steroid treatment duration, LGE status, and myocardial fibrosis burden, as assessed by the number of LGE positive (LGE+) LV segments.
Results: We analyzed 469 CMR studies from 99 DMD patients with ≥4 CMRs. Patient age at time of CMR ranged from 6.6 to 29.4 (median 12.3) years. There were 146 (31.1%) LGE+ studies and 59 studies (12.6%) that demonstrated depressed LVEF (LVEF<55%). An age-only model demonstrated that LVEF declined 0.58±0.10%/yr (p<0.0001, r2=0.067). Univariate modeling showed significant associations between LVEF and steroid treatment duration, presence of LGE, and number of LGE+ LV segments; multivariate modeling showed that LVEF declined by 0.93±0.09% for each LGE+ LV segment (p<0.0001, r2=0.171), while age and steroid treatment duration were no longer significant. The number of LGE+ LV segments increased with age by 1.2 segments/year (95% confidence interval 1.1-1.2), and steroid treatment partially mitigated this increase (interaction term β=-0.01±0.005, p=0.010).
Conclusions: Progressive myocardial fibrosis, as imaged by LGE on CMR, is a strong marker for the decline in LVEF in DMD patients. Steroid treatment partially attenuates the age-related increase in myocardial fibrosis burden.
15 - (J.Appl.Physiol, 2014) Sulforaphane alleviates muscular dystrophy in mdx mice by activation of Nrf2
Chengcao Sun, Cuili Yang, Ruilin Xue, Shujun Li, Ting Zhang, Lei Pan, Xuejiao Ma, Liang Wang, and Dejia Li - China
Sulforaphane (SFN), one of the most important isothiocyanates in the human diet, is known to have chemo-preventive and antioxidant activities in different tissues via activation of NF-E2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). However, its effects on muscular dystrophy remain unknown. This work was undertaken to evaluate the effects of SFN on Duchenne muscular dystrophy (DMD). 4-week-old mdx mice were treated with SFN by gavage (2 mg/kg body weight per day for 8 weeks), and our results demonstrated that SFN treatment increased the expression and activity of muscle phase II enzymes NQO1 and HO-1 with Nrf2 dependent manner. SFN significantly increased skeletal muscle mass, muscle force (~30%), running distance (~20%) and GSH/GSSG ratio (~3.2 folds) of mdx mice, and decreased the activities of plasma creatine phosphokinase (CK) (~45%) and lactate dehydrogenase (LDH) (~40%), gastrocnemius hypertrophy (~25%), myocardial hypertrophy (~20%) and MDA levels (~60%). Further, SFN treatment also reduced the central nucleation (~40%), fiber size variability, inflammation and improved the sarcolemmal integrity of mdx mice. Collectively, these results show that SFN can improve muscle function, pathology and protect dystrophic muscle from oxidative damage in mdx mice through Nrf2 signaling pathway, which indicate Nrf2 may represent a new therapeutic target for muscular dystrophy.
15 - (Neuroscience Meeting, 2014) Effects of omega-3 therapy in the cardiomyopathy of the mdx mice, at later stages of the disease
A. F. MAURICIO, J. A. PEREIRA, H. SANTO NETO, M. J. MARQUES - Brazil
Duchenne muscular dystrophy is the most common and severe dystrophynopathy in childhood characterized by absence of dystrophin, with progressive muscle wasting and cardiorespiratory failure. In the absence of dystrophin there is sarcolemma instability, increased calcium influx and myonecrosis. Cardiomyopathy is one of the most frequent causes of death in DMD. In the mdx mice model of DMD, signs of cardiomyopathy are first seen around 9 months of age. We investigated the effects of omega-3 therapy in the mdx cardiomyopathy, at later stages of disease (13 months of age). Mdx mice (8 months of age) received fish oil containing eicosapentaenoic acid and docosahexanoic acid (300mg/kg via gavage, 3 days a week), for 5 months. Control mdx received nujol in an equivalent dose and period. Control mdx showed elevated (3 times) serum levels of CK-MB, an indicator of heart necrosis, compared with normal C57BL/10. Omega-3 reduced CK-MB (119±20 UI in mdx-nujol vs. 86±20 UI in mdx-omega-3). In control mdx, electrocardiogram analysis indicated alterations in the amplitudes of some waves, a decrease in the R/S ratio and a significant increase in the cardiomyopathy index. Omega-3 ameliorated some of these heart functional parameters. No changes in heart fibrosis area were seen in omega-3-mdx, with higher levels of fibrosis in the right ventricule (16±3% in mdx-nujol vs. 13±3% in mdx-omega-3). The levels of TNF-a (proinflammatory cytokine), TGF-b (profibrotic factor) and metaloproteinases(MMP)-9 and MMP-2 were all increased in the heart of control mdx in comparison to normal C57BL/10. Omega-3 significantly reduced the levels of TNF-a (1.5±0.4 in mdx-nujol vs. 1.1±0.03 in mdx-omega-3) and MMP-9 (1.3±0.1 in mdx-nujol vs. 1.1±0.09 in mdx-omega-3), with a tendency to reduce TGF-b (1.9±0.3 in mdx-nujol vs. 1.7±0.4 in mdx-omega-3; p > 0.05, Anova). The present results demonstrate that omega-3 is effective against cardiomyopathy in the mdx mouse, at later stages of the disease, being able to improve functional parameters and to regulate molecular markers (TNF-a, TGF-b and MMPs) of dystrophy progression, therefore deserving future investigation in DMD clinical trials.
SEPTEMBER
27 - ATALUREN TREATMENT OF PATIENTS WITH NONSENSE MUTATION DYSTROPHINOPATHY
6 - Experimental Studies - Abstracts in World Muscle Society Meeting, Germany, October 2014
4 - Human Trials DMD/BMD - Abstracts in World Muscle Society Meeting, Germany, October 2014
3 - Exon Skipping - Abstracts in World Muscle Society Meeting, Germany, October 2014
AUGUST
30 - (The American Journal of Pathology,2014) Dietary Phosphorus Overload Aggravates the Phenotype of the Dystrophin-Deficient mdx mouse
Eiji Wada, Mizuko Yoshida, Yoriko Kojima, Ikuya Nonaka, Kazuya Ohashi, Yosuke Nagata, Masataka Shiozuka, Munehiro Date, Tetsuo Higashi, Ichizo Nishino, Ryoichi Matsuda - Japan
Duchenne muscular dystrophy is a lethal X-linked disease with no effective treatment. Progressive muscle degeneration, increased macrophage infiltration, and ectopic calcification are characteristic features of the mdx mouse, a murine model of Duchenne muscular dystrophy. Because dietary phosphorus/phosphate consumption is increasing and adverse effects of phosphate overloading have been reported in several disease conditions, we examined the effects of dietary phosphorus intake in mdx mice phenotypes. On weaning, control and mdx mice were fed diets containing 0.7, 1.0, or 2.0 g phosphorus per 100 g until they were 90 days old. Dystrophic phenotypes were evaluated in cryosections of quadriceps and tibialis anterior muscles, and maximal forces and voluntary activity were measured. Ectopic calcification was analyzed by electron microscopy to determine the cells initially responsible for calcium deposition in skeletal muscle. Dietary phosphorus overload dramatically exacerbated the dystrophic phenotypes of mdx mice by increasing inflammation associated with infiltration of M1 macrophages. In contrast, minimal muscle necrosis and inflammation were observed in exercised mdx mice fed a low-phosphorus diet, suggesting potential beneficial therapeutic effects of lowering dietary phosphorus intake on disease progression. To our knowledge, this is the first report showing that dietary phosphorus intake directly affects muscle pathological characteristics of mdx mice. Dietary phosphorus overloading promoted dystrophic disease progression in mdx mice, whereas restricting dietary phosphorus intake improved muscle pathological characteristics and function.
30 - Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice
JULY
4 - (Annals of Neurology, 2014) Sildenafil Does Not Improve Cardiomyopathy in Duchenne/Becker Muscular Dystrophy
Thompson, Bing He, Genila Bibat, Gihan Tennekoon, Stuart D. Russell, Karl H. Schuleri, Albert C. Lardo, David A. Kass, Richard E. Thompson, Daniel P. Judge and Kathryn R. Wagner - USA
Objective: Duchenne and Becker muscular dystrophy (DBMD) are allelic disorders caused by mutations in dystrophin. Adults with DBMD develop life-threatening cardiomyopathy. Inhibition of phosphodiesterase 5 (PDE5) improves cardiac function in mouse models of DBMD. To determine if the PDE5-inhibitor sildenafil benefits human dystrophinopathy, we conducted a randomized, double-blind, placebo-controlled trial (ClinicalTrials.gov, number NCT01168908).
Methods: Adults with DBMD and cardiomyopathy (ejection fraction ≤50%) were randomized to receive sildenafil (20mg three times daily) or placebo for 6 months. All subjects received an additional 6 months of open-label sildenafil. The primary endpoint was change in left ventricular end-systolic volume (LVESV) on cardiac MRI. Secondary cardiac endpoints, skeletal muscle function, and quality of life were also assessed.
Results: An interim analysis (performed after 15 subjects completed the blinded phase) revealed that 29% (4/14) of subjects had a ≥10% increase in LVESV after 6 months of sildenafil compared to 13% (1/8) of subjects receiving placebo. Subjects with LVESV >120ml at baseline were more likely to worsen at 12 months regardless of treatment assignment (p=0.035). Due to the higher number of subjects worsening on sildenafil, the Data and Safety Monitoring Board recommended early termination of the study. There were no statistically significant differences in outcome measures between treatment arms.
Interpretation: Due to the small sample size, comparisons between groups must be interpreted with caution. However, this trial suggests that sildenafil is unlikely to improve cardiac function in adults with DBMD
4 - (Annals of Neurology, 2014) Effect of sildenafil on skeletal and cardiac muscle in Becker muscular dystrophy
N Witting, C Kruuse, B Nyhuus, KP Prahm, G Citirak, SJ Lundgaard, S von Huth, N Vejlstrup, U Lindberg, TO Krag and J Vissing - Dennark
Background; patients with Becker and Duchenne muscular dystrophies, (BMD and DMD) lack neuronal nitric oxide synthase (nNOS). nNOS mediates physiological sympatholysis, thus ensuring adequate blood supply to working muscle. In mice lacking dystrophin, restoration of nNOS effects by a phosphodiesterase 5 (PDE5) inhibitor (sildenafil), improves skeletal and cardiac muscle performance. Sildenafil also improves blood flow in patients with BMD. We therefore hypothesized that sildenafil would improve blood flow, maximal work capacity and heart function in patients with BMD.
Methods; a randomised, double-blind, placebo-controlled crossover design with two 4-week periods of treatment, separated by 2-week wash-out was used. We assessed brachial artery blood flow during maximal handgrip exercise, six minutes walk test, maximal oxidative capacity, life quality, and cardiac function was evaluated by MRI at rest and during maximal handgrip exercise. Muscle nNOS and PDE5 was tested with western blotting in 5 patients
Findings; sixteen patients completed all skeletal muscle evaluations and 13 the cardiac MRI investigations. Sildenafil had no effect on any of the outcome parameters. No serious adverse effects were recorded. PDE5 and nNOS were deficient in 5/5 biopsies
Interpretation; despite positive evidence from animal models of dystrophinopathy and physiological findings in patients with BMD, this double-blind, placebo-controlled clinical study showed no effect of sildenafil on blood flow, maximal work capacity and heart function in adults with BMD. This discrepancy may be explained by a significant down-regulation of PDE5 in muscle
JUNE
28 - (Exp Physiol, Jun 2014) Amitriptyline is efficacious in ameliorating muscle inflammation and depressive symptoms in the mdx mouse model of Duchenne muscular dystrophy
Jennifer Manning, Rebecca Kulbida, Prerana Rai, Lindsay Jensen, Judith Bouma, Sanjay P. Singh, Dervla O’Malley, and Deniz Yilmazer-Hanke - Ireland and USA
Mutations in the structural protein dystrophin underlie muscular dystrophies characterized by progressive deterioration of muscle function. Dystrophin deficient mdx mice are considered a model for Duchenne Muscular Dystrophy (DMD). DMD sufferers are also susceptible to mood disorders such as depression and anxiety. Therefore, the study objectives are to investigate the effects of the tricyclic antidepressant, amitriptyline on mood, learning, central cytokine expression and skeletal muscle inflammation in mdx mice. Amitriptyline‐induced effects (10 mg kg−1 daily SC injections, 25 days) on the behaviour of mdx mice were investigated using the open field arena and tail suspension tests. The effects of chronic amitriptyline treatment on inflammatory markers were studied in the muscle and plasma of mdx mice and mood‐associated monoamine and cytokine concentrations were measured in the amygdala, hippocampus, prefrontal cortex, striatum, hypothalamus and midbrain. Mdx mice exhibited increased levels of anxiety and depressive‐like behaviour compared to wild‐type mice. Amitriptyline‐treatment had anxiolytic and anti‐depressant effects in mdx mice associated with elevations in serotonin levels in the amygdala and hippocampus. Inflammation in mdx skeletal muscle tissue was also reduced following amitriptyline treatment as indicated by decreased immune cell infiltration of muscle and lower levels of the pro‐inflammatory cytokines tumour necrosis factor‐alpha (TNF‐α) and interleukin‐6 (IL‐6) in the forelimb flexors. IL‐6 mRNA expression was remarkably reduced in the amygdala of mdx mice by chronic amitriptyline treatment. Positive effects of amitriptyline on mood, in addition to its anti‐inflammatory effects in skeletal muscle, may make it an attractive therapeutic option for individuals with DMD.
19 - (Stem Cells, 2014;32(7): 1817-30) Ex Vivo Gene Editing of the Dystrophin Gene in Muscle Stem Cells Mediated by Peptide Nucleic Acid Single Stranded Oligodeoxynucleotides Induces Stable Expression of Dystrophin in a Mouse Model for Duchenne Muscular Dystrophy
Farnoosh Nik-Ahd and Carmen Bertoni - USA
Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level. In this study, we show that PNA-ssODNs can target and correct muscle satellite cells (SCs), a population of stem cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SCs transfected with correcting PNA-ssODNs were able to engraft and to restore dystrophin expression. The number of dystrophin-positive fibers was shown to significantly increase over time. Expression was confirmed to be the result of the activation of a subpopulation of SCs that had undergone repair as demonstrated by immunofluorescence analyses of engrafted muscles using antibodies specific to full-length dystrophin transcripts and by genomic DNA analysis of dystrophin-positive fibers. Furthermore, the increase in dystrophin expression detected over time resulted in a significant improvement in muscle morphology. The ability of transplanted cells to return into quiescence and to activate upon demand was confirmed in all engrafted muscles following injury. These results demonstrate the feasibility of using gene editing strategies to target and correct SCs and further establish the therapeutic potential of this approach to permanently restore dystrophin expression into muscle of DMD patients.
MAY
24 - PTC Therapeutics Receives Positive Opinion from CHMP for Translarna™ (ataluren)
9 - Cardiac management of ventilator-assisted individuals with duchenne muscular dystrophy
APRIL
26 - Annual Meeting of American Society of Gene Therapy - May 2014
6 - (Experimental Biology, 2014) Short-term naproxcinod treatment ameliorates functional muscle ischemia in dystrophin-deficient mdx mice
Liang Li, Daniela Miglietta, and Gail Thomas - Italy and USA
Impaired nitric oxide (NO) signaling in the dystrophin-deficient muscles of patients with Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD renders the diseased muscles susceptible to functional ischemia, which enhances both fatigue and exercise-induced muscle injury. We therefore asked if short-term treatment with naproxcinod, a NO-donating naproxen, would prevent functional muscle ischemia and restore normal blood flow regulation in the exercising muscles of mdx mice. Norepinephrine (NE) evoked similar decreases in femoral vascular conductance (FVC) in resting and contracting hindlimbs of vehicle-treated mdx mice, indicating functional muscle ischemia (ΔFVC contraction/rest: 0.93 ± 0.16, n=5). Naproxcinod (40 mg/kg, po) modestly reduced muscle ischemia when given as a single dose and markedly ameliorated it when treatment was extended to 1 wk (ΔFVC contraction/rest: 0.67 ± 0.18 and 0.39 ± 0.08, respectively; n=9 each), while treatment with equimolar naproxen was without effect (n=5). Daily treatment with a lower dose of naproxcinod (20 mg/kg) was equally effective in preventing functional ischemia in mdx and nNOS null mice (ΔFVC contraction/rest: 0.39 ± 0.05, n=10 and 0.31 ± 0.02, n=4, respectively). These findings suggest that naproxcinod’s ability to donate NO rather than inhibit cyclooxygenase is the main mechanism by which the drug improves muscle blood flow regulation in mdx mice.
6 - (Experimental Biology, 2014) Effect of chronic quercetin supplementation on dystrophic cardiac pathology in mdx mice
Joshua Selsby, Christopher Ballman, and John Quindry -USA
Duchenne Muscular Dystrophy (DMD) includes cardiac complications with 10-40% of deaths resulting from cardiac pathologies. In absence of a cure, we are exploring clinically viable interventions to counter cardiac complications associated with DMD. Quercetin (Q) is a SIRT-1/PGC-1α activator that up-regulates antioxidant genes, mitochondrial biogenesis, and decreases inflammation when taken by nutraceutical means. Thus, we investigated 0.2% Q dietary enrichment to counter cardiac abnormalities in mdx mice, a widely used model for DMD. Mice were randomly treated from either 3 weeks or 3 months for a 6 month intervention (3w6m or 3m6m, n=10/group). Control (C) mice consumed rodent chow for equal time periods (n=10/group). At the end of the study, hearts were excised, weighed, and frozen (-80 C°), cut into 10µm sections for immunohistochemistry, or prepped for western blotting (WB). Body weight did not change. Relative and absolute heart weights were decreased in Q3m6m mice (p=.03,p=.021). WB targets for mitochondrial biogenesis (Cytochrome C, p=.045) and antioxidant expression (SOD2, p=.012) were increased in Q3w6m mice. Indications of remodeling and fibrosis (MMP9, p=.043, hematoxylin and eosin, p=.001) were improved in Q-fed mice while TGFβ-1 expression was unchanged. 6 months of Q feeding improved long term markers and appears to effectively attenuate dystrophic cardiac pathology. Based on findings from younger mice, improvement in mitochondrial content and antioxidant activity may precede these outcomes. Results reveal a promising intervention to complications of dystrophic cardiac pathology. Additional experiments are needed to confirm these findings with more reductionist approaches.
6 - (Experimental Biology, 2014) Dietary quercetin enrichment improves respiratory function in mdx mice
Joshua Selsby, Christopher Ballman, and John Quindry -USA
Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency and results in progressive muscle injury culminating in death due to respiratory or cardiac failure. We have established that gene transfer of the exercise mimetic, PGC-1α, will decrease disease severity and improve muscle function in skeletal muscle taken from mdx mice, the mouse model of DMD. While this approach is effective and repeatable, at present it is not readily translatable. Hence, the purpose of this project was to determine the extent to which oral delivery of quercetin, a PGC-1α activator, would improve respiratory function in mdx mice. To address this purpose baseline respiratory function was measured in vivo in two month old C57 and mdx mice. After measurement, the mdx group was randomly assigned into groups receiving a control diet (mdx) or diet enriched with 0.2% quercetin (mdx-Q). Respiratory function was measured again at four and six months of age. By six months of age we found that a 16% (p<0.05) increase in respiratory frequency and a 12% (p<0.05) increase in tidal volume caused a near 30% (p<0.05) increase in minute ventilation in mdx-Q compared to mdx. Further, peak inspiratory flow was increased 20% (p<0.05) and peak expiratory flow was increased 26% (p<0.05) in mdx-Q compared to mdx. These data suggest strongly that dietary enrichment of the orally available PGC-1α activator, quercetin, attenuated muscle injury and improved respiratory function in a mouse model of Duchenne muscular dystrophy.
6 - (Experimental Biology, 2014) ATP signaling complex is altered in muscular dystrophy and was partly recovered after nifedipine treatment
Denisse Valladares, Francisco Altamirano, Carlos Henrquez-Olgun, Alexis Daz-Vegas, Giomar Intriago, Ariel Contreras-Ferrat, Sonja Buvinic, Jos Lpez, Paul Allen, and Enrique Jaimovich -Chile
Duchenne muscular dystrophy (DMD) is a common genetic disorder characterized by a severe muscle wasting caused by the absence of dystrophin. In mdx muscle fibers, we have shown that basal ATP release is increased and that high extracellular ATP is a pro-apoptotic stimuli. We also have shown that Dihydropyridine receptors (DHPR) are needed for ATP release through pannexin-1(Pnx1) channels. The aim of this work was to study the potential therapeutic effect of DHPR blockage by nifedipine in DMD. We used muscle fibers isolated from six-week-old normal and mdx mice that were treated with daily intraperitoneal injections of 1mg/Kg nifedipine for 1-week. We studied differences in the interaction between DHPR and Pnx1by proximity ligation assay. In mdx fibers there was an increase in the number and volume of positive particles but not in the intensity of the positive reaction when compared to normal fibers. After the treatment with nifedipine, both number and volume of the positive reaction particles was normalized. This result correlates well with a decrease in both ATP release and intracellular resting Ca2+ concentration in mdx fibers after the treatment. Moreover, ATP signaling was no longer a pro-apoptotic stimuli. Finally, nifedipine treatment increased muscle strength assessed by both the inverted grip-hanging and forced swimming test. These data suggest that dihydropyridines can be used as a therapeutical tool to reduce muscle damage observed in dystrophic muscles.
6 - (Experimental Biology, 2014) Dietary sodium nitrate supplementation restores blood flow regulation in Becker muscular
Michael Nelson, Florian Rader, Xiu Tang, Thomas Stabler, Jason Allen, and Ronald Victor - USA
Chronic administration of a nitric oxide-donating drug fully corrects abnormal blood flow regulation in the mdx mouse model of dystrophinopathy. As a first step to clinical translation, we treated 6 men with Becker muscular dystrophy with a single oral dose of sodium nitrate- a simple, exogenous inorganic nitric oxide donor. We measured reflex vasoconstriction (ΔHb02, near infrared spectroscopy; evoked by lower body negative pressure) at rest and during rhythmic handgrip exercise, before and 3 hours after ~8.4 mmol of oral sodium nitrate. Before treatment, blood flow regulation was impaired in BMD, because handgrip exercise caused no attenuation of reflex vasoconstriction (ΔHbO2:-17±2 vs. -16±2 %, rest vs. HG). Remarkably, sodium nitrate supplementation more than doubled plasma nitrate and nitrite levels, and restored exercise-induced attenuation of reflex vasoconstriction (ΔHbO2: -18±1 vs. -11±2; P <.001, rest vs. HG). Moreover, the exercise-induced increase in forearm muscle blood flow, as measured by Doppler ultrasound, nearly tripled after sodium nitrate supplementation (Δ from baseline: 102 + 19% before treatment compared to 271 + 39 % after treatment), despite having no effect on basal flow. Taken together, these data indicate that oral sodium nitrate can recapitulate the normal regulation of blood flow in exercising dystrophic skeletal muscle, which is normally achieved by sarcolemmal nNOS.
MARCH
22 - Poloxomer 188 has a deleterious effect on dystrophic skeletal muscle function
8 - Resveratrol improves muscle function but not oxidative capacity in young mdx mice
8 - Therapeutic Effects of Mouse Adipose-derived Stem Cells and Losartan in the Skeletal Muscle of Injured Mdx mice
3 - Efficacy of fetal stem cells in Duchenne muscular dystrophy therapy
FEBRUARY
28 - Selected abstracts from the Annual Meeting of American Academy of Neurology - April 26 2014
JANUARY
31 - (Hum. Mol. Genet., Jan 2014) Long-term treatment with naproxcinod significantly improves skeletal and cardiac disease phenotype in the mdx mouse model of dystrophy
Kitipong Uaesoontrachoon, James L. Quinn, Kathleen S. Tatem, Jack H. Van der Meulen, Qing Yu, Aditi Phadke, Brittany K. Miller, Heather Gordish-Dressman, Ennio Ongini, Daniela Miglietta, and Kanneboyina Nagaraju - USA
In Duchenne muscular dystrophy (DMD) patients and the mouse model of DMD, mdx, dystrophin deficiency causes a decrease and mislocalization of muscle-specific neuronal nitric oxide synthase (nNOSμ), leading to functional impairments. Previous studies have shown that nitric oxide (NO) donation associated with anti-inflammatory action has beneficial effects in dystrophic mouse models. In this study, we have systematically investigated the effects of naproxcinod, an NO-donating naproxen derivative, on the skeletal and cardiac disease phenotype in mdx mice. Four-week-old mdx and C57BL/10 mice were treated with four different concentrations (0, 10, 21, and 41 mg/kg) of naproxcinod and 0.9 mg/kg of prednisolone in their food for 9 months. All mice were subjected to twice-weekly treadmill sessions, and functional and behavioral parameters were measured at 3, 6, and 9 months of treatment. In addition, we evaluated in vitro force contraction, optical imaging of inflammation, echocardiography, and blood pressure at the 9-month endpoint prior to sacrifice. We found that naproxcinod treatment at 21 mg/kg resulted in significant improvement in hindlimb grip strength and a 30% decrease in inflammation in the fore- and hindlimbs of mdx mice. Furthermore, we found significant improvement in heart function, as evidenced by improved fraction shortening, ejection fraction, and systolic blood pressure. In addition, the long-term detrimental effects of prednisolone typically seen in mdx skeletal and heart function were not observed at the effective dose of naproxcinod. In conclusion, our results indicate that naproxcinod has significant potential as a safe therapeutic option for the treatment of muscular dystrophies.
25 - Fibronectin is a serum biomarker for Duchenne muscular dystrophy
10 - (Muscle & Nerve, 2014) CINRG Duchenne Natural History Study demonstrates insufficient diagnosis and treatment of cardiomyopathy in Duchenne muscular dystrophy
Christopher Spurney, Reiko Shimizu, Lauren P. Hache, Hanna Kolski, , Heather Gordish-Dressman, Paula R. Clemens, and the CINRG Investigators
Introduction: Cardiomyopathy is a common cause of morbidity and death in patients with Duchenne muscular dystrophy (DMD).
Methods: A cross-sectional analysis of clinical data from a multi-institutional,
international CINRG DMD Natural History Study of 340 DMD patients aged 2 to 28 years. Cardiomyopathy was defined as shortening fraction (SF) <28% or ejection fraction (EF) <55%.
Results: 231 participants reported a prior clinical echocardiogram study, and 174 had data for SF or EF. The prevalence of cardiomyopathy was 27% (47/174), and it was significantly associated with age and clinical stage. The association of cardiomyopathy with age and clinical stage was not changed by glucocorticoid use as a covariate (P>0.68).
In patients with cardiomyopathy, 57 % (27/47) reported not taking any cardiac
medications. Cardiac medications were used in 12% (15/127) of patients without cardiomyopathy.
Discussion: Echocardiograms were underutilized, and cardiomyopathy was undertreated in this DMD natural history cohort.