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Duchenne muscular dystrophy (DMD) is an X-linked muscle disease with weakness, loss of ambulation, and premature death. DMD patients have reduced bone health, including decreased femur length (FL), density, and fractures. The mdx mouse model has paradoxically greater FL, density, and strength, positively correlating with muscle mass. Bone morphology has not been extensively studied in the genetic homolog, golden retriever muscular dystrophy (GRMD). The aim of this study was to compare bone and muscle characteristics in GRMD dogs to understand their relationship to muscle function and density. We hypothesized that GRMD bone measurements would be altered similarly to DMD and would correlate with muscle strength and density. Eighteen variably aged and mixed gender dogs (6 each dystrophic, carrier, normal) were studied by computed tomography (CT) and bone measures were compared with various muscle functional measurements. FL, density, and volume and muscle density of several pelvic limb muscles were assessed. Dystrophic dogs showed some boney and muscle density changes on CT analysis compared to carriers and normal dogs. In GRMD, bone measurements were highly correlated with several other functional outcome measures, including eccentric contraction decrement, hip joint angle, and muscle volume/density. This novel experiment demonstrates an impact of dystrophy on bone outcome measures and provides observations on their correlation with functional outcomes. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is an X-linked muscle disease with weakness, loss of ambulation, and premature death. DMD patients have reduced bone health, including decreased femur length (FL), density, and fractures. The mdx mouse model has paradoxically greater FL, density, and strength, positively correlating with muscle mass. Bone morphology has not been extensively studied in the genetic homolog, golden retriever muscular dystrophy (GRMD). The aim of this study was to compare bone and muscle characteristics in GRMD dogs to understand their relationship to muscle function and density. We hypothesized that GRMD bone measurements would be altered similarly to DMD and would correlate with muscle strength and density. Eighteen variably aged and mixed gender dogs (6 each dystrophic, carrier, normal) were studied by computed tomography (CT) and bone measures were compared with various muscle functional measurements. FL, density, and volume and muscle density of several pelvic limb muscles were assessed. Dystrophic dogs showed some boney and muscle density changes on CT analysis compared to carriers and normal dogs. In GRMD, bone measurements were highly correlated with several other functional outcome measures, including eccentric contraction decrement, hip joint angle, and muscle volume/density. This novel experiment demonstrates an impact of dystrophy on bone outcome measures and provides observations on their correlation with functional outcomes.

Abstract Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene and loss of the protein dystrophin, which ultimately leads to myofiber membrane fragility and necrosis, with eventual muscle atrophy and contractures. Affected boys typically die in their second or third decade due to either respiratory failure or cardiomyopathy. Among the developed therapeutic strategies for DMD, gene therapy approaches partially restore micro-dystrophin or quasi-dystrophin expression. However, despite extensive attempts to develop definitive therapies for DMD, the standard of care remains corticosteroid, which has only palliative benefits. Animal models have played a key role in studies of DMD pathogenesis and treatment development. The golden retriever muscular dystrophy (GRMD) dog displays a phenotype aligning with the progressive course of DMD. Therefore, canine studies may translate better to humans. Recent studies suggested that nicotinamide adenine dinucleotide (NAD+) cellular content could be a critical determinant for striated muscle function. We showed here that NAD+ content was decreased in the striated muscles of GRMD, leading to an alteration of one of NAD+ co-substrate enzymes, PARP-1. Moreover, we showed that boosting NAD+ content using nicotinamide (NAM), a natural NAD+ precursor, modestly reduces aspects of striated muscle disease. Collectively, our results provide mechanistic insights into DMD.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene and loss of the protein dystrophin, which ultimately leads to myofiber membrane fragility and necrosis, with eventual muscle atrophy and contractures. Affected boys typically die in their second or third decade due to either respiratory failure or cardiomyopathy. Among the developed therapeutic strategies for DMD, gene therapy approaches partially restore micro-dystrophin or quasi-dystrophin expression. However, despite extensive attempts to develop definitive therapies for DMD, the standard of care remains corticosteroid, which has only palliative benefits. Animal models have played a key role in studies of DMD pathogenesis and treatment development. The golden retriever muscular dystrophy (GRMD) dog displays a phenotype aligning with the progressive course of DMD. Therefore, canine studies may translate better to humans. Recent studies suggested that nicotinamide adenine dinucleotide (NAD+) cellular content could be a critical determinant for striated muscle function. We showed here that NAD+ content was decreased in the striated muscles of GRMD, leading to an alteration of one of NAD+ co-substrate enzymes, PARP-1. Moreover, we showed that boosting NAD+ content using nicotinamide (NAM), a natural NAD+ precursor, modestly reduces aspects of striated muscle disease. Collectively, our results provide mechanistic insights into DMD. [ABSTRACT FROM AUTHOR]

There is a great demand for accurate non-invasive measures to better define the natural history of disease progression or treatment outcome in Duchenne muscular dystrophy (DMD) and to facilitate the inclusion of a large range of participants in DMD clinical trials. This review aims to investigate which MRI sequences and analysis methods have been used and to identify future needs. Medline, Embase, Scopus, Web of Science, Inspec, and Compendex databases were searched up to 2 November 2019, using keywords magnetic resonance imaging and Duchenne muscular dystrophy. The review showed the trend of using T1w and T2w MRI images for semi-qualitative inspection of structural alterations of DMD muscle using a diversity of grading scales, with increasing use of T2map, Dixon, and MR spectroscopy (MRS). High-field (>3T) MRI dominated the studies with animal models. The quantitative MRI techniques have allowed a more precise estimation of local or generalized disease severity. Longitudinal studies assessing the effect of an intervention have also become more prominent, in both clinical and animal model subjects. Quality assessment of the included longitudinal studies was performed using the Newcastle-Ottawa Quality Assessment Scale adapted to comprise bias in selection, comparability, exposure, and outcome. Additional large clinical trials are needed to consolidate research using MRI as a biomarker in DMD and to validate findings against established gold standards. This future work should use a multiparametric and quantitative MRI acquisition protocol, assess the repeatability of measurements, and correlate findings to histologic parameters.

Stem cell-based therapies are a promising approach for the treatment of degenerative muscular diseases; however, clinical trials have shown inconclusive and even disappointing results so far. Noninvasive cell monitoring by medicine imaging could improve the understanding of the survival and biodistribution of cells following injection. In this study, we assessed the canine sodium iodide symporter (cNIS) reporter gene as an imaging tool to track by single-photon emission computed tomography (SPECT/CT) transduced canine myoblasts after intramuscular (IM) administrations in dogs. cNIS-expressing cells kept their myogenic capacities and showed strong 99 mTc-pertechnetate (99 mTcO4−) uptake efficiency both in vitro and in vivo. cNIS expression allowed visualization of cells by SPECT/CT along time: 4 h, 48 h, 7 days, and 30 days after IM injection; biopsies collected 30 days post administration showed myofiber’s membranes expressing cNIS. This study demonstrates that NIS can be used as a reporter to track cells in vivo in the skeletal muscle of large animals. Our results set a proof of concept of the benefits NIS-tracking tool may bring to the already challenging cell-based therapies arena in myopathies and pave the way to a more efficient translation to the clinical setting from more accurate pre-clinical results.

Abstract Background Accelerometric analysis of gait abnormalities in golden retriever muscular dystrophy (GRMD) dogs is of limited sensitivity, and produces highly complex data. The use of discriminant analysis may enable simpler and more sensitive evaluation of treatment benefits in this important preclinical model. Methods Accelerometry was performed twice monthly between the ages of 2 and 12 months on 8 healthy and 20 GRMD dogs. Seven accelerometric parameters were analysed using linear discriminant analysis (LDA). Manipulation of the dependent and independent variables produced three distinct models. The ability of each model to detect gait alterations and their pattern change with age was tested using a leave-one-out cross-validation approach. Results Selecting genotype (healthy or GRMD) as the dependent variable resulted in a model (Model 1) allowing a good discrimination between the gait phenotype of GRMD and healthy dogs. However, this model was not sufficiently representative of the disease progression. In Model 2, age in months was added as a supplementary dependent variable (GRMD_2 to GRMD_12 and Healthy_2 to Healthy_9.5), resulting in a high overall misclassification rate (83.2%). To improve accuracy, a third model (Model 3) was created in which age was also included as an explanatory variable. This resulted in an overall misclassification rate lower than 12%. Model 3 was evaluated using blinded data pertaining to 81 healthy and GRMD dogs. In all but one case, the model correctly matched gait phenotype to the actual genotype. Finally, we used Model 3 to reanalyse data from a previous study regarding the effects of immunosuppressive treatments on muscular dystrophy in GRMD dogs. Our model identified significant effect of immunosuppressive treatments on gait quality, corroborating the original findings, with the added advantages of direct statistical analysis with greater sensitivity and more comprehensible data representation. Conclusions Gait analysis using LDA allows for improved analysis of accelerometry data by applying a decision-making analysis approach to the evaluation of preclinical treatment benefits in GRMD dogs.

Introduction: Golden retriever muscular dystrophy (GRMD) is a spontaneous X-linked canine model of Duchenne muscular dystrophy that resembles the human condition. Muscle percentage index (MPI) is proposed as an imaging biomarker of disease severity in GRMD.Methods: To assess MPI, we used MRI data acquired from nine GRMD samples using a 4.7 T small-bore scanner. A machine learning approach was used with eight raw quantitative mapping of MRI data images (T1m, T2m, two Dixon maps, and four diffusion tensor imaging maps), three types of texture descriptors (local binary pattern, gray-level co-occurrence matrix, gray-level run-length matrix), and a gradient descriptor (histogram of oriented gradients).Results: The confusion matrix, averaged over all samples, showed 93.5% of muscle pixels classified correctly. The classification, optimized in a leave-one-out cross-validation, provided an average accuracy of 80% with a discrepancy in overestimation for young (8%) and old (20%) dogs.Discussion: MPI could be useful for quantifying GRMD severity, but careful interpretation is needed for severe cases. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disease. The disease is due to mutations in the DMD gene that encodes for a large intracellular protein called dystrophin. Dystrophin plays a critical role in linking the internal cytoskeleton of the striated muscle cell with the extracellular matrix as well as having cell signalling functions. In its absence muscle contraction is associated with cycles of damage, repair, inflammation and fibrosis with eventual loss of muscle and replacement with fat. Experiments in animal models of DMD have generated a number of different approaches to the induction of dystrophin including viral vector mediated delivery of a recombinant dystrophin gene, antisense oligonucleotide mediated exon-skipping to restore the open reading frame in the dystrophin mRNA, read-through of premature stop mutations, genome modification using CRISPR-Cas9 or cell based transfer of a functional dystrophin gene. In all cases, it will be important to understand how much dystrophin expression is required for a clinically effective therapy and this review examines the data from humans and animal models to estimate the percentage of endogenous dystrophin that is likely to have significant clinical benefit. While there are a number of important caveats to consider, including the appropriate outcome measures, this review suggests that approximately 20% of endogenous levels uniformly distributed within the skeletal muscles and the heart may be sufficient to largely prevent disease progression. [ABSTRACT FROM AUTHOR]

Introduction: Golden retriever muscular dystrophy (GRMD), an X-linked recessive disorder, causes similar phenotypic features to Duchenne muscular dystrophy (DMD). There is currently a need for a quantitative and reproducible monitoring of disease progression for GRMD and DMD.Methods: To assess severity in the GRMD, we analyzed texture features extracted from multi-parametric MRI (T1w, T2w, T1m, T2m, and Dixon images) using 5 feature extraction methods and classified using support vector machines.Results: A single feature from qualitative images can provide 89% maximal accuracy. Furthermore, 2 features from T1w, T2m, or Dixon images provided highest accuracy. When considering a tradeoff between scan-time and computational complexity, T2m images provided good accuracy at a lower acquisition and processing time and effort.Conclusions: The combination of MRI texture features improved the classification accuracy for assessment of disease progression in GRMD with evaluation of the heterogenous nature of skeletal muscles as reflection of the histopathological changes. Muscle Nerve 59:380-386, 2019. [ABSTRACT FROM AUTHOR]

Abstract Since respiratory insufficiency is the main cause of death in patients affected by Duchenne Muscular Dystrophy (DMD), the present study aims at establishing a new non-invasive method to evaluate the clinical parameters of respiratory conditions of experimental models affected by DMD. With this purpose in mind, we evaluated the cardiorespiratory clinical conditions, the changes in the intercostal muscles, the diaphragmatic mobility, and the respiratory cycles in Golden Retriever Muscular Dystrophy (GRMD) employing ultrasonography (US). A control group consisting of dogs of the same race, but not affected by muscular dystrophy, were used in this study. The results showed that inspiration, expiration and plateau movements (diaphragm mobility) were lower in the affected group. Plateau phase in the affected group was practically non-existent and showed that the diaphragm remained in constant motion. Respiratory rate reached 15.5 per minute for affected group and 26.93 per minute for the control group. Expiration and inspiration movements of intercostal muscles reached 8.99mm and 8.79mm, respectively, for control group and 7.42mm and 7.40mm, respectively, for affected group. Methodology used in the present analysis proved to be viable for the follow-up and evaluation of the respiratory model in GRMD and may be adapted to other muscular dystrophy experimental models.

Purpose: Metabolic dysfunction in Duchenne muscular dystrophy (DMD) is characterized by reduced glycolytic and oxidative enzymes, decreased and abnormal mitochondria, decreased ATP, and increased oxidative stress. We analyzed glucose metabolism as a potential disease biomarker in the genetically homologous golden retriever muscular dystrophy (GRMD) dog with molecular, biochemical, and in vivo imaging.Procedures: Pelvic limb skeletal muscle and left ventricle tissue from the heart were analyzed by mRNA profiling, qPCR, western blotting, and immunofluorescence microscopy for the primary glucose transporter (GLUT4). Physiologic glucose handling was measured by fasting glucose tolerance test (GTT), insulin levels, and skeletal and cardiac positron emission tomography/X-ray computed tomography (PET/CT) using the glucose analog 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG).Results: MRNA profiles showed decreased GLUT4 in the cranial sartorius (CS), vastus lateralis (VL), and long digital extensor (LDE) of GRMD vs. normal dogs. QPCR confirmed GLUT4 downregulation but increased hexokinase-1. GLUT4 protein levels were not different in the CS, VL, or left ventricle but increased in the LDE of GRMD vs. normal. Microscopy revealed diffuse membrane expression of GLUT4 in GRMD skeletal but not cardiac muscle. GTT showed higher basal glucose and insulin in GRMD but rapid tissue glucose uptake at 5 min post-dextrose injection in GRMD vs. normal/carrier dogs. PET/ CT with [18F]FDG and simultaneous insulin stimulation showed a significant increase (p = 0.03) in mean standard uptake values (SUV) in GRMD skeletal muscle but not pelvic fat at 5 min post-[18F]FDG /insulin injection. Conversely, mean cardiac SUV was lower in GRMD than carrier/normal (p < 0.01).Conclusions: Altered glucose metabolism in skeletal and cardiac muscle of GRMD dogs can be monitored with molecular, biochemical, and in vivo imaging studies and potentially utilized as a biomarker for disease progression and therapeutic response. [ABSTRACT FROM AUTHOR]

In the translational process of developing innovative therapies for DMD (Duchenne muscular dystrophy), the last preclinical validation step is often carried out in the most relevant animal model of this human disease, namely the GRMD (Golden Retriever muscular dystrophy) dog. The disease in GRMD dogs mimics human DMD in many aspects, including the inter-individual heterogeneity. This last point can be seen as a drawback for an animal model but is inherently related to the disease in GRMD dogs closely resembling that of individuals with DMD. In order to improve the management of this inter-individual heterogeneity, we have screened a combination of biomarkers in sixty-one 2-month-old GRMD dogs at the onset of the disease and a posteriori we addressed their predictive value on the severity of the disease. Three non-invasive biomarkers obtained at early stages of the disease were found to be highly predictive for the loss of ambulation before 6 months of age. An elevation in the number of circulating CD4+CD49dhi T cells and a decreased stride frequency resulting in a reduced spontaneous speed were found to be strongly associated with the severe clinical form of the disease. These factors can be used as predictive tests to screen dogs to separate them into groups with slow or fast disease progression before their inclusion into a therapeutic preclinical trial, and therefore improve the reliability and translational value of the trials carried out on this invaluable large animal model. These same biomarkers have also been described to be predictive for the time to loss of ambulation in boys with DMD, strengthening the relevance of GRMD dogs as preclinical models of this devastating muscle disease.

Introduction: Osteopontin (OPN) polymorphisms are associated with muscle size and modify disease progression in Duchenne muscular dystrophy (DMD). We hypothesized that OPN may share a molecular network with myostatin (MSTN).Methods: Studies were conducted in the golden retriever (GRMD) and mdx mouse models of DMD. Follow-up in-vitro studies were employed in myogenic cells and the mdx mouse treated with recombinant mouse (rm) or human (Hu) OPN protein.Results: OPN was increased and MSTN was decreased and levels correlated inversely in GRMD hypertrophied muscle. RM-OPN treatment led to induced AKT1 and FoxO1 phosphorylation, microRNA-486 modulation, and decreased MSTN. An AKT1 inhibitor blocked these effects, whereas an RGD-mutant OPN protein and an RGDS blocking peptide showed similar effects to the AKT inhibitor. RMOPN induced myotube hypertrophy and minimal Feret diameter in mdx muscle.Discussion: OPN may interact with AKT1/MSTN/FoxO1 to modify normal and dystrophic muscle. Muscle Nerve 56: 1119-1127, 2017. [ABSTRACT FROM AUTHOR]

Background: Accelerometric analysis of gait abnormalities in golden retriever muscular dystrophy (GRMD) dogs is of limited sensitivity, and produces highly complex data. The use of discriminant analysis may enable simpler and more sensitive evaluation of treatment benefits in this important preclinical model.Methods: Accelerometry was performed twice monthly between the ages of 2 and 12 months on 8 healthy and 20 GRMD dogs. Seven accelerometric parameters were analysed using linear discriminant analysis (LDA). Manipulation of the dependent and independent variables produced three distinct models. The ability of each model to detect gait alterations and their pattern change with age was tested using a leave-one-out cross-validation approach.Results: Selecting genotype (healthy or GRMD) as the dependent variable resulted in a model (Model 1) allowing a good discrimination between the gait phenotype of GRMD and healthy dogs. However, this model was not sufficiently representative of the disease progression. In Model 2, age in months was added as a supplementary dependent variable (GRMD_2 to GRMD_12 and Healthy_2 to Healthy_9.5), resulting in a high overall misclassification rate (83.2%). To improve accuracy, a third model (Model 3) was created in which age was also included as an explanatory variable. This resulted in an overall misclassification rate lower than 12%. Model 3 was evaluated using blinded data pertaining to 81 healthy and GRMD dogs. In all but one case, the model correctly matched gait phenotype to the actual genotype. Finally, we used Model 3 to reanalyse data from a previous study regarding the effects of immunosuppressive treatments on muscular dystrophy in GRMD dogs. Our model identified significant effect of immunosuppressive treatments on gait quality, corroborating the original findings, with the added advantages of direct statistical analysis with greater sensitivity and more comprehensible data representation.Conclusions: Gait analysis using LDA allows for improved analysis of accelerometry data by applying a decision-making analysis approach to the evaluation of preclinical treatment benefits in GRMD dogs. [ABSTRACT FROM AUTHOR]

Because respiratory failure remains a major issue in Duchenne Muscular Dystrophy patients, respiratory muscles are a key target of systemic therapies. In the Golden Retriever Muscular Dystrophy (GRMD) dogs, the disease shows strong clinical and histological similarities with the human pathology, making it a valuable model for preclinical therapeutic trials. We report here the first nuclear magnetic resonance (NMR) imaging anatomical study of the diaphragm in GRMD dogs and healthy controls. Both T1- and T2-weighted images of the diaphragm of seven healthy and thirteen GRMD dogs, from 3 to 36 months of age, were acquired on a 3 tesla NMR scanner. Abnormalities of texture and shape were revealed and consisted of increases in signal intensity on T2-weighted images and in signal heterogeneity on both T1- and T2-weighted images of the dystrophic diaphragm. These abnormalities were associated with a significant thickening of the muscle and we identified a clear 8-mm-threshold distinguishing clinically preserved GRMD dogs from those more severely affected. In this study, we demonstrated the feasibility of NMR imaging of the diaphragm and depicted several anatomical and mesoscopic anomalies in the dystrophic diaphragm. NMR imaging of the diaphragm shows a promise as an outcome measure in preclinical trials using GRMD dogs. [ABSTRACT FROM AUTHOR]

A Distrofia Muscular de Duchenne (DMD) é uma miopatia severa de caráter recessivo ligada ao cromossomo X e o modelo animal de estudo mais relevante é o Golden Retriever Muscular Dystrophy (GRMD). Além das severas alterações que ocorrem na musculatura estriada, muitos estudos mostram que outras estruturas, inclusive viscerais, podem se mostrar alteradas nesta patologia. Desta forma, este trabalho objetivou análisar e comparar possíveis alterações estruturais e funcionais do rim em cães GRMD. Neste modelo de estudo, foi possível observar a presença das faces convexa e côncava, do hilo renal e dos pólos craniais e caudais dos rins. O órgão mostrou-se envolto por uma cápsula fibrosa. Em um corte sagital do órgão, notou-se a presença das regiões cortical e medular e da pelve renal. Na análise microscópica foi possível identificar a zona medular e cortical com suas estruturas: os corpúsculos renais formados pelo glomérulo e pela cápsula de Bowman, os túbulos contorcidos proximais e distais, os ductos coletores, vasos sanguíneos e os segmentos das Alças de Henle. As dosagens séricas de creatinina e uréia encontram-se dentro dos limites de normalidade. Desta forma, de acordo com os nossos resultados, podemos concluir que os animais afetados estudados, não apresentaram alterações estruturais ou funcionais dos rins, o que nos permitir sugerir que apesar da ingestão hídrica comprometida, a estrutura renal, mantem- se preservada nos animais GRMD.

A proposta deste estudo foi avaliar se existem alterações nos padrões hematológicos e bioquímicos de cadelas da raça Golden Retriever portadoras do gene da distrofia muscular progressiva em comparação aos valores obtidos em cadelas não portadoras de mesma raça e idade. Foram analisados 33 animais, distribuídos em dois grupos, um composto por 19 cadelas Golden Retrievers não portadoras (GRNP) e outro composto por 14 cadelas Golden Retrievers portadoras do gene da distrofia muscular (GRP). Os dois grupos foram submetidos aos mesmos testes hematológicos e bioquímicos, com a mesma frequência e durante o mesmo intervalo de tempo. Apesar de existir diferença estatisticamente significativa entre os grupos para alguns parâmetros hematológicos avaliados, todos os resultados obtidos estavam de acordo com os valores de referência utilizados. Na avaliação dos parâmetros bioquímicos séricos a dosagem de ALT no grupo GRNP ficou levemente acima da média, porém sem grandes significados clínicos A CK também apresentou níveis elevados no grupo GRP, devido à degeneração e necrose muscular característicos da doença, as alterações encontradas nessa análise já eram esperadas. Os demais parâmetros não se alteraram.

Background: Duchenne muscular dystrophy (DMD) causes progressive muscle degeneration, cardiomyopathy and respiratory failure in approximately 1/5,000 boys. Golden Retriever muscular dystrophy (GRMD) resembles DMD both clinically and pathologically. Like DMD, GRMD exhibits remarkable phenotypic variation among affected dogs, suggesting the influence of modifiers. Understanding the role(s) of genetic modifiers of GRMD may identify genes and pathways that also modify phenotypes in DMD and reveal novel therapies. Therefore, our objective in this study was to identify genetic modifiers that affect discrete GRMD phenotypes. Results: We performed a linear mixed-model (LMM) analysis using 16 variably-affected dogs from our GRMD colony (8 dystrophic, 8 non-dystrophic). All of these dogs were either full or half-siblings, and phenotyped for 19 objective, quantitative biomarkers at ages 6 and 12 months. Each biomarker was individually assessed. Gene expression profiles of 59 possible candidate genes were generated for two muscle types: the cranial tibialis and medial head of the gastrocnemius. SNPs significantly associated with GRMD biomarkers were identified on multiple chromosomes (including the X chromosome). Gene expression levels for candidate genes located near these SNPs correlated with biomarker values, suggesting possible roles as GRMD modifiers. Conclusions: The results of this study enhance our understanding of GRMD pathology and represent a first step toward the characterization of GRMD modifiers that may be relevant to DMD pathology. Such modifiers are likely to be useful for DMD treatment development based on their relationships to GRMD phenotypes. [ABSTRACT FROM AUTHOR]

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Duchenne muscular dystrophy (DMD) is an X-linked myopathy resulting in progressive weakness and wasting of all the striated muscles including the respiratory muscles. The consequences are loss of ambulation before teen ages, cardiac involvement and breathing difficulties, the main cause of death. A cure for DMD is not currently available. In the last decades the survival of patients with DMD has improved because the natural history of the disease can be changed thanks to a more comprehensive therapeutic approach. This comprises interventions targeted to the manifestations and complications of the disease, particularly in the respiratory care. These include: 1) pharmacological intervention, namely corticosteroids and idebenone that significantly reduce the decline of spirometric parameters; 2) rehabilitative intervention, namely lung volume recruitment techniques that help prevent atelectasis and slows the rate of decline of pulmonary function; 3) scoliosis treatment, namely steroid therapy that is used to reduce muscle inflammation/degeneration and prolong ambulation in order to delay the onset of scoliosis, being an additional contribution to the restrictive lung pattern; 4) cough assisted devices that improve airway clearance thus reducing the risk of pulmonary infections; and 5) non-invasive mechanical ventilation that is essential to treat nocturnal hypoventilation, sleep disordered breathing, and ultimately respiratory failure. Without any intervention death occurs within the first 2 decades, however, thanks to this multidisciplinary therapeutic approach life expectancy of a newborn with DMD nowadays can be significantly prolonged up to his fourth decade. This review is aimed at providing state-of-the-art methods and techniques for the assessment and management of respiratory function in DMD patients. [ABSTRACT FROM AUTHOR]

This study aimed to characterise canine flow cytometry semen analysis, as well as seminal reactive oxygen species dosage using the Golden Retriever breed as model of study. Moreover, we searched for the influence of muscular dystrophy in Golden Retriever dogs on semen parameters. Thirty-seven semen samples were obtained from healthy Golden Retrievers ( n = 15) and from muscular dystrophy affected dogs ( n = 22). Sperm-rich fractions were analysed by standardised breeding soundness examination in addition to the assay of fluorescence assisted cell sorting for acrosome integrity, mitochondrial activity and DNA fragmentation. Volume of ejaculate, per cent of motile spermatozoa and vigour were similar between groups; there were no differences in the per cent of minor and major defects. Integrity of acrosomal membrane, mitochondrial potential and sperm DNA fragmentation had no significant differences between groups either. Animals from control group had higher concentration of spontaneous seminal oxidative species in comparison with affected animals. Dogs affected by dystrophy had seminal parameters similar to those observed in healthy dogs except for the lower concentration of oxidative species. Future studies aiming to establish reference values for canine seminal parameters should be considered preferably with distinction of breeds. [ABSTRACT FROM AUTHOR]

Abstract: The goal of this study was to assess whether magnetic resonance imaging (MRI) biomarkers can quantify disease progression in golden retriever muscular dystrophy (GRMD) via a natural history study. The proximal pelvic limbs of ten GRMD and eight normal dogs were scanned at 3, 6, and 9–12months of age. Several MRI imaging and texture analysis biomarkers were quantified in seven muscles. Almost all MRI biomarkers readily distinguished GRMD from control dogs; however, only selected biomarkers tracked with longitudinal disease progression. The biomarkers that performed best were full-length muscle volume and a texture analysis biomarker, termed heterogeneity index. The biceps femoris, semitendinosus and cranial sartorius muscles showed differential progression in GRMD versus control dogs. MRI features in GRMD dogs showed dynamic progression that was most pronounced over the 3- to 6-month period. Volumetric biomarkers and water map values correlated with histopathological features of necrosis/regeneration at 6-months. In conclusion, selected MRI biomarkers (volume and heterogeneity index) in particular muscles (biceps femoris, semitendinosus, and cranial sartorius) adjusted for age effect allow distinction of differential longitudinal progression in GRMD dogs. These biomarkers may be used as surrogate outcome measures in preclinical GRMD trials. [Copyright &y& Elsevier]

This investigation tested the hypothesis that carriers of golden retriever muscular dystrophy (GRMD), a genetically homologous condition of Duchenne muscular dystrophy (DMD), have quantifiable abnormalities in myocardial function, structure, or cardiac rhythm. Eleven GRMD carriers and four matched controls had cardiac evaluations and postmortem examinations. 24-h ECG Holter monitoring disclosed ventricular ectopy in 10 of 11 carriers and 2 of 4 controls. Conventional echocardiography failed to demonstrate significant differences between carriers and controls in systolic function. All carriers had multi-focal, minimal to marked myofiber necrosis, fibrosis, mineralization, inflammation, and/or fatty change in their hearts. Immunohistochemistry revealed a mosaic dystrophin deficiency in scattered cardiac myofibers in all carriers. No controls had cardiac histologic lesions; all had uniform dystrophin staining. Despite cardiac mosaic dystrophin expression and degenerative cardiac lesions, GRMD carriers at up to 3 years of age could not be distinguished statistically from normal controls by echocardiography or 24-h Holter monitoring. [ABSTRACT FROM AUTHOR]

Many anomalies exist in the resting 31P muscle spectra of boys with Duchenne muscular dystrophy (DMD) but few have been reported in Golden Retriever muscular dystrophy (GRMD), the closest existing animal model for DMD. Because GRMD is recommended for preclinical evaluation of therapies and quantitative outcome measures are needed, we investigated anomalies of 31P NMRS in tibial cranial and biceps femoris muscles from 14 GRMD compared to 9 control (CONT) dogs. Alterations observed in DMD children - low phosphocreatine and high phospho-monoesters and -diesters - were all found in GRMD but increased pH was not. More surprisingly, inorganic phosphate (Pi) appeared to present a prominent splitting with an enhanced Pib resonance at 0.3 ppm downfield of Pia. Assuming that both resonances are Pi, the pH for Pia in GRMD corresponded to a physiological intracellular pHa (6.97 ± 0.05), while pHb approached the extracellular range (7.27 ± 0.10) and correlated with pHa in GRMD (R2 = 0.65). Both Pia and Pib were elevated compared to CONT and Pia increased with age for GRMD (R2 = 0.48, p < 0.001). Magnetisation transfer experiments between γATP and Pi were conducted to better characterise Pi pools. Equal T1 relaxation times for Pib and Pia did not support a mitochondrial origin of Pib. We suggest that Pib could originate from degenerating hypercontracted cells that have a leaky membrane and inadequate cell homeostasis and pH regulation. Pib showed minimal chemical exchange in all dogs, while the exchange rate of Pia was reduced in GRMD and might extraneously reflect low glycolytic activity in DMD. Taken together, the ensemble of 31P NMRS alterations identifies muscle dysfunction and could provide useful biomarkers of therapeutic efficacy. Furthermore, among these, two might relate more specifically to dystrophic processes and merit further investigation: one is the existence of the enhanced alkaline Pib pool; the other, mechanisms by which membrane disruption might increase phosphodiesters in dystrophy. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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Abstract: Duchenne muscular dystrophy is a neuromuscular degenerative disorder caused by the absence of dystrophin protein. It is characterized by progressive muscle weakness and cycles of degeneration/regeneration accompanying chronic muscle damage and repair. Canine models of muscular dystrophy, including the dystrophin-deficient golden retriever muscular dystrophy (GRMD), are the most promising animal models for evaluation of potential therapies, however canine-specific molecular tools are limited. In particular, few immune reagents for extracellular epitopes marking canine satellite cells (muscle stem cells) are available. We generated an antibody to the satellite cell marker syndecan-4 that identifies canine satellite cells. We then characterized isolated satellite cells from GRMD muscle and wildtype muscle by several in vitro metrics, and surprisingly found no significant differences between the two populations. We discuss whether accumulated adverse changes in the muscle environment rather than cell-intrinsic defects may be implicated in the eventual failure of satellite cell efficacy in vivo. [Copyright &y& Elsevier]

Abstract: Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease that affects boys and leads to early death. In the quest for new treatments that improve the quality of life and in the search for a possible definitive cure, the use of animal models plays undoubtedly an important role. Therefore, a number of different mammalian models for DMD have been described. Much knowledge on the molecular mechanisms underlying the disease has arisen from studies in these animals. However, the use of different models does not often allow a direct comparison of results obtained in preclinical trials and therefore hinders a straightforward translational research. In the frame of “TREAT-NMD”, a European Network of Excellence addressing the fragmentation in the assessment and treatment of neuromuscular diseases, we compare here the currently used mammalian animal models for DMD with the aim of selecting and recommending the most appropriate ones for preclinical efficacy testing of new therapeutic strategies. [Copyright &y& Elsevier]

Abstract: The Golden Retriever Muscular Dystrophy dog lacks dystrophin. Disease progression in this model shares many similarities with the Duchenne muscular dystrophy, both from anatomico pathological and clinical standpoints. The model is increasingly used in pre-clinical trials but needs to be further investigated, particularly with reference to the evaluation of therapies. The aim of this study was to identify quantitative indices that would help characterize the dystrophic dog non-invasively using NMR imaging. Two-month-old dystrophic dogs and healthy control animals were scanned at 4T. Standard T2- and T1-weighted images, fat-saturated T1-weighted images pre- and post-gadolinium chelate injection were acquired and kinetics of muscle enhancement were studied over a 2-h period. Several indices were found to be abnormally high in dystrophic dogs: the T2-weighted/T1-weighted signal ratio, T2-weighted image heterogeneity and maximal signal enhancement post-gadolinium. These may be proposed to evaluate muscle structural alterations non-invasively in this disease. [Copyright &y& Elsevier]

Manipulation of pre-mRNA splicing by antisense oligonucleotides (AOs) offers considerable potential for a number of genetic disorders. One of these is Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene typically result in premature termination of translation that causes a loss of functional protein. AOs can induce exon skipping such that the mutation is by-passed and the reading frame restored, producing an internally deleted protein similar to that found in the milder Becker muscular dystrophy. To date, this approach has been applied to the mdx mouse model in vitro and in vivo and in human myoblast cultures. Here, we report the application of AO-directed exon skipping to induce dystrophin expression in vitro in a canine model of DMD, golden retriever muscular dystrophy (GRMD). The efficacy of 2′-O-methyl phosphorothioate (2OMe), phosphorodiamidate morpholino oligomers (PMOs) and peptide-linked PMOs (PMO-Pep) to induce dystrophin expression was assessed. The 2OMe chemistry was only effective for short-term induction of corrected transcript and could not induce detectable dystrophin protein. The PMO chemistry generally induced limited exon skipping at only high concentrations; however, a low level of dystrophin protein was produced in treated cells. Use of the PMO-Pep, applied here for the first time to a DMD model, was able to induce high and sustained levels of exon skipping and induced the highest level of dystrophin expression with no apparent adverse effects upon the cells. The induction of dystrophin in the GRMD model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials.Gene Therapy (2006) 13, 1373–1381. doi:10.1038/sj.gt.3302800; published online 25 May 2006 [ABSTRACT FROM AUTHOR]

Abstract: The Golden Retriever Muscular Dystrophy (GRMD) dog is the closest animal counterpart of Duchenne muscular dystrophy in humans and has, for this reason, increasingly been used in preclinical therapeutic trials for this disease. The aim of this study was to describe the abnormalities in canine dystrophic muscle non-invasively, quantitatively, thoroughly and serially by means of NMR imaging. Thoracic and pelvic limbs of five healthy and five GRMD dogs were imaged in a 3T NMR scanner at 2, 4, 6 and 9months of age. Standard and fat-saturated T 1-, T 2- and proton-density-weighted images were acquired. A measurement of T 1 and a two-hour kinetic study of muscle enhancement after gadolinium-chelate injection were also performed. Ten out of the 15 indices evaluated differed between healthy and GRMD dogs. The maximal relative enhancement after gadolinium injection and the proton-density-weighted/T 2-weighted signal ratio were the most discriminating indices. Inter-muscle heterogeneity was found to vary significantly for most of the indices. The body of data that has been acquired here will help in designing and interpreting preclinical trials using dystrophin-deficient dogs. [Copyright &y& Elsevier]