Like Duchenne muscular dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog model of DMD is characterized by muscle necrosis, progressive paralysis, and pseudohypertrophy in specific ...skeletal muscles. This severe GRMD phenotype includes moderate atrophy of the biceps femoris (BF) as compared to unaffected normal dogs, while the long digital extensor (LDE), which functions to flex the tibiotarsal joint and serves as a digital extensor, undergoes the most pronounced atrophy. A recent microarray analysis of GRMD identified alterations in genes associated with lipid metabolism and energy production.
We, therefore, undertook a non-targeted metabolomics analysis of the milder/earlier stage disease GRMD BF muscle versus the more severe/chronic LDE using GC-MS to identify underlying metabolic defects specific for affected GRMD skeletal muscle.
Untargeted metabolomics analysis of moderately-affected GRMD muscle (BF) identified eight significantly altered metabolites, including significantly decreased stearamide (0.23-fold of controls, p = 2.89 × 10
), carnosine (0.40-fold of controls, p = 1.88 × 10
), fumaric acid (0.40-fold of controls, p = 7.40 × 10
), lactamide (0.33-fold of controls, p = 4.84 × 10
), myoinositol-2-phosphate (0.45-fold of controls, p = 3.66 × 10
), and significantly increased oleic acid (1.77-fold of controls, p = 9.27 × 10
), glutamic acid (2.48-fold of controls, p = 2.63 × 10
), and proline (1.73-fold of controls, p = 3.01 × 10
). Pathway enrichment analysis identified significant enrichment for arginine/proline metabolism (p = 5.88 × 10
, FDR 4.7 × 10
), where alterations in L-glutamic acid, proline, and carnosine were found. Additionally, multiple Krebs cycle intermediates were significantly decreased (e.g., malic acid, fumaric acid, citric/isocitric acid, and succinic acid), suggesting that altered energy metabolism may be underlying the observed GRMD BF muscle dysfunction. In contrast, two pathways, inosine-5'-monophosphate (VIP Score 3.91) and 3-phosphoglyceric acid (VIP Score 3.08) mainly contributed to the LDE signature, with two metabolites (phosphoglyceric acid and inosine-5'-monophosphate) being significantly decreased. When the BF and LDE were compared, the most significant metabolite was phosphoric acid, which was significantly less in the GRMD BF compared to control and GRMD LDE groups.
The identification of elevated BF oleic acid (a long-chain fatty acid) is consistent with recent microarray studies identifying altered lipid metabolism genes, while alterations in arginine and proline metabolism are consistent with recent studies identifying elevated L-arginine in DMD patient sera as a biomarker of disease. Together, these studies demonstrate muscle-specific alterations in GRMD-affected muscle, which illustrate previously unidentified metabolic changes.
Duchenne muscular dystrophy (DMD) is an X-linked inherited myopathy that causes progressive skeletal and cardiac muscle disease. Heart lesions were described in the earliest DMD reports, and ...cardiomyopathy is now the leading cause of death. However, diagnostics and treatment for cardiomyopathy have lagged behind those for appendicular and respiratory skeletal muscle disease. Most animal model studies have been done in the mdx mouse, which has a relatively mild form of cardiomyopathy. Dogs with the genetically homologous condition, Golden Retriever muscular dystrophy (GRMD), develop progressive cardiomyopathy analogous to that seen in DMD. Previous descriptive studies of GRMD cardiomyopathy have mostly been limited to selective sampling of the hearts from young dogs.
We systematically assessed cardiac lesions in 31 GRMD and carrier dogs aged 3 to 76 months and a separate cohort of 2-10-year-old normal hounds. Both semi-quantitative lesion scoring and quantitation of the cross-sectional area of fibrosis distinguished dogs with GRMD disease from normal dogs. The carriers generally had intermediate involvement but had even greater fibrosis than GRMD dogs. Fatty infiltration was the most prominent feature in some older GRMD dogs. Vascular hypertrophy was increased in GRMD dogs and correlated positively with lesion severity. Purkinje fiber vacuolation was also increased but did not correlate with lesion severity. Histopathologic changes correlated with late gadolinium enhancement on cardiac MRI.
These features are generally compatible with those of DMD and further validate GRMD as a useful model to study cardiomyopathy pathogenesis and treatment. Additionally, the nature of some degenerative lesions suggests that functional hypoxia or non-thrombotic ischemia may contribute to disease progression.
ABSTRACT
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
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Duchenne muscular dystrophy (DMD) is an X-linked human disorder in which absence of the protein dystrophin causes degeneration of skeletal and cardiac muscle. For the sake of treatment development, ...over and above definitive genetic and cell-based therapies, there is considerable interest in drugs that target downstream disease mechanisms. Drug candidates have typically been chosen based on the nature of pathologic lesions and presumed underlying mechanisms and then tested in animal models. Mammalian dystrophinopathies have been characterized in mice (mdx mouse) and dogs (golden retriever muscular dystrophy GRMD). Despite promising results in the mdx mouse, some therapies have not shown efficacy in DMD. Although the GRMD model offers a higher hurdle for translation, dogs have primarily been used to test genetic and cellular therapies where there is greater risk. Failed translation of animal studies to DMD raises questions about the propriety of methods and models used to identify drug targets and test efficacy of pharmacologic intervention. The mdx mouse and GRMD dog are genetically homologous to DMD but not necessarily analogous. Subcellular species differences are undoubtedly magnified at the whole-body level in clinical trials. This problem is compounded by disparate cultures in clinical trials and preclinical studies, pointing to a need for greater rigor and transparency in animal experiments. Molecular assays such as mRNA arrays and genome-wide association studies allow identification of genetic drug targets more closely tied to disease pathogenesis. Genes in which polymorphisms have been directly linked to DMD disease progression, as with osteopontin, are particularly attractive targets.
ABSTRACT
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
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, which codes for the dystrophin protein. While progress has been made in defining the molecular basis and pathogenesis of DMD, ...major gaps remain in understanding mechanisms that contribute to the marked delay in cardiac compared to skeletal muscle dysfunction.
To address this question, we analyzed cardiac and skeletal muscle tissue microarrays from golden retriever muscular dystrophy (GRMD) dogs, a genetically and clinically homologous model for DMD. A total of 15 dogs, 3 each GRMD and controls at 6 and 12 months plus 3 older (47-93 months) GRMD dogs, were assessed.
GRMD dogs exhibited tissue- and age-specific transcriptional profiles and enriched functions in skeletal but not cardiac muscle, consistent with a "metabolic crisis" seen with DMD microarray studies. Most notably, dozens of energy production-associated molecules, including all of the TCA cycle enzymes and multiple electron transport components, were down regulated. Glycolytic and glycolysis shunt pathway-associated enzymes, such as those of the anabolic pentose phosphate pathway, were also altered, in keeping with gene expression in other forms of muscle atrophy. On the other hand, GRMD cardiac muscle genes were enriched in nucleotide metabolism and pathways that are critical for neuromuscular junction maintenance, synaptic function and conduction.
These findings suggest differential metabolic dysfunction may contribute to distinct pathological phenotypes in skeletal and cardiac muscle.
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-
18
Ffluoro-
d
-glucose (
18
FFDG).
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
18
FFDG 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-
18
FFDG /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.
Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease for which there is no cure and limited treatment options. Prednisone is currently the first line treatment option for DMD and ...studies have demonstrated that it improves muscle strength. Although prednisone has been used for the treatment of DMD for decades, the mechanism of action of this drug remains unclear. Recent studies have shown that the α7β1 integrin is a major modifier of disease progression in mouse models of DMD and is therefore a target for drug-based therapies. In this study we examined whether prednisone increased α7β1 integrin levels in mdx mouse and GRMD dog models and myogenic cells from humans with DMD. Our results show that prednisone promotes an increase in α7 integrin protein in cultured myogenic cells and in the muscle of mdx and GRMD animal models of DMD. The prednisone-mediated increase in α7 integrin was associated with increased laminin-α2 in prednisone-treated dystrophin-deficient muscle. Together, our results suggest that prednisone acts in part through increased merosin in the muscle basal lamina and through sarcolemmal stabilization of α7β1 integrin in dystrophin-deficient muscle. These results indicate that therapies that target an increase in muscle α7β1 integrin, its signaling pathways and/or laminin could be therapeutic in DMD.
Highlights • GRMD dogs walked shorter absolute and height-adjusted distances on the 6MWT at 6 and 12 months of age. • 6MWT results correlated with other functional outcome parameters for all dogs, ...but not GRMD alone. • The percent change in CK after the 6MWT was greater in GRMD versus normal/carrier dogs at 6 months. • A 80% increase in distance walked or 55% decrease in CK would be necessary to achieve a power of 0.80 if six GRMD dogs were assessed at 6 months.
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