Peter P. Nghiem

Canine Models of Duchenne Muscular Dystrophy and Their Use in Therapeutic Strategies

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which the loss of dystrophin causes progressive degeneration of skeletal and cardiac muscle. Potential therapies that carry substantial risk, such as gene- and cell-based approaches, must first be tested in animal models, notably the mdx mouse and several dystrophin-deficient breeds of dogs, including golden retriever muscular dystrophy (GRMD). Affected dogs have a more severe phenotype, in keeping with that of DMD, so may better predict disease pathogenesis and treatment efficacy. Various phenotypic tests have been developed to characterize disease progression in the GRMD model. These biomarkers range from measures of strength and joint contractures to magnetic resonance imaging. Some of these tests are routinely used in clinical veterinary practice, while others require specialized equipment and expertise. By comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Potential treatments for DMD may be broadly categorized as molecular, cellular, or pharmacologic. The GRMD model has increasingly been used to assess efficacy of a range of these therapies. A number of these studies have provided largely general proof-of-concept for the treatment under study. Others have demonstrated efficacy using the biomarkers discussed. Importantly, just as symptoms in DMD vary among patients, GRMD dogs display remarkable phenotypic variation. Though confounding statistical analysis in preclinical trials, this variation offers insight regarding the role that modifier genes play in disease pathogenesis. By correlating functional and mRNA profiling results, gene targets for therapy development can be identified.

The Paradox of Muscle Hypertrophy in Muscular Dystrophy

Mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophy in humans and syndromes in mice, dogs, and cats. Affected humans and dogs have progressive disease that leads primarily to muscle atrophy. Mdx mice progress through an initial phase of muscle hypertrophy followed by atrophy. Cats have persistent muscle hypertrophy. Hypertrophy in humans has been attributed to deposition of fat and connective tissue (pseudohypertrophy). Increased muscle mass (true hypertrophy) has been documented in animal models. Muscle hypertrophy can exaggerate postural instability and joint contractures. Deleterious consequences of muscle hypertrophy should be considered when developing treatments for muscular dystrophy.

Pharmacologic Management of Duchenne Muscular Dystrophy: Target Identification and Preclinical Trials

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.

Sparing of the dystrophin-deficient cranial sartorius muscle is associated with classical and novel hypertrophy pathways in GRMD dogs

Both Duchenne and golden retriever muscular dystrophy (GRMD) are caused by dystrophin deficiency. The Duchenne muscular dystrophy sartorius muscle and orthologous GRMD cranial sartorius (CS) are relatively spared/hypertrophied. We completed hierarchical clustering studies to define molecular mechanisms contributing to this differential involvement and their role in the GRMD phenotype. GRMD dogs with larger CS muscles had more severe deficits, suggesting that selective hypertrophy could be detrimental. Serial biopsies from the hypertrophied CS and other atrophied muscles were studied in a subset of these dogs. Myostatin showed an age-dependent decrease and an inverse correlation with the degree of GRMD CS hypertrophy. Regulators of myostatin at the protein (AKT1) and miRNA (miR-539 and miR-208b targeting myostatin mRNA) levels were altered in GRMD CS, consistent with down-regulation of myostatin signaling, CS hypertrophy, and functional rescue of this muscle. mRNA and proteomic profiling was used to identify additional candidate genes associated with CS hypertrophy. The top-ranked network included α-dystroglycan and like-acetylglucosaminyltransferase. Proteomics demonstrated increases in myotrophin and spectrin that could promote hypertrophy and cytoskeletal stability, respectively. Our results suggest that multiple pathways, including decreased myostatin and up-regulated miRNAs, α-dystroglycan/like-acetylglucosaminyltransferase, spectrin, and myotrophin, contribute to hypertrophy and functional sparing of the CS. These data also underscore the muscle-specific responses to dystrophin deficiency and the potential deleterious effects of differential muscle involvement.

OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages.

What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full‐length human OPN‐a isoform is the most pro‐inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD‐integrin‐dependent manner. OPN‐a upregulates expression of tenascin‐C (TNC), a known Toll‐like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro‐inflammatory effects of OPN‐a, suggesting that a potential mechanism of OPN action is by promoting TNC–TLR4 signalling.

Whole genome sequencing reveals a 7 base-pair deletion in DMD exon 42 in a dog with muscular dystrophy

Dystrophin is a key cytoskeletal protein coded by the Duchenne muscular dystrophy (DMD) gene located on the X-chromosome. Truncating mutations in the DMD gene cause loss of dystrophin and the classical DMD clinical syndrome. Spontaneous DMD gene mutations and associated phenotypes occur in several other species. The mdx mouse model and the golden retriever muscular dystrophy (GRMD) canine model have been used extensively to study DMD disease pathogenesis and show efficacy and side effects of putative treatments. Certain DMD gene mutations in high-risk, the so-called hot spot areas can be particularly helpful in modeling molecular therapies. Identification of specific mutations has been greatly enhanced by new genomic methods. Whole genome, next generation sequencing (WGS) has been recently used to define DMD patient mutations, but has not been used in dystrophic dogs. A dystrophin-deficient Cavalier King Charles Spaniel (CKCS) dog was evaluated at the functional, histopathological, biochemical, and molecular level. The affected dog’s phenotype was compared to the previously reported canine dystrophinopathies. WGS was then used to detect a 7 base pair deletion in DMD exon 42 (c.6051-6057delTCTCAAT mRNA), predicting a frameshift in gene transcription and truncation of dystrophin protein translation. The deletion was confirmed with conventional PCR and Sanger sequencing. This mutation is in a secondary DMD gene hotspot area distinct from the one identified earlier at the 5′ donor splice site of intron 50 in the CKCS breed.

Use of the six-minute walk test to characterize golden retriever muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which loss of the dystrophin protein causes progressive skeletal/cardiac muscle degeneration and death within the third decade. For clinical trials and supportive animal studies, DMD disease progression and response to treatment must be established using outcome parameters (biomarkers). The 6-minute walk test (6MWT), defined as the distance an individual can walk in 6 minutes, is commonly used in DMD clinical trials and has been employed in dogs to characterize cardiac and respiratory disease severity. Building on methods established in DMD and canine clinical studies, we assessed the 6MWT in dogs with the DMD genetic homolog, golden retriever muscular dystrophy (GRMD). Twenty-one cross-bred golden retrievers were categorized as affected (DMD mutation and GRMD phenotype), carrier (female heterozygous for DMD mutation and no phenotype), and normal (wild type DMD gene and normal phenotype). When compared to grouped normal/carrier dogs, GRMD dogs walked shorter height-adjusted distances at 6 and 12 months of age and their distances walked declined with age. Percent change in creatine kinase after 6MWT was greater in GRMD versus normal/carrier dogs at 6 months, providing another potential biomarker. While these data generally support use of the 6MWT as a biomarker for preclinical GRMD treatment trials, there were certain limitations. Results of the 6MWT did not correlate with other outcome parameters for GRMD dogs when considered alone and an 80% increase in mean distance walked would be necessary to achieve satisfactory power.

Osteopontin is Linked with AKT, FoxO1, and Myostatin in Skeletal Muscle Cells.

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

A novel canine model for Duchenne muscular dystrophy (DMD): single nucleotide deletion in DMD gene exon 20

BackgroundBoys with Duchenne muscular dystrophy (DMD) have DMD gene mutations, with associated loss of the dystrophin protein and progressive muscle degeneration and weakness. Corticosteroids and palliative support are currently the best treatment options. The long-term benefits of recently approved compounds such as eteplirsen and ataluren remain to be seen. Dogs with naturally occurring dystrophinopathies show progressive disease akin to that of DMD. Accordingly, canine DMD models are useful for studies of pathogenesis and preclinical therapy development. A dystrophin-deficient, male border collie dog was evaluated at the age of 5xa0months for progressive muscle weakness and dysphagia.Case presentationDramatically increased serum creatine kinase levels (41,520xa0U/L; normal range 59–895xa0U/L) were seen on a biochemistry panel. Histopathologic changes characteristic of dystrophinopathy were seen. Dystrophin was absent in the skeletal muscle on immunofluorescence microscopy and western blot. Whole genome sequencing, polymerase chain reaction, and Sanger sequencing revealed a frameshift, single nucleotide deletion in canine DMD exon 20, position 27,626,466 (c.2841delT mRNA), resulting in a stop codon six nucleotides downstream. Semen was archived for future line perpetuation.ConclusionsThis spontaneous canine dystrophinopathy occurred due to a novel mutation in the minor DMD mutation hotspot (between exons 2 through 20). Perpetuating this line could allow for preclinical testing of genetic therapies targeted to this area of the DMD gene.

Glucose Metabolism as a Pre-clinical Biomarker for the Golden Retriever Model of Duchenne Muscular Dystrophy

PurposeMetabolic 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.ProceduresPelvic 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).ResultsMRNA 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 5xa0min post-dextrose injection in GRMD vs. normal/carrier dogs. PET/ CT with [18F]FDG and simultaneous insulin stimulation showed a significant increase (pu2009=u20090.03) in mean standard uptake values (SUV) in GRMD skeletal muscle but not pelvic fat at 5xa0min post-[18F]FDG /insulin injection. Conversely, mean cardiac SUV was lower in GRMD than carrier/normal (pu2009<u20090.01).ConclusionsAltered 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.