Chronic Administration of a Leupeptin-Derived Calpain Inhibitor Fails to Ameliorate Severe Muscle Pathology in a Canine Model of Duchenne Muscular Dystrophy

Calpains likely play a role in the pathogenesis of Duchenne muscular dystrophy (DMD). Accordingly, calpain inhibition may provide therapeutic benefit to DMD patients. In the present study, we sought to measure benefit from administration of a novel calpain inhibitor, C101, in a canine muscular dystrophy model. Specifically, we tested the hypothesis that treatment with C101 mitigates progressive weakness and severe muscle pathology observed in young dogs with golden retriever muscular dystrophy (GRMD). Young (6-week-old) GRMD dogs were treated daily with either C101 (17 mg/kg twice daily oral dose, n = 9) or placebo (vehicle only, n = 7) for 8 weeks. A battery of functional tests, including tibiotarsal joint angle, muscle/fat composition, and pelvic limb muscle strength were performed at baseline and every 2 weeks during the 8-week study. Results indicate that C101-treated GRMD dogs maintained strength in their cranial pelvic limb muscles (tibiotarsal flexors) while placebo-treated dogs progressively lost strength. However, concomitant improvement was not observed in posterior pelvic limb muscles (tibiotarsal extensors). C101 treatment did not mitigate force drop following repeated eccentric contractions and no improvement was seen in the development of joint contractures, lean muscle mass, or muscle histopathology. Taken together, these data do not support the hypothesis that treatment with C101 mitigates progressive weakness or ameliorates severe muscle pathology observed in young dogs with GRMD

Investigating a Possible Treatment of Duchene Muscular Dystrophy with a Novel Calpain Inhibitor [abstract]

Abstract only availableFaculty Mentor: Dr. Martin K. Childers, Physical Medicine & RehabilitationDuchene Muscular Dystrophy (DMD) is the most common lethal X-linked recessive muscle disease, affecting nearly one out of every 3,500 newborn males.  Symptoms appear before age three and by eleven, most children are unable to walk.  Few live past the age of 25.The genetic disorder is caused by a mutation in the dystrophin gene, eradicating the body's ability to produce the cytoskeletal protein, dystrophin.  In normal muscle cells, dystrophin is part of a molecular complex that adds mechanical integrity to the sarcolemma by linking the cytoskeleton to the extracellular matrix.  When the complex is disrupted, as in the case of DMD, the membrane is easily torn during regular muscle use.  Damage to the membrane causes aberrant influxes of Ca++, initiating a cascade of devastating molecular events in the sarcomere.  Elevated Ca++ over activates a family of proteases known as calpains. Calpains cleave proteins at specific sites.  Over-active calpains are thought to contribute to pathology in DMD.  Compounds that hinder calpain activity present a possible treatment for the disease.  A novel protease inhibitor has shown promising results in preliminary investigations in mice and this study was proposed to further explore the compound's effect on gene expression in canine muscle. An Affymetrix canine microarray was used to compare mRNA expression between normal dogs, dogs with golden retriever muscular dystrophy (GRMD), and inhibitor-treated GRMD dogs.  By comparing these expression levels, we are able to speculate whether calpain inhibitor treatment is able to mitigate aberrant gene expression in GRMD dogs.  Analysis of raw data is ongoing.  Further study is required to determine if mRNA levels equate with the protein expression levels using PCR, Western Blotting, or other methods

Respiratory dysfunction in unsedated dogs with golden retriever muscular dystrophy

Golden retriever muscular dystrophy (GRMD) is a well-established model of Duchenne muscular dystrophy. The value of this model would be greatly enhanced with practical tools to monitor progression of respiratory dysfunction during treatment trials. Arterial blood gas analysis, tidal breathing spirometry, and respiratory inductance plethysmography (RIP) were performed to determine if quantifiable abnormalities could be identified in unsedated, untrained, GRMD dogs. Results from 11 dogs with a mild phenotype of GRMD and 11 age-matched carriers were compared. Arterial blood gas analysis was successfully performed in all dogs, spirometry in 21 of 22 (95%) dogs, and RIP in 18 of 20 (90%) dogs. Partial pressure of carbon dioxide and bicarbonate concentration were higher in GRMD dogs. Tidal breathing peak expiratory flows were markedly higher in GRMD dogs. Abnormal abdominal motion was present in 7 of 10 (70%) GRMD dogs. Each technique provided objective, quantifiable measures that will be useful for monitoring respiratory function in GRMD dogs during clinical trials while avoiding the influence of sedation on results. Increased expiratory flows and the pattern of abdominal breathing are novel findings, not reported in people with Duchenne muscular dystrophy, and might be a consequence of hyperinflation

Genetic myostatin decrease in the golden retriever muscular dystrophy model does not significantly affect the ubiquitin proteasome system despite enhancing the severity of disease

Recent studies suggest that inhibiting the protein myostatin, a negative regulator of skeletal muscle mass, may improve outcomes in patients with Duchenne muscular dystrophy by enhancing muscle mass. When the dystrophin-deficient golden retriever muscular dystrophy (GRMD) dog was bred with whippets having a heterozygous mutation for the myostatin gene, affected GRMD dogs with decreased myostatin (GRippets) demonstrated an accelerated physical decline compared to related affected GRMD dogs with full myostatin. To examine the role of the ubiquitin proteasome and calpain systems in this accelerated decline, we determined the expression of the muscle ubiquitin ligases MuRF1, Atrogin-1, RNF25, RNF11, and CHIP: the proteasome subunits PSMA6, PSMB4, and PSME1: and calpain 1/2 by real time PCR in the cranial sartorius and vastus lateralis muscles in control, affected GRMD, and GRippet dogs. While individual affected GRMD and GRippet dogs contributed to an increased variability seen in ubiquitin ligase expression, neither group was significantly different from the control group. The affected GRMD dogs demonstrated significant increases in caspase-like and trypsin-like activity in the cranial sartorius; however, all three proteasome activities in the GRippet muscles did not differ from controls. Increased variability in calpain 1 and calpain 2 expression and activity in the affected GRMD and GRippet groups were identified, but no statistical differences from the control group were seen. These studies suggest a role of myostatin in the disease progression of GRMD, which does not significantly involve key components of the ubiquitin proteasome and calpain systems involved in the protein quality control of sarcomere and other structural skeletal muscle proteins

Eccentric contractions induce rapid isometric torque drop in dystrophin-deficient dogs

We tested the hypothesis that eccentric contractions (ECCs) rapidly induce greater-than-normal isometric torque drop in dystrophin-deficient golden retriever muscular dystrophy (GRMD) muscles. ECCs were imposed by forcibly stretching activated muscles. The results indicate that isometric torque drop was greater in GRMD versus controls (P < 0.0001). Our findings support the hypothesis that ECCs induce greater-than-normal isometric torque drop in GRMD muscles. The magnitude of ECC-induced isometric torque loss may be an ideal clinical endpoint in the GRMD model

NBD delivery improves the disease phenotype of the golden retriever model of Duchenne muscular dystrophy

Abstract Background Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene and afflicts skeletal and cardiac muscles. Previous studies showed that DMD is associated with constitutive activation of NF-κB, and in dystrophin-deficient mdx and utrophin/dystrophin (utrn -/- ;mdx) double knock out (dko) mouse models, inhibition of NF-κB with the Nemo Binding Domain (NBD) peptide led to significant improvements in both diaphragm and cardiac muscle function. Methods A trial in golden retriever muscular dystrophy (GRMD) canine model of DMD was initiated with four primary outcomes: skeletal muscle function, MRI of pelvic limb muscles, histopathologic features of skeletal muscles, and safety. GRMD and wild type dogs at 2 months of age were treated for 4 months with NBD by intravenous infusions. Results were compared with those collected from untreated GRMD and wild type dogs through a separate, natural history study. Results Results showed that intravenous delivery of NBD in GRMD dogs led to a recovery of pelvic limb muscle force and improvement of histopathologic lesions. In addition, NBD-treated GRMD dogs had normalized postural changes and a trend towards lower tissue injury on magnetic resonance imaging. Despite this phenotypic improvement, NBD administration over time led to infusion reactions and an immune response in both treated GRMD and wild type dogs. Conclusions This GRMD trial was beneficial both in providing evidence that NBD is efficacious in a large animal DMD model and in identifying potential safety concerns that will be informative moving forward with human trials

River ecosystem conceptual models and non‐perennial rivers: A critical review

Conceptual models underpin river ecosystem research. However, current models focus on continuously flowing rivers and few explicitly address characteristics such as flow cessation and drying. The applicability of existing conceptual models to nonperennial rivers that cease to flow (intermittent rivers and ephemeral streams, IRES) has not been evaluated. We reviewed 18 models, finding that they collectively describe main drivers of biogeochemical and ecological patterns and processes longitudinally (upstream-downstream), laterally (channel-riparian-floodplain), vertically (surface water-groundwater), and temporally across local and landscape scales. However, perennial rivers are longitudinally continuous while IRES are longitudinally discontinuous. Whereas perennial rivers have bidirectional lateral connections between aquatic and terrestrial ecosystems, in IRES, this connection is unidirectional for much of the time, from terrestrial-to-aquatic only. Vertical connectivity between surface and subsurface water occurs bidirectionally and is temporally consistent in perennial rivers. However, in IRES, this exchange is temporally variable, and can become unidirectional during drying or rewetting phases. Finally, drying adds another dimension of flow variation to be considered across temporal and spatial scales in IRES, much as flooding is considered as a temporally and spatially dynamic process in perennial rivers. Here, we focus on ways in which existing models could be modified to accommodate drying as a fundamental process that can alter these patterns and processes across spatial and temporal dimensions in streams. This perspective is needed to support river science and management in our era of rapid global change, including increasing duration, frequency, and occurrence of drying.info:eu-repo/semantics/publishedVersio

Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures

Abstract Background Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. Methods To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn +/−) whippets. A total of four GRippets (dystrophic and Mstn +/−), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. Results Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no differential expression between GRMD and GRippet dogs. Satellite cell exhaustion was not observed in GRippets up to 3 years of age. Conclusions Partial myostatin loss may exaggerate selective muscle hypertrophy or atrophy/hypoplasia in GRMD dogs and worsen contractures. While muscle imbalance is not a feature of myostatin inhibition in mdx mice, findings in a larger animal model could translate to human experience with myostatin inhibitors

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. We and others have developed various phenotypic tests 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. While some of these studies have largely provided 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. While 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