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

Coyotes Go “Bridge and Tunnel”: A Narrow Opportunity to Study the Socio-ecological Impacts of Coyote Range Expansion on Long Island, NY Pre- and Post-Arrival

Currently, Long Island, NY is without a breeding population of northeastern coyote (Canis latras var.), yet recent evidence of dispersing individuals on the island, coupled with the “dogged” momentum of coyote range expansion across North America, suggests a Long Island coyote population is close at hand. We highlighted the fleeting opportunity to takes advantage of this natural experiment by developing a multidisciplinary research framework to investigate the ecological and social impacts of the coyote, pre- and post- range expansion. We reviewed coyote spatial ecology, community ecology, and human dimensions research and identified three components of future investigation: predicting future occupancy, monitoring colonization, testing hypotheses of trophic cascades by leveraging and expanding existing ecological data, and exploring attitudes towards coyotes to better understand and mitigate human-wildlife conflicts. Each proposed component will integrate for a comprehensive investigation to advance theory and applied management of northeastern coyotes

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

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

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

Spatial and temporal variation in Arctic freshwater chemistry—Reflecting climate-induced landscape alterations and a changing template for biodiversity

Freshwater chemistry across the circumpolar region was characterised using a pan-Arctic data set from 1,032 lake and 482 river stations. Temporal trends were estimated for Early (1970-1985), Middle (1986-2000), and Late (2001-2015) periods. Spatial patterns were assessed using data collected since 2001.Alkalinity, pH, conductivity, sulfate, chloride, sodium, calcium, and magnesium (major ions) were generally higher in the northern-most Arctic regions than in the Near Arctic (southern-most) region. In particular, spatial patterns in pH, alkalinity, calcium, and magnesium appeared to reflect underlying geology, with more alkaline waters in the High Arctic and Sub Arctic, where sedimentary bedrock dominated.Carbon and nutrients displayed latitudinal trends, with lower levels of dissolved organic carbon (DOC), total nitrogen, and (to a lesser extent) total phosphorus (TP) in the High and Low Arctic than at lower latitudes. Significantly higher nutrient levels were observed in systems impacted by permafrost thaw slumps.Bulk temporal trends indicated that TP was higher during the Late period in the High Arctic, whereas it was lower in the Near Arctic. In contrast, DOC and total nitrogen were both lower during the Late period in the High Arctic sites. Major ion concentrations were higher in the Near, Sub, and Low Arctic during the Late period, but the opposite bulk trend was found in the High Arctic.Significant pan-Arctic temporal trends were detected for all variables, with the most prevalent being negative TP trends in the Near and Sub Arctic, and positive trends in the High and Low Arctic (mean trends ranged from +0.57%/year in the High/Low Arctic to -2.2%/year in the Near Arctic), indicating widespread nutrient enrichment at higher latitudes and oligotrophication at lower latitudes.The divergent P trends across regions may be explained by changes in deposition and climate, causing decreased catchment transport of P in the south (e.g. increased soil binding and trapping in terrestrial vegetation) and increased P availability in the north (deepening of the active layer of the permafrost and soil/sediment sloughing). Other changes in concentrations of major ions and DOC were consistent with projected effects of ongoing climate change. Given the ongoing warming across the Arctic, these region-specific changes are likely to have even greater effects on Arctic water quality, biota, ecosystem function and services, and human well-being in the future

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