Levels of α7 integrin and laminin-α2 are increased following prednisone treatment in the mdx mouse and GRMD dog models of Duchenne muscular dystrophy

SUMMARY 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

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

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest follows every 10 contractions with a total of 30 contractions performed

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

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

Maternal choline supplementation in a sheep model of first trimester binge alcohol fails to protect against brain volume reductions in peripubertal lambs

Fetal alcohol spectrum disorder (FASD) is a leading potentially preventable birth defect. Poor nutrition may contribute to adverse developmental outcomes of prenatal alcohol exposure, and supplementation of essential micronutrients such as choline has shown benefit in rodent models. The sheep model of first-trimester binge alcohol exposure was used in this study to model the dose of maternal choline supplementation used in an ongoing prospective clinical trial involving pregnancies at risk for FASD. Primary outcome measures included volumetrics of the whole brain, cerebellum, and pituitary derived from magnetic resonance imaging (MRI) in 6-month-old lambs, testing the hypothesis that alcohol-exposed lambs would have brain volume reductions that would be ameliorated by maternal choline supplementation. Pregnant sheep were randomly assigned to one of five groups – heavy binge alcohol (HBA; 2.5 g/kg/treatment ethanol), heavy binge alcohol plus choline supplementation (HBC; 2.5 g/kg/treatment ethanol and 10 mg/kg/day choline), saline control (SC), saline control plus choline supplementation (SCC; 10 mg/kg/day choline), and normal control (NC). Ewes were given intravenous alcohol (HBA, HBC; mean peak BACs of ~280 mg/dL) or saline (SC, SCC) on three consecutive days per week from gestation day (GD) 4–41; choline was administered on GD 4–148. MRI scans of lamb brains were performed postnatally on day 182. Lambs from both alcohol groups (with or without choline) showed significant reductions in total brain volume; cerebellar and pituitary volumes were not significantly affected. This is the first report of MRI-derived volumetric brain reductions in a sheep model of FASD following binge-like alcohol exposure during the first trimester. These results also indicate that maternal choline supplementation comparable to doses in human studies fails to prevent brain volume reductions typically induced by first-trimester binge alcohol exposure. Future analyses will assess behavioral outcomes along with regional brain and neurohistological measures

Kinematics of gait in Golden Retriever Muscular Dystrophy

The goal of this study was to quantify the two-dimensional kinematics of pathologic gait during overground walking at a self-selected speed at the stifle (knee) and hock (ankle) joints in six Golden Retriever Muscular Dystrophy (GRMD) dogs and six carrier littermates (controls). We found that GRMD dogs walked significantly slower than controls (p < 0.01). At the stifle joint, both groups displayed similar ROM (range of motion), but compared to controls, GRMD dogs walked with the stifle joint relatively more extended. At the hock joint, GRMD dogs displayed less ROM (range of motion) and walked with the joint relatively less flexed compared to controls. We controlled for gait speed in all analyses, so the differences we observed in joint kinematics between groups cannot be attributed solely to the slower walking speed of the GRMD dogs. This is the first kinematic study of gait in the GRMD dog, an important step in using this model in pre-clinical trials

Computed tomography assessment of peripubertal craniofacial morphology in a sheep model of binge alcohol drinking in the first trimester

Identification of facial dysmorphology is essential for the diagnosis of fetal alcohol syndrome (FAS); however, most children with fetal alcohol spectrum disorders (FASD) do not meet the dysmorphology criterion. Additional objective indicators are needed to help identify the broader spectrum of children affected by prenatal alcohol exposure. Computed tomography (CT) was used in a sheep model of prenatal binge alcohol exposure to test the hypothesis that quantitative measures of craniofacial bone volumes and linear distances could identify alcohol-exposed lambs. Pregnant sheep were randomly assigned to four groups: heavy binge alcohol, 2.5 g/kg/day (HBA); binge alcohol, 1.75 g/kg/day (BA); saline control (SC); and normal control (NC). Intravenous alcohol (BA; HBA) or saline (SC) infusions were given three consecutive days per week from gestation day 4-41, and a CT scan was performed on postnatal day 182. The volumes of eight skull bones, cranial circumference, and 19 linear measures of the face and skull were compared among treatment groups. Lambs from both alcohol groups showed significant reduction in seven of the eight skull bones and total skull bone volume, as well as cranial circumference. Alcohol exposure also decreased four of the 19 craniofacial measures. Discriminant analysis showed that alcohol-exposed and control lambs could be classified with high accuracy based on total skull bone volume, frontal, parietal, or mandibular bone volumes, cranial circumference, or interorbital distance. Total skull volume was significantly more sensitive than cranial circumference in identifying the alcohol-exposed lambs when alcohol-exposed lambs were classified using the typical FAS diagnostic cutoff of ≤10th percentile. This first demonstration of the usefulness of CT-derived craniofacial measures in a sheep model of FASD following binge-like alcohol exposure during the first trimester suggests that volumetric measurement of cranial bones may be a novel biomarker for binge alcohol exposure during the first trimester to help identify non-dysmorphic children with FASD

Polystyrene-coated micropallets for culture and separation of primary muscle cells

Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures’ surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact-printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells