Summary BACKGROUND Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. RESULTS PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. CONCLUSIONS Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. GENERAL SIGNIFICANCE This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy.
Caution should be exercised when interpreting fibre size profiles in shipped samples. Equine muscle biopsy samples are optimally shipped in dry gauze, sealed in plastic containers and shipped on ice packs to be processed within 24 h and can thus be interpreted by the receiving laboratory with minimal artefact.
A glutamic acid to lysine (E40K) residue substitution in superoxide dismutase 1 (SOD1) is associated with canine degenerative myelopathy: the only naturally occurring large animal model of amyotrophic lateral sclerosis (ALS). The E40 residue is highly conserved across mammals, except the horse, which naturally carries the (dog mutant) K40 residue. Here we hypothesized that in vitro expression of mutant dog SOD1 would recapitulate features of human ALS (ie, SOD1 protein aggregation, reduced cell viability, perturbations in mitochondrial morphology and membrane potential, reduced ATP production, and increased superoxide ion levels); further, we hypothesized that an equivalent equine SOD1 variant would share similar perturbations in vitro, thereby explain horses' susceptibility to certain neurodegenerative diseases. As in human ALS, expression of mutant dog SOD1 was associated with statistically significant increased aggregate formation, raised superoxide levels (ROS), and altered mitochondrial morphology (increased branching (form factor)), when compared to wild-type dog SOD1-expressing cells. Similar deficits were not detected in cells expressing the equivalent horse SOD1 variant. Our data helps explain the ALS-associated cellular phenotype of dogs expressing the mutant SOD1 protein and reveals that species-specific sequence conservation does not necessarily predict pathogenicity. The work improves understanding of the etiopathogenesis of canine degenerative myelopathy.
This publication is a compilation of all Research Abstracts presented at the Ninth International Conference on Equine Exercise Physiology. Unlike previous ICEEP conferences there will not be a conference proceedings of full length manuscripts. These abstracts succinctly summarise a wide array of investigations relevant to the equine athlete, and will be useful to veterinarians and others involved in management of horses used for sport, work and competition. The abstracts encompass the topics of Applied PhysiologyIntroduction: The ability to accurately assess equine oxygen consumption (VO2) under field conditions has been limited by the need for unrestricted gas exchange.Methods: Two variations of a mask and an associated electronics control module (ECM) were designed to enable breath-by-breath measurement of airflows with two 8.0 cm diameter pneumotachometers located 7.5 cm in front of each narus and connected to differential pressure transducers mounted on the outside of the mask. The ECM was comprised of electronics for signal filtering to the flow transducers, an oxygen sensing cell, and an analog-to-digital converter all powered by a lithium-ion battery. The battery also powered a pump connected to gas sampling ports between the nares and pneumotachometers. Airflow and oxygen content of inspired and expired gases were recorded through the ECM and electronically transferred to a notebook. VO2 was determined from these recordings by an operator using a customized software analysis program. One mask encased the lower head (E). The other left the jaw free so horse could wear a bit and be ridden (R). Multiple treadmill exercise tests were undertaken by 6 horses to measure VO2max and blood gases. Each mask was worn twice and results compared to those from an open flow-through system (O) by 2-way RMANOVA (P<0.05). Utility of the system was evaluated using the intraclass correlation coefficient of 4 independent raters.Results: Blood gases and VO2max (152.0 ± 4.0 (mean ± SEM; O), 147.7 ± 4.3 (E), 150.7 ± 3.3 (R) ml/(kg.min) were not different between masks. VO2 measures were reproducible for each mask. Agreement between the 4 raters was excellent (intraclass correlation coefficient = 0.99).Conclusions: Masks capable of measuring VO2 during field exercise were developed, tested and found accurate by multiple users.Ethical Animal Research: Studies performed were approved by the Institution's Animal Care and Use Committee (protocol #3807). Sources of funding: Institutional sources. Competing interests: Washington State University has filed notice of its intent to apply for a patent for facemask used in this study.Introduction: Exertional myopathies are a common cause of exercise intolerance in the equine athlete, and Polysaccharide Storage Myopathy (PSSM) is a widely described muscular disease. It is characterized by an accumulation of abnormal glycogen in myofibers due to a genetic defect in the skeletal muscle glycogen synthase (GYS1) enzyme. We hypothesized that energy production through oxidative phosphorylation (OXP...
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