Expression and Regulation of Glycogen Synthase in Equine Type 1 Polysaccharide Storage Myopathy

Abstract

IntroductionEquine Type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense, gain‐of‐function mutation in the equine skeletal muscle glycogen synthase gene (GYS1). Affected horses have excessive glycogen storage, amylopectate‐like inclusions in their muscle and higher muscle glycogen synthase activities (GS) than controls. GS is normally allosterically‐regulated by glucose‐6‐phosphate and enzyme activity is reduced by phosphorylation. We hypothesised that the higher GS activity in PSSM1‐affected horses might be due either to 1) increased expression or reduced degradation of the mutant enzyme or 2) due to reduced phosphorylation.MethodsSemimembranosus muscle samples were biopsied from 4 homozygotes (HH), 13 heterozygotes (RH) and 12 control (RR) Belgian Draught horses. Samples were analysed for glycogen content and GS activity. Western immunoblotting was used to examine expression of total and phosphorylated forms of GS, GSK3β and GLUT4. Data was analysed using non‐parametric ANOVA statistical tests with post‐hoc Dunn's multiple comparison tests.ResultsPSSM1 affected heterozygotes and homozygotes had significantly higher muscle glycogen contents than controls (RR vs. RH P = 0.005, RR vs. HH P = 0.041) and GS activity was significantly higher in PSSM1‐affected homozygotes than in heterozygotes and control horses (P = 0.03). Although there was no difference in total GS expression between groups, there was significantly more expression of GS phosphorylated at site 2 + 2a in PSSM1‐affected horses in comparison with controls (P = 0.009).ConclusionsOur data suggest that increased GS activity in PSSM1‐affected horses is not associated with increased expression or degradation of the mutant enzyme and occurs despite increased phosphorylation of GS at site 2 + 2a. Alternative mechanisms, including aberrant ligand binding might account for the increased GS activity in PSSM1‐affected muscle.Ethical Animal ResearchMuscle biopsy samples were collected according to ethical approval from the local Institutional Animal Care and Use Committee. Explicit owner informed consent for participation in this study is not stated. Sources of funding: Petplan Charitable Trust and The Royal Veterinary College. Competing interests: none.