Metabolomic Study of Duchene Muscular Dystrophy in Canine Models and In- Silico Analysis of Dystrophin-B-Dystroglycan interaction.

Abstract

Duchene muscular dystrophy (DMD) is a devastating genetic disease of adolescents that causes disabilities in early teens and death in late teens or early twenties. It is the worst and severest form of disease among several different types of muscular dystrophies. Researches at genomic and proteomic levels have increased the molecular understanding of the disease. Lesser work, however, has been done on metabolomic front. Several animal models of Duchene muscular dystrophy including Caenorhabditis Elegans, Zebra fish, murine, feline and canine models have been established, bred and maintained for research purposes of which dogs serve as the best models because of their anatomical, physiological and pathological similarities with human subjects. Serum level studies of metabolome in human patients of DMD have identified several differentially present metabolites in the blood of the affected individuals but neither in humans nor in animals direct studies have been done on muscle metabolome of whole organism. In the current study, we have carried out metabolomic analysis on six months control vs Golden Retriever muscular dystrophy (GRMD) affected dogs using GC/MS to discover underlying metabolic defects. The metabolomic analysis was performed directly on two of the pelvic limb muscles; the more proximal biceps femoris muscle and the more distal long digital extensor muscle which is more severely affected than bicep femoris muscle. Our hypothesis was that the two muscles would have different metabolome. The partial least square discriminant analysis identified 15 metabolites to be significantly altered each in bicep femoris (BF) and long digital extensor (LDE) muscles out of which six metabolites {significantly decreased stearamide (0.23-fold of controls), carnosine (0.40-fold of controls), fumaric acid (0.40-fold of controls), and significantly increased oleic acid (1.77- fold of controls), glutamic acid (2.48-fold of controls) and proline(1.73-fold of controls)} were found to be t-test significant in bicep femoris while two metabolites inosine-5’- monophosphate (0.68-fold of controls) and 3-phosphoglycericacid (0.30-fold of controls) were t-test significant in long digital extensor muscles. Pathway enrichment analysis identified significant enrichment for arginine/proline metabolism (p = 5.88×10−4, FDR 4.7×10−2), where alterations in L-glutamic acid, proline, and carnosine were found. Additionally, multiple Krebs cycle intermediates were significantly decreased (e.g., malic VII acid, fumaric acid, citric/iso citric acid, and succinic acid), suggesting that altered energy metabolism maybe underlying the observed GRMD BF muscle dysfunction. Also, significant alterations were found in nucleic acid metabolism. Some of the results of our study support the previous findings in serum metabolome of human patients of DMD while others are novel to this study suggesting the need to further evaluate those metabolites for their role in dystrophic muscles. These newly identified unevenly produced metabolites in GRMD dogs as compared to healthy controls may also serve as potential biomarkers for the disease in future. Dystrophin, the key protein in which mutations or defects principally lead to Duchene muscular dystrophy, interacts with several other plasma membrane proteins to form dystrophin associated protein complex (DAPC). Beta-dystroglycan is an important protein of dystrophin associated protein complex whose failure to interact with dystrophin may also lead to muscular dystrophies. It has been proposed that post translational modifications of two of the beta-dystroglycan’s tyrosine residues present in its C-terminal domain may disrupt this interaction between dystrophin and beta-dystroglycan. These tyrosine residues are phosphorylated in adhesion dependent manner that disrupts dystrophin-β-dystroglycan interaction. In this study, we have performed molecular docking analysis of dystrophin with wild type, phosphorylated and mutated variants of β-DG to pinpoint the actual nature of this interaction at molecular level. We have discovered significant structural and conformational changes in β-DG molecule caused by mutations and tyrosine phosphorylation that alter the nature and site of its interaction with dystrophin. Our results not only support the previous findings but also bring to attention previously unreported discoveries about the nature of this interaction and behavior of different β-DG variants with dystrophin WW, EF-hand and ZZ domains.