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. 2013 Jul;23(7):833-43.
doi: 10.1093/glycob/cwt020. Epub 2013 Mar 20.

N-Glycolylneuraminic Acid Deficiency Worsens Cardiac and Skeletal Muscle Pathophysiology in α-Sarcoglycan-Deficient Mice

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Free PMC article

N-Glycolylneuraminic Acid Deficiency Worsens Cardiac and Skeletal Muscle Pathophysiology in α-Sarcoglycan-Deficient Mice

Paul T Martin et al. Glycobiology. .
Free PMC article

Abstract

Roughly 3 million years ago, an inactivating deletion occurred in CMAH, the human gene encoding CMP-Neu5Ac (cytidine-5'-monophospho-N-acetylneuraminic acid) hydroxylase (Chou HH, Takematsu H, Diaz S, Iber J, Nickerson E, Wright KL, Muchmore EA, Nelson DL, Warren ST, Varki A. 1998. A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence. Proc Natl Acad Sci USA. 95:11751-11756). This inactivating deletion is now homozygous in all humans, causing the loss of N-glycolylneuraminic acid (Neu5Gc) biosynthesis in all human cells and tissues. The CMAH enzyme is active in other mammals, including mice, where Neu5Gc is an abundant form of sialic acid on cellular membranes, including those in cardiac and skeletal muscle. We recently demonstrated that the deletion of mouse Cmah worsened the severity of pathophysiology measures related to muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy (Chandrasekharan K, Yoon JH, Xu Y, deVries S, Camboni M, Janssen PM, Varki A, Martin PT. 2010. A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy. Sci Transl Med. 2:42-54). Here, we demonstrate similar changes in cardiac and skeletal muscle pathology and physiology resulting from Cmah deletion in α-sarcoglycan-deficient (Sgca(-/-)) mice, a model for limb girdle muscular dystrophy 2D. These experiments demonstrate that loss of mouse Cmah can worsen disease severity in more than one form of muscular dystrophy and suggest that Cmah may be a general genetic modifier of muscle disease.

Keywords: dystroglycan; limb girdle; muscular dystrophy; sarcoglycan; sialic acid.

Figures

Fig. 1.
Fig. 1.
Neu5Gc immunostaining in Cmah-deficient α-sarcoglycan (Sgca)-deficient mouse skeletal muscle. Affinity purified chicken IgY-specific for Neu5Gc was used to immunostain gastrocnemius skeletal muscle from 6-week-old WT, Cmah−/−, Sgca−/−, and Cmah−/−Sgca−/− mice. Non-immune IgY control antisera were used to demonstrate specificity for Neu5Gc. Arrows show the rare cells stained for Neu5Gc in Cmah−/−Sgca−/− muscle. Bar is 100 μm for top four panels and 50 μm for bottom two panels.
Fig. 2.
Fig. 2.
Expression of Neu5Gc in macrophages and satellite cells of Cmah−/−Sgca−/− muscle. Neu5Gc was co-stained with CD68, a marker of macrophages, embryonic myosin (eMyosin), a marker of regenerating muscle and Pax7, a marker of satellite cells, in Cmah−/−Sgca−/− muscle. Control muscle shown is stained with non-immune chicken IgY (for Neu5Gc) and secondary antibody alone (anti-mouse) and is representative of all control conditions. Bar is 100 μm for all panels.
Fig. 3.
Fig. 3.
Staining of muscle biopsies from LGMD2D patients. Three different LGMD2D biopsies were stained with hematoxylin and eosin (H and E), with affinity purified chicken IgY-specific for Neu5Gc (Neu5Gc) or with non-immune chicken IgY control antiserum (Control). Arrows indicate staining with the Neu5Gc antiserum. Bar is 200 μm for upper row and 100 μm for bottom two rows.
Fig. 4.
Fig. 4.
Altered skeletal muscle pathology in Cmah−/−Sgca−/− mice. Hematoxylin and eosin (H and E) staining of the gastrocnemius muscle (Gastroc) in 8-month-old (mo) Sgca−/− and Cmah−/−Sgca−/ mice (upper panels). EBD uptake after a 45-min period of exercise (middle panels). Mason's trichrome staining of the diaphragm muscle (lower panels). Collagen stains blue with trichrome stain. Bar is 50 μm in upper and lower panels and 200 μm in middle panels.
Fig. 5.
Fig. 5.
Altered cardiac muscle pathology in Cmah−/−Sgca−/− mice. Mason's trichrome staining of WT, Cmah−/−, Sgca−/− and Cmah−/−Sgca−/− heart muscles at 4 months of age. Collagen stains blue with trichrome stain, whereas muscle stains red. Bar is 50 μm for all panels.
Fig. 6.
Fig. 6.
Increased dye uptake and loss of muscle tissue in Cmah−/−Sgca−/− mice. (A) Mice were injected with Evan's blue dye and subjected to walking for 45 min prior to analysis of muscles for dye uptake. The percentage of myofibers or cardiomyocytes with dye was compared with the total number of myofibers or cardiomyocytes present. (B) The percentage of the muscle area taken up by non-muscle tissue was quantified. Such non-muscle tissue resulted from the presence of necrotic foci of mononuclear cells as well as fat and extracellular matrix deposition. Errors are SD for n = 5–6 animals per condition in (A) and (B). **P < 0.01, ***P < 0.001, comparing Sgca−/−Cmah−/− to Sgca−/−.
Fig. 7.
Fig. 7.
Altered cardiac and skeletal muscle physiology in Cmah−/−Sgca−/− mice. (A) Maximal developed force of isolated cardiac trabecular muscles, relative to the cross-sectional area, at various frequencies within the physiological range. (B) Developed force of isolated cardiac trabecular muscles in response to different doses of the β-agonist isoproterenol. (C) Specific force of isolated diaphragm muscle. (D) Relative force loss during repeated eccentric contractions in isolated EDL muscles over 10 repetitive stimulations. WT, CMP-Neu5Ac hydroxylase-deficient (Cmah−/−), α-sarcoglycan-deficient (Sgca−/−) and CMP-Neu5Ac hydroxylase-deficient and α-sarcoglycan-deficient (Cmah−/−Sgca−/−). Errors are SEM for n = 6–12 muscles per condition.
Fig. 8.
Fig. 8.
Anti-Neu5Gc serum antibody titers in Cmah−/−Sgca−/− mice. Neu5Gc-specific antibody titers were measured by ELISA, comparing 1 μg spots of Cmah−/− (Neu5Gc-free) muscle glycoprotein and WT (Neu5Gc-rich) muscle glycoprotein, along with mouse Ab antibody standard curves. (A) SNA, a sialic acid binding lectin, bound glycoproteins from WT and Cmah−/− muscle glycoproteins equally well, whereas an anti-Neu5Gc-specific antibody bound only WT muscle glycoprotein and not Cmah−/− muscle glycoprotein. Errors are SD for n = 3–4 mice per condition. (B) Serum anti-Neu5Gc-specific antibody titers in individual Cmah−/−Sgca−/− mice.

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