Increased histopathology in Cmah−/−mdx skeletal and cardiac muscle. (A) 3 month-old (mo) heart sections were stained with hematoxylin and eosin. Cmah−/−mdx heart muscle had large mononuclear infiltrates normally not seen in age-matched mdx heart muscle. (B) 2 mo mdx and Cmah−/−mdx skeletal muscle (quadriceps) was stained with modified Mason’s Trichrome. Fibrosis, replacement of muscle tissue with collagen (blue), was evident in some regions of the Cmah−/−mdx quadriceps muscle, while it was never observed in mdx muscle at this age. (C) 6 mo diaphragm muscle was stained with hematoxylin and eosin. Cmah−/−mdx diaphragm showed increased fibrosis relative to mdx. (D) Evan’s blue dye uptake in 2 mo quadriceps muscle was increased, after exercise, in Cmah−/−mdx muscle relative to mdx. (E) Areas of muscle damage were quantified for skeletal muscles and heart muscles. No significant damage was measured in any WT or Cmah−/− muscle. Bar is 200μm in A, B and C and 100μm in C. Errors are SEM for n=6-12 per condition in E.

Expression of Neu5Gc in normal and dystrophic mouse and human muscles. (A) N-glycolylneuraminic acid (Neu5Gc) differs from N-acetylneuraminic acid (Neu5Ac) by an additional oxygen at the 5 position of this sialic acid (modified from(112)). Because mice express a functional Cmah gene, they incorporate sialic acids (Sias) at the outer ends of glycolipids and glycoproteins that is usually either Neu5Gc or Neu5Ac. Humans and Cmah−/− mice, by contrast, contain an inactivating deletion in Cmah, and therefore do not express Neu5Gc on the surface of glycoconjugates but instead have increased Neu5Ac. Skeletal muscles (B) and cardiac muscles (C) were immunostained with a Neu5Gc-specific antibody. (D) DMD and Cmah−/−mdx skeletal muscles were co-stained for Neu5Gc (green) and embryonic myosin (eMyosin, red), which marks regenerating myofibers/myoblasts. Bar is 100μm in B, 200μm in C, and 50μm in D. Abbreviations: WT, wild type; DMD, Duchenne muscular dystrophy; IM, inflammatory myopathy (inclusion body myositis); N-glycolylneuraminic acid, Neu5Gc; embryonic myosin, eMyosin.

Anti-Neu5Gc titers and complement deposition and killing in Cmah−/−mdx mice. (A) Serum antibody titers to Neu5Gc-containing glycoproteins were compared to titers against Neu5Ac-containing glycoproteins to determine Neu5Gc-specific antibody titers. Skeletal muscles proteins for wild type or Cmah−/− skeletal muscles were purified using Maackia amurensus agglutinin (MAA), which purifies α2-3-linked sialylglycoproteins, or Sambucus nigra agglutinin (SNA), which purifies α2-6-linked sialylglycoproteins (see S3C). Only Cmah−/−mdx mouse serum showed measurable Neu5Gc-specific titers, and only toα2-6-linked glycoprotein. (B) Serum from Cmah−/− mdx and mdx mice was compared for complement-mediated cell killing of Neu5Gc-expressing myoblasts and myotubes, from wild type (WT) mice, and Neu5Gc-deficient myoblasts and myotubes, from Cmah−/− mice. (C) Quantification of the area of muscle deposited with activated C5b-9 complement. Levels were elevated in Cmah−/−mdx gastronemius (Gastroc), quadriceps (Quad), tibialis anterior (TA), diaphragm (Diaph) and heart relative to mdx. (D) Increased staining of activated C5b-9 complement in Cmah−/− mdx and DMD quadriceps relative to mdx or normal human muscle. Errors are SEM for n=6-12 in A-C. *P<0.05, **P<0.01, ***P<0.001

Increased mortality and functional deficits in Cmah−/−mdx skeletal and cardiac muscle. (A) Cmah−/−mdx mice showed a highly significant decrease in lifespan relative to mdx, Cmah−/−, and wild type (WT) mice (P<0.001 for Cmah−/−mdx vs. all others). (B) By 8 months of age, Cmah−/−mdx showed a 70% reduction in time able to walk on a constant speed rotorod for 5 minutes relative to mdx littermates (P<0.001), while Cmah−/− mice did not show a significant decrease relative to WT (P>0.05). (C) Developed tension (specific force in mN/mm²) of isolated cardiac trabeculae muscles was reduced in 8 month-old Cmah−/−mdx relative to mdx. (D) Specific force of diaphragm muscle was reduced in 8 month-old Cmah−/−mdx animals relative to mdx. (E) Evan’s blue dye uptake into myofibers was increased after exercise in the gastrocnemius (Gastroc), quadriceps (Quad), tibialis anterior (TA), triceps, and diaphragm muscles of 2 month-old Cmah−/−mdx mice relative to mdx. Cmah−/− and WT muscles showed no dye uptake in any muscle. (F) Normalized specific force during repeated eccentric contractions (left) in the extensor digitorum longus muscle was reduced in Cmah−/−mdx compared to mdx, as was maximal specific force. Errors are SEM for n=75-100 animals per condition in A, 12-25 in B, 5-9 in C, 3-6 in D, 5-6 in E, and 11-12 in F. *P<0.05, **P<0.01, ***P<0.001

Measures of immune response in Cmah−/−mdx muscle. (A) mRNA expression for markers of muscle inflammation (IL-1β, MCP-1, and MIP-1a) and macrophage/monocyte infiltration (CD68) were elevated in 2 month-old (mo) and 5mo mdx and Cmah−/−mdx skeletal muscles, relative to wild type. 5mo MIP-1a levels for WT and Cmah−/− were undetectable. (B) mRNA expression of MCP-1 and CD68 were elevated in 5 mo Cmah−/−, mdx, and Cmah−/−mdx heart. (C) 5 mo Cmah−/−, mdx and Cmah−/−mdx hearts all showed elevations in some markers of cardiac hypertrophy (P<0.05 for all vs. WT for CD68 and MCP-1). (D) Number of cells per unit muscle area expressing CD4, CD8, or CD68 was quantified in 2 mo skeletal muscles and heart. (E) ELISPOT assays for stimulation by Concanavalin A (ConA) of T cell interleukin 4 or interferon gamma were compared. No stimulant is buffer alone. (F) Stimulation Index was calculated to assess T cell proliferation in response to ConA as compared to no stimulant (buffer alone). Abbreviations: ANP, atrial natriuretic peptide; AT1a, angiotensin II receptor, type 1a; BNP, B-type natriuretic peptide; Col1(α1), collagen type I, alpha 1; Col3(α1) collagen type III, alpha 1; ET1, endothelin 1; Serca2, sarco/endoplasmic reticulum calcium transporting ATPase 2; TNFα, tumor necrosis factor alpha; IL1β, interleukin 1 beta; MCP-1, monocyte chemoattractant protein 1 (also chemokine (C-C motif) ligand 2 (Ccl2)); MIP-1a, macrophage inflammatory protein 1a (also chemokine (C-C motif) ligand 3 (Ccl3)); RANTES, regulation upon activation, normally T-expressed, and presumably secreted (also chemokine (C-C motif) ligand 5 (Ccl5)). Errors are SEM for n=9-12 in A-C, n=6 in D-F. *P<0.05, **P<0.01, ***P<0.001

Molecular changes resulting from loss of Neu5Gc expression. (A) Loss of Neu5Gc (Cmah−/−) on α dystroglycan (αDG) led to decreased laminin α2 (LNα2) binding (P<0.001 for WT vs. Cmah−/− at 20nM). Affinity purified recombinant G1-G5 domain of laminin α2 was used as the ligand. (B-D) Neu5Gc(α2-3 or α2-6)Galβ1-4GlcNAc (3′ or 6′SLN)-biotin-polyacrylamide (PAA-glycan) had significantly higher binding to laminin α2 (LNα2, B), laminin α4 (LNα4, C) and laminin α5 (LNα5, D) than did Neu5Ac (α2-3 or α2-6)Galβ1-4GlcNAc-biotin-PAA (P<0.001 for Neu5Gc vs. Neu5Ac for all α2,3- or α2,6 Sia-linked comparisons at 500nM). Galα1-3Galβ1-4GlcNAc (GalLN)-PAA, by contrast, showed only very weak binding to laminins. (E) Immunoblot analysis of DAG protein changes in skeletal muscle and heart. Expected molecular weights for proteins are: Dystrophin (Dys)-427kDa, Utrophin (Utrn)-400kDa, α dystrobrevin (αDbn) 2-65kD, α dystrobrevin 3–4)(αDbn 3-4)-44kDa, α dystroglycan (αDG)-160kDa, β dystroglycan (βDG)-43kDa, α sarcoglycan (αSG)-50kDa, β sarcoglycan (βSG)-43kDa, γ sarcoglycan (γSG)-35kDa, δ sarcoglycan (δSG)-35kDa, mouse IgG (55kDa), mouse IgM* (70kDa), Actin-42kDa. (F-G) Gene expression changes in skeletal muscle and heart at 5 months of age. Abbreviations: Collagen IV, α1 chain (Col4(α1)), Collagen IV, α2 chain (Col4(α2)), laminin (LN) α2, α4, and α5, dystroglycan (Dag1), Integrin (Int) α7 or β1. (H-I) Protein expression was quantified by densitometric scanning of Western blots in skeletal muscle or heart. Abbreviations: Collagen I (Col1), Collagen III (Col3), ATPase, Ca²⁺ transporting, cardiac muscle, slow twitch 2 (Serca2). (J) Western blots of heart muscle protein show elevated collagen 1 and 3 expression in Cmah−/−, mdx, and Cmah−/−mdx cardiac muscle and reduced Serca2 in Cmah−/−mdx muscle. Expected molecular weights are: Col1(precursor) 140-210kDa, Col3 110-140kDa, Serca2 100kDa (K) 2 month-old heart or skeletal muscle (gastrocnemius) mRNA was compared using microarrays (n=3 per condition). Genes that were significantly up or down-regulated by 2-fold or more (P<0.05 between Cmah−/−, mdx, or Cmah−/−mdx and wild type) were identified and subdivided based on whether they overlapped between groups or not. Errors are SEM for n=6-8 per condition in A-D and n=9-10 in F-I.