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First published online 28 March 2006
doi: 10.1242/jcs.02857

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Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex

University of Washington School of Medicine, Department of Neurology Box 357720, Seattle, WA 98195-7720, USA

View larger version (52K): [in a new window]   Fig. 1. Generation of Dp116/mdx4cv transgenic mice. (A) Design of the Dp116 expression construct with the domain structure of Dp116 compared with other dystrophin isoforms and mini-genes. NT, N-terminal domain; H, hinge; W, WW domain; CR, cysteine rich domain; CT, C-terminal domain; DgBD, dystroglycan-binding domain; HSA, human skeletal -actin promoter; in, hybrid HSA/SV40 vp1 intron; F, Flag epitope tag; pA, SV40 polyadenylation sequence. (B) Western analysis using a monoclonal antibody specific for the dystrophin C-terminal domain (Dys-2) shows expression of Dp116 in diaphragm (D) and quadriceps (Q) muscles from three independent lines of Dp116/mdx4cv transgenic mice. Samples from control C57Bl/6 mice show full-length dystrophin in diaphragm and quadriceps, and Dp116 in peripheral nerve (N). (C) Immunofluorescence staining with antisera to the dystrophin N-terminal domain detects full-length dystrophin only in control C57Bl/6 muscles. Polyclonal antibodies to the dystrophin C-terminal domain or the Flag epitope demonstrate uniform expression of the Dp116 transgene in quadriceps and diaphragm muscles (line 2197).

View larger version (83K): [in a new window]   Fig. 2. Soleus muscle is dramatically affected at an early age in Dp116/mdx4cv transgenic mice. Complete cross sections of hematoxylin- and eosin-stained soleus muscles from Dp116/mdx4cv (a',b') and mdx4cv (a,b) mice at both 3 weeks (a,a') and 4 weeks (b,b') of age are shown. At 3 weeks the mdx4cv muscle has small areas of degeneration but minimal numbers of fibers with central nuclei. At this age, Dp116 transgenic muscle shows extensive degeneration and inflammation with clusters of regenerated fibers containing central nuclei (arrow). At 4 weeks the mdx4cv muscle has many successfully regenerated fibers (arrow) and large areas of normal tissue. By contrast, the transgenic muscle at 4 weeks has little normal muscle tissue remaining. Bar, 500 µm.

View larger version (109K): [in a new window]   Fig. 3. Histopathology of Dp116/mdx4cv transgenic muscle is more severe than that of mdx4cv. Hematoxylin and eosin stained sections from the diaphragm muscle at 4 weeks of age and diaphragm, quadriceps, and soleus muscles at 12 weeks age are shown for C57Bl/6, mdx4cv, and Dp116/mdx4cv mice. Widespread necrosis of muscle fibers and mononuclear cell infiltrates are more prominent in the diaphragm and soleus muscles of Dp116/mdx4cv transgenic mice at all ages compared with mdx4cv. The quadriceps muscles from Dp116/mdxcv and mdx4cv mice were not noticeably different at 12 weeks. Immunofluorescence staining of serial sections of 12 week soleus muscles with anti-CD11b antibody demonstrates infiltration of inflammatory cells in mdx4cv and transgenic/mdx4cv muscles. Inflammation in the transgenic/mdx4cv soleus muscle was widespread and diffuse, in contrast to the focal pattern in mdx4cv soleus muscle. Bars, 100 µm.

View larger version (67K): [in a new window]   Fig. 4. Overexpression of Dp116 on a wild-type background has dominant-negative effects. (A) Cross sections of quadriceps muscle from Dp116 transgenic mice and C57Bl/6 controls. Immunofluorescence staining with antibodies specific to the dystrophin N-terminal domain and the Flag epitope demonstrate decreased expression of full-length dystrophin but high levels of Dp116 on the sarcolemma of transgenic muscles. Hematoxylin and eosin staining shows increased numbers of muscle fibers with central nuclei in the transgenic quadriceps. Bar, 100 µm. (B) Western analysis using a monoclonal antibody specific for the dystrophin C-terminus confirms reduced amounts of full-length dystrophin protein in various skeletal muscles of transgenic mice compared with controls. An identical gel stained in parallel with Coomassie Blue is shown as a loading control. D, diaphragm; Q, quadriceps; S, soleus; TA, tibialis anterior; N, peripheral nerve.

View larger version (17K): [in a new window]   Fig. 5. Quantitative assessment of histopathology in tibialis anterior muscles of 12-week-old mice. (A) Percentage of fibers with central nuclei. (B) Box plots show variance in the muscle fiber diameter. Boxes represent the middle quartiles from the 25th to 75th percentiles, bars represent high and low values (statistical outliers are shown as circles). Each data set is composed of >1000 fibers of n=3-6 mice each. ANOVA statistical tests were performed on measurements of % central nuclei and the median fiber diameters. *P<0.01, statistical difference compared with C57Bl/6; #P<0.01, statistical difference compared with mdx4cv; P<0.05, statistical difference compared with C57Bl/6.

View larger version (109K): [in a new window]   Fig. 6. Expression of dystrophin-associated proteins is restored in Dp116/mdx4cv transgenic muscle. (A) Immunofluorescence staining of quadriceps muscles with antibodies specific for ß-dystroglycan, -sarcoglycan, ß-sarcoglycan, -sarcoglycan, -dystrobrevin 2, and 1-syntrophin. Bar, 100 µm. (B) Western blots from KCl-washed microsomes using antibodies against the dystrophin C-terminus (Dys-2), ß-dystroglycan, ß-sarcoglycan, -sarcoglycan, -dystrobrevin 2, and 1-syntrophin. An identical gel stained in parallel with Coomassie Blue is shown as a loading control. c, C57Bl/6; m, mdx4cv; tg, Dp116/mdx4cv.

View larger version (67K): [in a new window]   Fig. 7. Expression of nNOS is not restored by expression of Dp116. (A) Immunofluorescence staining of quadriceps muscles with a polyclonal antibody against nNOS. Dp116/mdx4cv and transgenic/mdx muscles expressing Dp260, H2-R19 (mini-dystrophin), and R4-23 (micro-dystrophin) are shown. Bar, 100 µm. (B) Western analysis of nNOS from pellet (microsomal) and supernatant (soluble) fractions of KCl-washed microsome preparations from C57Bl/6 (1), mdx4cv (2), Dp116/mdx4cv (3), and R4-23/mdx (4) skeletal muscles. An identical gel stained in parallel with Coomassie Blue is shown as a loading control.

View larger version (55K): [in a new window]   Fig. 8. Dp116 expression displaces utrophin from the sarcolemma in soleus muscle. Immunofluorescence staining of quadriceps and soleus muscles using an antibody specific for the utrophin A isoform. The split panels shown for Dp116/mdx4cv quadriceps muscle represent extremes in utrophin staining. Bar, 100 µm.