Volume 90, Issue 4, Pages 729-738 (August 1997) Skeletal and Cardiac Myopathies in Mice Lacking Utrophin and Dystrophin: A Model for Duchenne Muscular Dystrophy  R.Mark Grady, Haibing Teng, Mia C Nichol, Jeanette C Cunningham, Robert S Wilkinson, Joshua R Sanes  Cell  Volume 90, Issue 4, Pages 729-738 (August 1997) DOI: 10.1016/S0092-8674(00)80533-4

Figure 1 Poor Growth and Kyphosis in mdx:utrn−/− Mice (a) Double mutants (open squares) are initially the same size as mdx (closed circles), utrn−/−, and wild-type (not shown) littermates but grow more slowly after 3 weeks of age. (b–d) Radiographs of 6- to 7-week-old mdx:utrn−/− (b), mdx (c), and wild-type mice (d), showing small size and pronounced kyphosis in the double mutant. Cell 1997 90, 729-738DOI: (10.1016/S0092-8674(00)80533-4)

Figure 2 Degeneration and Regeneration Is Transient in mdx Muscle but Persists in mdx:utrn−/− Muscle Sections from tibialis anterior muscles of 2-week-old (a and b), 4-week-old (c and d), or 10-week-old (e–j) mice were stained with hematoxylin and eosin. Necrosis is prominent in both mdx and mdx:utrn−/− muscle at 4 weeks, and >90% of fibers show central nuclei characteristic of regeneration (g and h) by 10 weeks. However, necrosis begins earlier and persists longer in the double mutants, leading to fibrosis (f). Neither necrosis nor regeneration is seen in wild-type or utrn−/− muscle at any age (i and j). Magnification: (a)–(f), (i), and (j), 4.8×; (g) and (h), 9.6×. Cell 1997 90, 729-738DOI: (10.1016/S0092-8674(00)80533-4)

Figure 3 Ongoing Regeneration in mdx:utrn−/− Muscle Sections from tibialis anterior muscles of 2-week-old (a and b), 4-week-old (c and d), or 10-week-old (e–h) mice were stained with a monoclonal antibody specific for embryonic and neonatal myosin heavy chains, which are expressed by newly regenerated muscle fibers. Regeneration is prominent in both mdx and mdx:utrn−/− muscle at 4 weeks but begins earlier and persists longer in the latter. No regeneration is seen in wild-type or utrn−/− muscle (i and j). Magnification, 4.8×. Cell 1997 90, 729-738DOI: (10.1016/S0092-8674(00)80533-4)

Figure 4 Subtle Abnormalities at mdx:utrn−/− Neuromuscular Junctions (a–d) En face views of endplates in 7- to 8-week-old mice, stained with rhodamine-α-bungarotoxin. mdx and mdx:utrn−/− endplates are subdivided into discrete boutons, probably as a consequence of myofiber degeneration and regeneration. (e and f) Electron microscopy shows that nerve terminals and Schwann cells are qualitatively similar in wild-type (e) and double-mutant (f) muscle, but that the density of junctional folds is reduced in the latter. (g) Quantitation of junctional fold density from 66 to 94 measurements per genotype. Magnification: (a)–(d), 20.8×; bar in (f) is 1 μm for (e) and (f). Asterisk, p < 0.01 versus wild type; double asterisk, p < 0.001 versus mdx by ANOVA. Bars, SEM. Cell 1997 90, 729-738DOI: (10.1016/S0092-8674(00)80533-4)

Figure 5 Disruption of the Dystrophin-Associated Complex at mdx:utrn−/− Synapses Sections from tibialis anterior muscles of 4- to 6-week-old mice were doubly stained with the indicated antibodies plus rhodamine- α-bungarotoxin. Synaptic sites, identified by bungarotoxin staining (not shown), are marked by arrowheads. Utrophin and dystrophin are, as expected, absent from double mutants (d and h). In addition, the synapse-specific β2-syntrophin is undetectable (x) and levels of β-dystroglycan (p) and dystrobrevin (t) are markedly reduced. However, rapsyn (l), agrin, and laminin-β2 (not shown) remain concentrated at synaptic sites in the double mutant. Magnification, 31.2×. Cell 1997 90, 729-738DOI: (10.1016/S0092-8674(00)80533-4)

Figure 6 Weakness and Contractile Abnormality in mdx:utrn−/− Muscle Sternomastoid muscles were attached to a force transducer and stimulated supramaximally through their nerve at 0.5 Hz to assess twitch tension (a–d) or at 100 Hz for 8 s to assess tetanic tension (e). (a) Representative tracings of single twitches. (b) Peak tension is reduced in double-mutant muscle. (c) Relaxation time is prolonged in double mutants. (d) Latency does not vary among genotypes. (e) Fatigue time, which reflects synaptic failure, does not vary among genotypes. Bars, SEM. Cell 1997 90, 729-738DOI: (10.1016/S0092-8674(00)80533-4)

Figure 7 Cardiomyopathy in mdx:utrn−/− Mice (a–d) Hematoxylin- and eosin-stained sections of ventricular muscle from 10-week-old mice of the indicated genotypes. Significant necrosis is present only in the double mutant. (e and f) Mice were injected with Evans blue, which binds to albumin and accumulates in cells with damaged membranes. Hearts were sectioned and viewed with fluorescence optics. Large patches of permeable myocytes were evident in the double mutant (f), but not in mdx (e), wild-type, or utrn−/− (not shown) hearts. (g) Hematoxylin- and eosin-stained section from the heart of a mouse that had been injected with Evans blue. Dye-filled (damaged) fibers, which appear purple (arrow), are grouped between areas of necrosis and areas of normal fibers (pink). Magnification: (a)–(d), 9.6×; (e) and (f), 4.8×; (g), 19.2×. Cell 1997 90, 729-738DOI: (10.1016/S0092-8674(00)80533-4)