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Comparative Study
. 1997 Oct 20;139(2):375-85.
doi: 10.1083/jcb.139.2.375.

Animal models for muscular dystrophy show different patterns of sarcolemmal disruption

V Straub  1 , J A Rafael, J S Chamberlain, K P Campbell
Affiliations
Comparative Study

Animal models for muscular dystrophy show different patterns of sarcolemmal disruption

V Straub et al. J Cell Biol. .

Abstract

Genetic defects in a number of components of the dystrophin-glycoprotein complex (DGC) lead to distinct forms of muscular dystrophy. However, little is known about how alterations in the DGC are manifested in the pathophysiology present in dystrophic muscle tissue. One hypothesis is that the DGC protects the sarcolemma from contraction-induced damage. Using tracer molecules, we compared sarcolemmal integrity in animal models for muscular dystrophy and in muscular dystrophy patient samples. Evans blue, a low molecular weight diazo dye, does not cross into skeletal muscle fibers in normal mice. In contrast, mdx mice, a dystrophin-deficient animal model for Duchenne muscular dystrophy, showed significant Evans blue accumulation in skeletal muscle fibers. We also studied Evans blue dispersion in transgenic mice bearing different dystrophin mutations, and we demonstrated that cytoskeletal and sarcolemmal attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. The extent of dye incorporation in transgenic mice correlated with the phenotypic severity of similar dystrophin mutations in humans. We furthermore assessed Evans blue incorporation in skeletal muscle of the dystrophia muscularis (dy/dy) mouse and its milder allelic variant, the dy2J/dy2J mouse, animal models for congenital muscular dystrophy. Surprisingly, these mice, which have defects in the laminin alpha2-chain, an extracellular ligand of the DGC, showed little Evans blue accumulation in their skeletal muscles. Taken together, these results suggest that the pathogenic mechanisms in congenital muscular dystrophy are different from those in Duchenne muscular dystrophy, although the primary defects originate in two components associated with the same protein complex.

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Figures

Figure 1
Figure 1
Macroscopic evaluation of Evans blue staining after intravenous dye injection into two 9-wk-old mdx littermates (mouse 1, a and b; mouse 2, c and d). The mice were fixed in a 8% formaldehyde solution 6 h after the dye injection. EBD staining in mouse 1 (a and b) showed staining mainly within the regions of the pelvic girdle muscles. In particular, the dye was incorporated in the gluteal and the femoral quadriceps muscles. Plasma membrane permeability for Evans blue varied from animal to animal and even from limb to limb in the same animal. In mouse 1, we found differences in both the intensity and the extent of stained regions in the left and right triceps brachii muscles (a and b, arrows). Besides dye uptake in proximal limb muscles, we also found discoloration of the external oblique muscle of the abdomen, the longest thoracic and lumbar muscles, and the cutaneous muscles of the trunk (c and d). Affected muscles were not stained homogeneously, but they typically showed blue strands according to damaged muscle fibers. Anterior tibial muscles in mdx mice were often macroscopically spared from dye staining (a and c).
Figure 2
Figure 2
EBD (a–e) and H&E staining (f) on 7-μm (a, b, e, and f) and 15-μm (c and d) skeletal muscle cryosections from 8- (a, b, e, and f) and 16-wk-old (c and d) intravenously (a, b, e, and f) and intraperitoneally (c and d) injected mdx mice. In some animals, the number of dye-positive fibers in the femoral quadriceps muscle was >70% (a). b shows a magnification of a in which the fascia demarcates a highly damaged muscle region from the unaffected adjacent muscle. Other muscles, including the intercostal muscles (c, white asterisks indicate rips) and gluteal muscles (d, longitudinal section) took up the dye. EBD staining in mdx diaphragm (e) demonstrated variation in the dye-positive fibers. Dye positive fibers in the corresponding H&E staining (f) revealed morphological features of normal fibers (asterisk) as well as of necrosis (arrowhead). Hypercontracted fibers did not necessarily indicate membrane damage, since some of them did not take up EBD (arrows). Bars, 100 μm.
Figure 3
Figure 3
EBD staining on 7-μm cryosections of mdx cardiac muscle. EBD-positive cardiomyocytes were detected in 8 out of 16 mdx mice but never in control animals. One intravenously injected 4-wk-old animal showed large areas of dye uptake into the myocardium (a), whereas in other mice, smaller regions of EBD-positive fibers were found in the ventricular walls (b). Cardiac dye uptake in intraperitoneally injected mdx mice (c, 10-mo-old animal) demonstrated that the fiber damage was caused by the underlying disease and not by a volume overload of intravenously administered dye, leading to a cardiac infarct. Bars, 100 μm.
Figure 4
Figure 4
Loss of membrane integrity is indicated by intracellular staining of serum proteins. EBD-positive fibers in skeletal muscle from mdx mice also showed positive staining with antibodies against albumin on the same section. (A) Double staining for EBD (a) and albumin (b) in an 8-wk-old mdx mouse. (B) Skeletal muscle of 10-wk-old mice that were not injected with EBD were studied for uptake of serum proteins into muscle fibers. Immunohistochemical staining of 7-μm femoral quadriceps cryosection from uninjected normal and mdx mice with antibodies against mouse IgG (a and b), mouse IgM (c and d), and mouse albumin (e and f) showed accumulation of the serum markers in mdx skeletal muscle. No difference of protein uptake into muscle fibers was detected between control and dy/dy mice. Bar, 50 μm.
Figure 4
Figure 4
Loss of membrane integrity is indicated by intracellular staining of serum proteins. EBD-positive fibers in skeletal muscle from mdx mice also showed positive staining with antibodies against albumin on the same section. (A) Double staining for EBD (a) and albumin (b) in an 8-wk-old mdx mouse. (B) Skeletal muscle of 10-wk-old mice that were not injected with EBD were studied for uptake of serum proteins into muscle fibers. Immunohistochemical staining of 7-μm femoral quadriceps cryosection from uninjected normal and mdx mice with antibodies against mouse IgG (a and b), mouse IgM (c and d), and mouse albumin (e and f) showed accumulation of the serum markers in mdx skeletal muscle. No difference of protein uptake into muscle fibers was detected between control and dy/dy mice. Bar, 50 μm.
Figure 5
Figure 5
EBD staining on 7-μm cryosection of skeletal muscle from four transgenic/mdx mice. The top portion of the picture shows a model of the normal dystrophin gene. The femoral quadriceps muscle of the Dp71 mouse (20 wk old) revealed the same amount of fiber damage in the EBD assay as the original mdx mutant. Mice with a deletion of dystrophin exons 71–74 (17 wk old) did not show significantly more dye uptake into the femoral quadriceps muscle than seen in control animals. The Δ17-48 transgenic/mdx mouse (9 wk old), which has a deletion in the rod domain of dystrophin, showed few dye-positive fibers in the quadriceps femoris muscle. The Δ3-7 transgenic/mdx mouse (10 wk old), which has a deletion in the amino-terminal actin-binding domain of dystrophin, showed dye-positive fibers in the diaphragm (Δ3-7), as well as in the quadriceps femoris muscle. Bar, 50 μm.
Figure 6
Figure 6
Macroscopic and microscopic evaluation of EBD staining after intravenous dye injection into 3-mo-old normal control (a and b), mdx (c and d), and dy/dy (e and f) mice. Uptake of dye into the hind legs of the mice was examined 6 h after the injection. The hind legs were fixed in a 8% formaldehyde solution. In contrast to mdx mice (c), dy/dy (e) or dy2J/dy2J mice never showed localized EBD uptake into skeletal muscles by visual inspection. Blue coloration of dy/dy and dy2J/dy2J mice observed by macroscopic evaluation was caused by dye uptake into the connective tissue, which is increased in these dystrophic animals. This finding was confirmed by fluorescence microscopy analysis of 7-μm cryosections from the quadriceps femoris muscle of injected animals. Grouped EBD-positive fibers were only detected in mdx mice (d), whereas cryosections from normal control mice (b), dy/dy (f), or dy2J/dy2J mice did not show dye uptake into groups of muscle fibers. On cryosections of dy/dy (f) mice, single necrotic fibers showed EBD staining by fluorescence microscopy. Bar, 50 μm.
Figure 7
Figure 7
EBD staining (a) and H&E staining (b) on 7-μm cryosections from a 7-wk-old dy/dy diaphragm. The few EBD-positive fibers in dy/dy skeletal muscle always showed necrotic features in the corresponding H&E stain. The nuclei of infiltrating immune cells were also detected by the EBD. Bars, 100 μm.
Figure 8
Figure 8
Immunohistochemical staining of 7-μm cryosection from normal human skeletal muscle, DMD skeletal muscle, and skeletal muscle from patients with laminin α2 chain–deficient CMD with antibodies against human IgG and IgM. The plasma proteins showed intracellular fiber staining in the quadriceps femoris muscle from DMD biopsies, indicating loss of membrane integrity. Positive staining of grouped fibers was only detected in DMD patients. The antibody against human IgG did not show the same staining intensity as the antibody against human IgM. Interestingly, not all IgG-positive fibers took up IgM molecules (arrow), indicating different sizes of membrane disruptions. This observation was confirmed on serial sections throughout the damaged area. Bar 50, μm.
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