Department of * Physiology and Biophysics and Department of Neurology, Howard Hughes Medical Institute, University of
Iowa College of Medicine, Iowa City, Iowa 52242; and § Department of Human Genetics, University of Michigan Medical School,
Ann Arbor, Michigan 48109
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 2-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.
MUTATIONS in several components of the dystrophin-glycoprotein complex (DGC)1 are known
to be involved in the pathogenesis of muscular
dystrophies (Ozawa et al., 1995 It has been proposed that the initial event in muscle cell
necrosis in DMD was the focal breakdown of the plasmalemma (Mokri and Engel, 1975 To study altered sarcolemmal permeability in dystrophic muscle fibers, we injected animal models for muscular
dystrophy with Evans blue dye (EBD). The tetrasodium
diazo salt Evans blue, also called T-1824, is a membrane-impermeant molecule that can be used in determining
blood volume (Reeve, 1957 For the tracer injection, we used mdx mice, transgenic/
mdx mice, dy/dy, and dy2J/dy2J mice. The mdx mouse is a
dystrophin-deficient animal model for X-linked DMD
(Bulfield et al., 1984 Furthermore, we examined the dy/dy and the dy2J/dy2J
mice. These murine diseases are inherited in an autosomal
recessive fashion and have been studied as models for human laminin Mice
Normal control, the original mdx mutant, and dy2J/dy2J mice were bred at
the University of Iowa from stocks originally obtained from the Jackson
ImmunoResearch Laboratories, Inc. (Bar Harbor, ME). All mice are on a
C57BL/10 background. The dy/dy mice were also obtained from the Jackson Laboratories. The transgenic/mdx mouse strains were maintained at
the University of Michigan. Control and disease animals were matched by
age and gender. The age range of the mice was between 4 wk and 1 yr. All
animal studies were authorized by the Animal Care Use and Review
Committee of the University at Iowa.
Evans Blue Injection
EBD was injected either into the tail vein of the mice or into the peritoneal cavity without anesthesia. For the assessment of EBD uptake into
muscle fibers, intravenous administration of the dye was preferred. Nonspecific coloration of the diaphragm and the abdominal muscles was
avoided by this route of injection. EBD was dissolved in PBS (0.15 M
NaCl, 10 mM phosphate buffer, pH 7.4) and sterilized by passage through
membrane filters with a 0.2-µm pore size. The concentration of the injected dye was 0.5 mg EBD/0.05 ml PBS. Animals were injected with 50 µl
of this solution per 10 g body wt. 3-6 h after injection, the mice were killed
by cervical dislocation. The skin of the mice was removed, and the animals
were visually inspected for dye uptake into skeletal muscles, indicated by
blue coloration. To better characterize dye uptake in distinct muscles of
injected animals, we investigated cryosections of the femoral quadriceps,
the sural triceps, the pectoral, the diaphragm, and the cardiac muscle. Additional skeletal muscle samples were taken if the muscle showed blue coloration. Muscle sections from EBD-injected animals were incubated in
ice-cold acetone at Antibodies
For immunofluorescence analysis of albumin, the AIAG3140 rabbit anti-
mouse albumin antibody conjugated to FITC (Accurate Chemical & Scientific Corp., Westbury, NY) was used at a concentration of 1:75. Staining
for Igs was performed with biotinylated anti-mouse IgG (H+L), biotinylated anti-mouse IgM, biotinylated anti-human IgG (H+L), and biotinylated anti-human IgM (Vector Laboratories). All anti-Ig antibodies were
used at a concentration of 1:500. A monoclonal antibody VIA42A3 against
dystrophin (Ervasti et al., 1990 Immunofluorescence Microscopy
Muscle tissue from DMD and CMD patients was obtained from diagnostic muscle biopsies. The clinical diagnosis of DMD was confirmed by complete absence of dystrophin expression in muscle tissue. The clinical diagnosis of CMD was confirmed by white matter changes on brain magnetic
resonance images and by reduction or deficiency of laminin Cryosections for IgG and IgM staining were preincubated for 30 min
with 5% BSA in PBS, followed by a 1-h incubation with biotin-labeled goat anti-mouse IgG or IgM antibodies. The sections were washed 3× for
5 min with 1% BSA/PBS, incubated with FITC-conjugated streptavidin, and after a final wash, mounted with Vectashield mounting medium (Vector Laboratories). For albumin staining, the sections were blocked with
1% gelatin in PBS for 15 min, washed in PBS + 0.2% gelatin, and incubated for 1 h in PBS + 1% normal goat serum with a rabbit anti-mouse albumin FITC-conjugated antibody. Histochemical examination of muscle
tissue was performed by hematoxylin and eosin (H&E) staining as described (Dubowitz, 1985 Loss of Sarcolemmal Integrity in mdx Mice
Within seconds after intravenous injection of EBD, discoloration of all animals was observed. A successful injection
of the dye was indicated by the blue coloration of ears and
paws. Control mice did not show dye uptake into their
skeletal muscles by visual inspection. In EBD-injected
control mice, the presence of EBD in the perivascular
spaces was seen microscopically when the muscle sections
were inspected without any washing steps. There were very few single fibers with a positive dye staining on sections of control animals, which were otherwise EBD negative (Table I).
Table I.
Analysis of EBD-injected Mice
; Straub and Campbell,
1997
). This oligomeric complex connects the subsarcolemmal cytoskeleton to the extracellular matrix (Ervasti and
Campbell, 1993
). The intracellular link of the DGC is the
membrane-associated cytoskeletal protein dystrophin, the
protein product of the Duchenne muscular dystrophy
(DMD) gene (Hoffman et al., 1987
). The high density of
dystrophin in the subsarcolemmal cytoskeleton (Ohlendieck and Campbell, 1991a
; Ohlendieck et al., 1991b
), its
homology to
-actinin and spectrin (Koenig et al., 1988
;
Dhermy, 1991
), its costameric organization (Porter et al.,
1992
; Straub et al., 1992
), and its expression at the myotendinous junction (Tidball, 1991
) strongly suggest that it plays an important structural role in muscle fibers. Dystrophin binds with its amino-terminal and rod domain to actin (Rybakova et al., 1996
) and with its carboxy terminus
to the integral membrane protein
-dystroglycan (Suzuki
et al., 1994
; Jung et al., 1995
). The peripheral membrane
glycoprotein
-dystroglycan, a receptor for the heterotrimeric basement membrane protein laminin-2, binds to
-dystroglycan and so completes the connection from the inside to the outside of the cell (Henry and Campbell, 1996
). Mutations in the LAMA2 gene, encoding the
2 chain of
laminin-2, have been characterized in a form of congenital
muscular dystrophy (CMD) linked to chromosome 6q
(Tomé et al., 1994
; Helbling-Leclerc et al., 1995
; Nissinen
et al., 1996
).
; Schmalbruch, 1975
; Carpenter and Karpati, 1979
; Weller et al., 1990
). Evidence
for leakage of intracellular contents out of dystrophic or
damaged muscle cells is provided by elevated serum levels
of muscle enzymes (Rosalki, 1989
) and growth factors (D'Amore et al., 1994
; Kaye et al., 1996
). Simultaneously,
this loss of sarcolemmal integrity allows influx of molecules into muscle fibers. In particular, elevated calcium
levels have been noted in dystrophin-deficient skeletal
muscle (Bodensteiner and Engel, 1978
; Gillis, 1996
). One
or both of these events may contribute to the pathogenesis
of muscular dystrophy.
). This in vivo tracer technique
provides information about certain structural and dynamic features of normal and pathological skeletal muscles (Matsuda et al., 1995
).
). It has been reported that the dystrophin mutation in the mdx mouse leads to an associated reduction of DGC components (Ohlendieck and Campbell,
1991b). We also injected four different transgenic/mdx
mice that have been shown to result in different skeletal
muscle phenotypes (Rafael et al., 1994
; Phelps et al., 1995
;
Corrado et al., 1996
; Rafael et al., 1996
). These mice enabled us to analyze regions of dystrophin that are critical
for maintaining sarcolemmal integrity. One of the transgenic animals expressed only the COOH-terminal isoform of dystrophin (Dp71), which is encoded by exons 63-79.
These Dp71 mice display proper localization of the DGC
at the sarcolemma, but still show a dystrophic phenotype
(Cox et al., 1994
; Greenberg et al., 1994
). The other three
transgenic/mdx mice we injected were generated by expressing "full length" dystrophin constructs, but with consecutive deletions within the amino terminal domain (
3-7;
Corrado et al., 1996
), the rod domain (
17-48; Phelps et al., 1995
), and the carboxy-terminal domain (
71-74; Rafael
et al., 1994
, 1996
). Mice missing exons 71-74 or exons 17-48
of the dystrophin gene display a markedly milder phenotype than mdx mice despite the expression of moderate
levels of dystrophin. In contrast to deletions of exons 71-74
or in the central rod domain, proteins with a deletion in
the actin-binding NH2 terminus must be expressed at high
levels to prevent a dystrophic phenotype (Corrado et al., 1996
).
2 chain-deficient CMD (Sunada et al., 1994
;
Xu et al., 1994b
), whereby the dy2J/dy2J mouse represents
the milder allelic variant of the dy/dy mouse (Sunada et
al., 1995
). The Lama2 gene on mouse chromosome 10 was closely mapped to the dy/dy locus (Sunada et al., 1994
),
and a single in-frame deletion eliminating 57 amino acids
was discovered in the
2 chain transcript in dy2J/dy2J mice
(Sunada et al., 1995
). A principal goal of our studies was to
determine whether deficiency of the extracellular ligand of the DGC, laminin-2, had similar effects on sarcolemmal
integrity as deficiency of its intracellular connecting link,
dystrophin.
Materials and Methods
20°C for 10 min, washed 3× 10 min with PBS, and
mounted with Vectashield mounting medium (Vector Laboratories, Inc.,
Burlingame, CA). By fluorescence microscopy analysis, EBD staining
showed a bright red emission. Fiber counts of EBD-positive muscle fibers were done independently by two investigators on 7-µm cryosections of
dye injected mice. All sections were examined and photographed under
an Axioplan fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY)
or a MRC-600 laser scanning confocal microscope (Bio Rad Laboratories, Hercules, CA).
) and a polyclonal rabbit antibody against
the
2 chain of laminin (Allamand et al., 1997
) were used for screening
purposes of mouse and patient samples. Biotinylated antibodies were detected using streptavidin conjugated to either Texas red or FITC (Vector Laboratories).
2 chain expression in muscle tissue. Normal human control muscle was received
from patients undergoing orthopedic surgery not related to neuromuscular disorders. Immunofluorescence microscopy of 7-, 15-, and 30-µm cryosections from skeletal and cardiac muscle was performed as described previously (Ohlendieck et al., 1991a
).
).
Results
In the mdx mutant, administered EBD always resulted
in blue discoloration of skeletal muscles (Fig. 1). This result was independent of the age of the animals used. The
accumulation of EBD tended to be more intense at certain
anatomical sites, forming a characteristic topographic pattern of staining. Areas of blue staining appeared mainly
within the regions of the proximal limb muscles, as well as
the pelvic and the shoulder girdle (Fig. 1). The dye was
particularly incorporated in gluteal, femoral quadriceps,
and the ischiocrural muscles (hind limbs) and sometimes
the pectoral and the triceps brachii muscles (forelimbs; Fig.
1). Affected muscles were not stained homogeneously, but
they typically showed blue strands representing damaged
muscle fibers. In a longitudinal alignment, these strands
could be seen throughout the entire length of the muscles
(Fig. 1). Although there was a general topographic distribution of the dye in skeletal muscles of mdx mice, permeability of a given muscle region varied from animal to animal and even between opposing limbs within the same
animal. We found differences in both the intensity and the
extent of stained regions in left and right limbs (Fig. 1 b).
In addition to the proximal limb muscles, we also found
discoloration of the external oblique muscle of the abdomen, the longest thoracic and lumbar muscles, the cutaneous muscle of the trunk (Fig. 1 c), and in intercostal muscles (Fig. 2 c). Anterior tibial muscles in mdx mice were
often spared from dye staining. In contrast, the diaphragm
of mdx mice showed areas of dye incorporation in all
tested animals (Fig. 2 e). Because of the variability in dye
accumulation and distribution, there was no significant difference in the staining pattern among mice that were 4-52
wk old.
In mdx mice, skeletal muscles that visually showed dye uptake were always EBD positive by fluorescence microscopy analysis (Fig. 2 a). In most of the cases, the intracellular staining was diffusely distributed across the myofiber cytoplasm, although sometimes the fiber periphery was more strongly stained than the center of the fiber. Furthermore, the intensity of the dye signal showed differences between fibers (Fig. 2, a-e). These findings were also detected on 15- and 30-µm cross-sections and on longitudinal sections (Fig. 2). Most of the EBD-positive fibers showed intense staining, whereas in some others, the signal was faint. Transverse cryosections of blue discolored muscles never showed EBD in all fibers of a section. Instead, the frequency of EBD fibers in a given muscle varied from animal to animal and from muscle to muscle, reflecting the results of visual inspection (Fig. 1). In transverse sections of the mdx femoral quadriceps muscle, the mean frequency of dye-positive fibers was >10% of the total fiber number (Table I), with a maximum of >70% positive fibers (Fig. 2 a).
In transverse sections, the dye-positive mdx fibers occurred either singly or, more often, in groups. The grouped
EBD-positive fibers were one of the most striking features
of dye staining in mdx mice (Fig. 2). Although EBD-positive fibers sometimes showed signs of necrosis in the H&E
staining, there was no clear correlation between dye-positive fibers and a uniform morphological feature (Fig. 2, e
and f). In contrast to a previous report (Matsuda et al.,
1995), we also found EBD-negative hypercontracted fibers besides positive hypercontracted fibers (Fig. 2, e and
f, arrows) and therefore considered these to have an intact
sarcolemma. Fibers with distinctive necrotic features in
H&E seemed to be always EBD positive, no matter which
animal was injected (Fig. 2, e and f, arrowheads). On longitudinal sections, we could demonstrate that dye uptake
was not confined to a small part of the fiber, but appeared
over long segments of the entire muscle cell (Fig. 2 d).
Evans Blue Staining in Cardiac Muscle of mdx Mice
We also examined cardiac muscle from all injected animals, since cardiac involvement is a common feature in
many forms of muscular dystrophy. Control mice never
showed EBD-positive fibers in cardiac muscle tissue. In
contrast, 8 out of 16 mdx mice had EBD-positive lesions in
the myocardium. In one 4-wk-old mouse, large areas of
the cardiac section showed EBD uptake into cardiac muscle fibers (Fig. 3 a), whereas in the other mice, ranging
from 6 to 52 wk of age, regions of variable size in the ventricular wall were affected (Fig. 3, b and c). On sections
stained with H&E, the EBD-positive areas showed characteristic features of myocardial damage, with a strong inflammatory component (data not shown). Myocardial
EBD staining in intraperitoneally injected mice (Fig. 3 c)
provided evidence that the fiber damage was caused by
the underlying disease and not by volume overload of intravenously administered dye, leading to a cardiac infarct.
Presence of Serum Proteins in Damaged Skeletal Muscle Fibers
The chief characteristic of the EBD is its ability to form a
tight complex with serum albumin within seconds after its
injection into the bloodstream (Reeve, 1957). In view of
the tight association between EBD and albumin, areas of
blue macroscopic staining were taken to represent regions
of albumin uptake into muscle fibers. To demonstrate that
EBD-positive muscle fibers in mdx mice do take up albumin, we stained cryosections for albumin. We could show
that the same fibers that took up the dye were also positive for albumin staining (Fig. 4 A).
To further demonstrate that serum proteins cross into damaged fibers, we stained skeletal muscle sections of uninjected mice with antibodies against albumin (65 kD), IgG (150 kD), and IgM (900 kD; Fig. 4 B). In all muscles examined, positive staining with anti-mouse serum albumin antibodies, anti-mouse IgG antibodies, and anti-mouse IgM antibodies was observed in the endo- and perimysium (Fig. 4 B). Control mice did not show intracellular fiber staining with these antibodies. On the other hand, mdx mice showed intracellular staining for all antibodies within a part of their muscle fibers (Fig. 4 B). The staining patterns for the serum markers were strikingly similar to the patterns of EBD deposition. Intracellular staining was diffusely distributed across the myofiber cytoplasm, although sometimes it appeared to be localized to the fiber periphery. Examination of serial sections showed that the pattern of intracellular staining was similar for albumin, IgG, and IgM (Fig. 4 B).
The Role of Muscle Fiber Membrane Damage in Transgenic/mdx Mice
To test the importance of distinct dystrophin domains for
maintaining sarcolemmal integrity, we injected EBD into
transgenic lines of mdx mice expressing different portions
of the dystrophin molecule (Fig. 5). Intravenous injection
of EBD into Dp71 mice led to incorporation of the tracer
into skeletal muscles by visual inspection. The intensity
and the extent of stained regions was similar to that of
mdx mice. In contrast, 17-48 transgenic/mdx mice, which
mimic the dystrophin mutation in an extremely mild case
of Becker muscular dystrophy (England et al., 1990
;
Phelps et al., 1995
), and
71-74 transgenic/mdx mice,
which have been reported to show no dystrophic phenotype up to at least 2 yr of age (Rafael et al., 1994
, 1996
),
did not demonstrate macroscopic dye uptake after intravenous injections. The
3-7 transgenic mdx mice, in which
the expression of a full-length dystrophin construct deleted for the amino-terminal, actin-binding domain improves the mdx pathology to a mild "Becker-like" phenotype (Corrado et al., 1996
), did take up EBD into skeletal
muscle fibers. However, sarcolemmal damage in
3-7
transgenic/mdx mice, as assessed by EBD incorporation,
appeared less severe than in the mdx or the Dp71 mice
(Fig. 5). The femoral quadriceps muscle and the diaphragm were the only muscles in which dye accumulation was visually observed in
3-7 transgenic/mdx mice.
Fluorescence microscopy analysis of Dp71 skeletal muscle cryosections revealed no difference in the amount of
EBD-positive muscle fibers between Dp71 and mdx mice
(Fig. 5). In the 17-48 transgenic/mdx mice and the
71-74
transgenic/mdx mice, fluorescent microscopy analysis revealed some EBD-positive fibers in the diaphragm and the
femoral quadriceps muscle, although never to the extent of the mdx (Fig. 5). In
3-7 transgenic/mdx mice, the number of EBD-positive fibers in macroscopically blue areas
of skeletal muscle was smaller, and the fibers were more
loosely distributed compared to sections of injected mdx
or Dp71 mice (Fig. 5).
dy/dy and dy2J/dy2J Mice Maintain Plasma Membrane Integrity
Interestingly, dy/dy and dy2J/dy2J mice did not reveal dye
incorporation into skeletal muscles (Fig. 6). Because of the
increase in connective tissue in dystrophic muscle, the
mice showed a blue aspect resulting from dye accumulation into their connective tissue. Otherwise, we did not detect any dye accumulation in the inspected muscles (Table I).
The sections from injected dy/dy and dy2J/dy2J mice
showed only occasional EBD-positive fibers in the diaphragm (Fig. 7) or the femoral quadriceps muscle (Fig. 6 f).
The low level of intracellular fiber staining was a constant
feature of the muscles we examined from dy/dy and dy2J/
dy2J mice (Table I). The dye-positive fibers always showed
necrotic features according to H&E staining (Fig. 7). They
appeared singly and never showed grouping, which is characteristic for mdx or Dp71 transgenic/mdx mice (Fig. 6).
There were no differences in the extent or intensity of dye
accumulation and distribution in skeletal muscles between
dy/dy and dy2J/dy2J mice. In contrast to mdx mice, we never
found EBD staining in the cardiac muscle of dy/dy and
dy2J/dy2J mice.
Different Patterns of Sarcolemmal
Disruption in Patients with DMD and Laminin 2
Chain-deficient CMD
To test whether the findings in the animal models could be
reproduced in patients with DMD and laminin 2 chain-
deficient CMD, we tested cryosections from diagnostic biopsies with antibodies against IgG and IgM. In three out
of eight needle biopsies from DMD patients, we found
IgG- and IgM-positive muscle fibers (Fig. 8). The globulin-positive fibers showed the same grouping pattern as that
described for the mdx mice. On normal human muscle sections, IgG and IgM were detected only in the endo- and
perimysium (Fig. 8). Interestingly, not all IgG-positive fibers in DMD patients showed IgM uptake into the cytoplasm (Fig. 8, arrow), possibly because of the different
sizes of the molecules. This finding was confirmed by looking at serial sections of the biopsy samples. In the biopsies
of eight patients with laminin
2 chain-deficient CMD, we
never found grouped fibers with positive staining for Igs.
Two of the CMD patients had a deletion in the LAMA2
gene similar to the dy2J mice and were previously characterized (Allamand et al., 1997
). Single fibers with an intracellular staining signal for Igs were detected on CMD biopsies and showed morphological features of necrosis.
Previous findings support the idea that one function of the
DGC is to provide mechanical reinforcement of the sarcolemma and to maintain membrane integrity during cycles
of contraction and relaxation (Weller et al., 1990; Clarke
et al., 1995
; Petrof et al., 1993
). To test the involvement of
sarcolemmal damage in the pathogenesis of muscle fiber
degeneration and necrosis, we injected animal models for
muscular dystrophy with EBD. Intracellular accumulation of the dye in skeletal muscle fibers indicated loss of sarcolemmal integrity due to plasma membrane disruptions.
As a further test of altered membrane permeability, we examined the intracellular deposition of serum proteins in
skeletal muscle fibers in the murine models and in patients
with DMD and CMD.
Our results demonstrated that focal plasma membrane
defects have actually occurred in vivo and not, as argued,
during biopsy (Bradley and Fulthorpe, 1978). The diffuse
intracellular distribution of the tracer implied a break in
the structural integrity of the surface membrane. Interestingly, sarcolemmal permeability in certain muscles, including the heart, not only appeared to vary from animal to animal, but also showed alterations between the same muscle groups in the limbs of one animal. These findings suggest
that in addition to the genetic disposition, sarcolemmal
damage is also a function of environmental influences,
such as activity in the time period between dye injection
and death. By intravenous and intraperitoneal injection of
EBD into mdx mice, we also demonstrated loss of membrane integrity in cardiomyocytes. These results show that
the tracer technique may be helpful in evaluating the distribution and pattern of pathologic lesions in cardiac muscle, and that it might be a useful tool in studies of cardiac
infarctions.
According to our findings and previous studies (Mokri
and Engel, 1975; Karpati and Carpenter, 1982
), we suggest
that EBD-positive fibers in mdx mice do not inevitably
lead to necrosis of the whole fiber, but often reflect degenerating fibers with a potential for regeneration. Segmental
loss of sarcolemmal integrity or plasma membrane disruptions confined to a small region of the cell will allow the
dye to cross into a muscle fiber and to diffuse along the
longitudinal axis (Fig. 2 d). In the mdx or Dp71 mice, most of the EBD-positive fibers presumably undergo a stage of
segmental necrosis and regeneration, and just a small
group of dye positive fibers will undergo full-length fiber
necrosis, depending on the amount and size of membrane
disruptions. With the tracer technique, we demonstrated
that in the femoral quadriceps muscle of mdx mice, >70%
of the myofibers can show EBD uptake (Fig. 2 a). In addition, we could demonstrate that a number of EBD-positive fibers showed normal morphology by H&E staining
(Fig. 2 f). It has been suggested that membrane defects allowed the influx of calcium-rich extracellular fluid into the
muscle cells that caused hypercontraction and also initiated fiber necrosis (Mokri and Engel, 1975
; Schmalbruch, 1975
). Our results, on the other hand, indicate that hypercontracted fibers did not generally show primary plasma
membrane defects, and they did not in and of themselves
cause secondary rupture of the muscle fiber plasma membrane (Fig. 2, e and f). The interesting observation of
grouped EBD fibers in cross-sections of mdx and Dp71
mice may reflect the fact that the fibers branch extensively (Bell and Conen, 1968
; Schmalbruch, 1984
; Head et al.,
1992
), and that the dye may diffuse into all branches of a
fiber if one branch loses sarcolemmal integrity. Some of
the faintly fluorescent muscle fibers may be segments adjacent to a damaged site from where the EBD diffused
into the surviving stumps.
Furthermore, we were able to demonstrate that EBD-positive fibers were rendered transiently or permanently permeable to extracellular serum albumin and other serum proteins. These findings were confirmed in uninjected animals and in patient samples with muscular dystrophy (Figs. 3 and 6). The different molecular weights and sizes of these serum markers allowed us to draw conclusions about the size of sarcolemmal disruptions in affected fibers. The 900 kD IgM molecules were able to cross into the same fibers as 65 kD albumin proteins. The accumulation of serum proteins in the sarcoplasm also indicates that they may play a role in the pathogenic mechanism that finally leads to cell death.
As assessed by intracellular uptake of EBD, the mdx
and Dp71 mice showed the most severe sarcolemmal disruptions. According to our results in the transgenic mice,
the rod domain of dystrophin and the COOH-terminal domain encoded by exons 71-74 do not seem to be as important for membrane integrity. Expression of dystrophin deleted for the actin-binding sites encoded by exons 3-7 at or above normal dystrophin levels in the 3-7 transgenic/mdx
mice resulted in a mild phenotype (Corrado et al., 1996
)
with relatively few EBD-positive fibers. The findings in
the transgenic mice were particularly interesting with respect to the Dp71 mouse. Recent studies on the interaction of dystrophin with F-actin identified a novel F-actin-
binding site near the middle of the dystrophin rod domain,
and a model was proposed in which dystrophin binds F-actin
in a manner analogous to actin side-binding proteins (Rybakova et al., 1996
). In contrast to the
3-7 transgenic/mdx
mouse, the Dp71 mouse lacks all known actin-binding domains of the dystrophin molecule, but is sufficient to restore the DGC at the sarcolemma. Our data indicate that
the preservation of the DGC at the sarcolemma is not sufficient to prevent membrane disruptions. Taken together, these results indicate that cytoskeletal and sarcolemmal
attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. If these connections
are altered, physiologic mechanical forces might already
overstress the fragile membrane structure and cause focal
plasma membrane disruptions. Pasternak et al. (1995)
have demonstrated that the lack of dystrophin causes a substantial reduction in the stiffness of living muscle cells. In their model, dystrophin acts as a molecular spring that
could redistribute stresses imposed locally on the sarcolemma over a wide area of the cell.
Most interestingly, the dy/dy and dy2J/dy2J mice showed
no signs of sarcolemmal damage despite their severe clinical phenotype. The fact that we did not find a more significant dye accumulation in skeletal muscles of dy or dy2J
mice as compared to normal controls indicated that plasma
membrane disruptions do not seem to play a role in muscle cell degeneration of these animals. In the dy2J mouse,
the deletion in the lama2 locus is located in the NH2-terminal domain IV, which is presumed to be involved in self-aggregation of laminin-2 heterotrimers, as in the case of
laminin 1 (Schittny and Yurchenco, 1990). This mutation
could disrupt the formation of the laminin-2 network in
the muscle basal lamina, which could weaken or eliminate
the linkage between the extracellular matrix and the muscle fiber surface. Our studies demonstrate that the laminin
alteration does not effect the plasma membrane integrity. This finding is supported by electron microscopic studies
suggesting that the muscular basement membrane in dy/dy
mice is fragmented but plasma membrane disruptions do
not occur (Xu et al., 1994a
). Similar findings have also
been reported in patients with congenital muscular dystrophy with laminin
2 chain deficiency (Minetti et al., 1996
).
The small size of the dy/dy and the dy2J/dy2J mice, their
severe clinical phenotype, and the fact that they do not
show dye accumulation in their skeletal muscles suggests
that laminin-2 may transmit a specific signal to the muscle
cell required for muscle function rather than play a mechanical role in maintaining membrane integrity. Laminin
molecules are known to be multifunctional, performing key roles in development, differentiation, and migration
through their ability to interact with cells via cell surface
receptors, including -dystroglycan. In their association
with the DGC, these molecules do not seem to influence
the mechanical functions of the sarcolemma.
Plasma membrane disruptions might be an alternative
way into and out of the sarcoplasm and subsequently serve
as a route for the release of growth factors (McNeil and
Khakee, 1992) and other myocyte-derived autocrine molecules. It was proposed that such cell-mediated processes
are one way to stimulate the adjustment of muscle fibers
to exercise and to repair membrane damage (Clarke et al.,
1995
; Kaye et al., 1996
). Release of growth factors in skeletal muscle might be directly coupled to plasma membrane
disruptions, whether induced by exercise or caused by reduced sarcolemmal stiffness (McNeil et al., 1989
; Muthukrishnan et al., 1991
). In this respect, plasma membrane
disruptions might be beneficial for skeletal muscle regeneration and initiate not only damage, but also the cellular processes necessary for its repair. The EBD-injected mice
strains that incorporated the dye into skeletal muscle fibers and hence had sarcolemmal disruptions all showed a
benign phenotype, hypertrophic muscle fibers, and a normal to increased body weight. Consistent with this hypothesis, the dy/dy and dy2J/dy2J mice, which show severe clinical symptoms, did not reveal major dye uptake, do not
show hypertrophic fibers, are small in size, and weigh less
than their wild-type littermates.
In conclusion, we demonstrate that loss of plasma membrane integrity in skeletal muscle fibers may play a primary role in the course of the Xp21 muscular dystrophies.
Membrane disruptions induced by mechanical forces may
provide a route into and out of the sarcoplasm distinct
from the conventional, membrane-bound routes of endo-
and exocytosis (McNeil and Khakee, 1992). In diseased muscle with a higher muscle membrane vulnerability, this
route may be of technical importance for introducing foreign therapeutic substances or even genes into damaged
muscle fibers. Sarcolemmal permeability is of principal interest with regard to its role in the genesis of muscle cell
degeneration and necrosis, and as this role becomes better
defined, in the prevention and treatment of disease.
Received for publication 4 June 1997 and in revised form 30 July 1997.
Address all correspondence to Kevin P. Campbell, Howard Hughes Medical Institute, 400 Eckstein Medical Research Building, The University of Iowa College of Medicine, Iowa City, Iowa 52442. Tel: (319) 335-7867. Fax: (319) 335-6957. e-mail: kevin-campbell{at}uiowa.eduWe gratefully acknowledge Rachelle H. Crosbie and Michael Henry for their comments on this manuscript and the helpful discussion. We thank Jane Lee for her support with the dy2J/dy2J mice.
This work was supported by the Muscular Dystrophy Association. Volker Straub is supported by a grant from the Deutsche Forschungsgemeinschaft. Kevin P. Campbell is an Investigator of the Howard Hughes Medical Institute. J.S. Chamberlain was supported by National Institutes of Health grant AR40864.
CMD, congenital muscular dystrophy; DGC, dystrophin-glycoprotein complex; DMD, Duchenne muscular dystrophy; EBD, Evans blue dye; H&E, hematoxylin and eosin.
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