Download Muscle fiber and motor end plate involvement in the

Original Articles
Muscle fiber and motor end plate
involvement in the extraocular muscles
of the myotonic mouse*
Bruce R. Pachter, Jacob Davidowitz, and Goodwin M. Breinin
The extraocular muscles of the C57BL/6Jdy2i myotonic mouse were studied by phase and
electron microscopy. The most susceptible ocular muscle was the levator palpebrae; the
other muscles manifested scattered abnormalities to varying degrees. Central nucleation and
fiber splitting were prominent. Junctional abnormalities consisted of a reduction in postjunctional folding, excessive numbers of axonal terminals on hypertrophic fibers, and the
presence of dense granules between axon and muscle. This study demonstrates the affection
of both muscle fiber and motor end plate in mouse myotonic dystrophy.
Key words: extraocular muscle, mouse myotonic dystrophy, muscle fiber alterations,
motor end plate alterations.
I
scribed changes compatible to those encountered in the skeletal musculature.
An animal model for the study of myotonic dystrophy has been suggested in
mouse Strain Re-129 dy 2j /dy 2j insofar as
these animals show myotonic physiologic
responses in addition to progressive muscle weakness and atrophy." Previous studies
of animals with this myotonic (dy 2j ) gene
have been limited to the peripheral musculature.07 In the present study, the extraocular muscles of another strain of mouse
(Bar Harbor C57BL/6Jdy2j) carrying this
same myotonic (dy 2j ) gene were examined
by light and electron microscopy. A variety
of abnormal changes were seen in the
muscle fibers and motor end plates of these
muscles.
n myotonic dystrophy the facial and
extraocular muscles typically become involved in addition to the limb musculature.1"' The morphology of extraocular muscles in myotonic dystrophy has rarely been
examined, and then only by light microscope.1- 4r> These studies utilized human
biopsy and autopsy specimens and de-
From the Department of Ophthalmology, New
York University Medical Center, New York,
N. Y.
This study has been supported by National Institutes of Health Grant EY-00309 from the
National Eye Institute.
Submitted for publication Sept. 16, 1974.
Reprint requests: Dr. B. R. Pachter, Department
of Ophthalmology, New York University Medical Center, New York, N. Y. 10016.
"Presented at the Spring Meeting of the Association for Research in Vision and Ophthalmology, April 1974, Sarasota, Fla.
Materials and methods
Five myotonic and five nonmyotonic littermate
controls of the Bar Harbor Mouse Strain C57BL/
418
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Extraocular muscles of myotonic mouse 419
Fig. 1. Two fibers, the result of fiber splitting, as verified in sequential sections, are seen by
electron microscopy. In one fiber, deep sarcolemmal imaginations (arrow) are observed,
some of which completely isolate an incipient daughter fiber (arrowhead). Levator palpebrae,
x4,500.
6Jdy2J, age 3 to 5 months, were examined. The
affected animals manifested atrophic hind limbs
which were extended and dragging. The upper
extremities were little affected and the eyes appeared to be normal. The limb musculature of
these animals was clinically examined by electro-
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myography and with tension measurements. These
muscles exhibited myotonic electrophysiologic and
tension responses typical of this disorder. The
control animals were normal in this respect.8 The
animals were perfused through the heart with
1 per cent paraformaldehyde-1 per cent glutaral-
420 Pachter, Davidowitz, and Breinin
Investigative Ophthalmology
June 1975
Fig. 2. Abnormal mitochondria with paracrystalline inclusions (arrow). Superior rectus,
xl8,000.
Fig. 3. Higher magnification of bracketed area in Fig. 2, as seen in a nearby section. A
triplicate lamellar structure is apparent. Its total thickness is about 208 A, with electron
transparent layers of about 104 A between the three dense layers. The inclusion appears
between the outer and inner mitochondrial membranes (arrows). Superior rectus, x80,000.
dehyde in phosphate buffer. The extraocular muscles, including the levator palpebrae, were freed
under a dissecting microscope, fixed in cold 4
per cent glutaraldehyde for 10 hours, postfixed in
1 per cent osmium tetroxide for two hours, dehydrated in graded alcohols, and embedded in
Epon 812. The embedded muscles were serially
sectioned transversely at 15 microns by steel knife
on a sliding microtome. These thick Epon sections were cleared for light microscopy by curing
a layer of Epon onto them within a sandwich
of polystyrene film"; the complete muscles could
thus be initially surveyed by phase contrast. Sections of interest were freed from the plastic
sandwich and remounted for further one micron and ultrathin sectioning.
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Results
In the nonmyotonic littermate control
animals, the muscles examined appeared
normal by light and electron microscopy.
In the myotonic animals, morphologic alterations were observed only in the muscle cells and innervation of fibers characterized by well delineated myofibrils
separated by abundant sarcoplasmic reticulum in both the I and A bands. These
cells have relatively complex myoneural
junctions, and vary widely in size and
mitochondrial content. Such fibers would
cover the range encompassed by the so-
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Extraocular muscles of myotonic mouse 421
Fig. 4. Paracrystalline inclusions (arrows) in the intracristae space of mitochondria. Such
abnormal mitochondria were found near the end plate of a fiber which contained den.se
granules. Superior rectus, x48,000.
called Type I and Type II fibers described
in the skeletal musculature.1"11 The former
appeared to be much more susceptible to
morphologic change. In a given section,
approximately 5 to 10 per cent gave evidence of being affected. The "morphologically slow" fibers, which are also found
in extraocular muscle,1- are not affected at
this stage of the disease process. Such
"slow" fibers have a sparse sarcoplasmic
reticulum in the A band, large or irregularly shaped myofibrils in the I band, and
relatively simple myoneural junctions
wherein the postjunctional folding is minimal or altogether absent. The levator palpebrae muscle was the most susceptible to
morphologic alteration; "slow" fibers were
not observed in this muscle.
Fiber splitting was a prominent finding;
the complexity of this process ranged from
that of simple focal separation and rejoining to cases where the daughter fibers
atrophied and terminated. Splitting fibers
often present an otherwise essentially normal ultrastructure (Fig. 1).
The mitochondria of occasional fibers
contained triplicate (Figs. 2 and 3) or
duplicate (Fig. 4) lamellar paracrystalline
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inclusions. These were located in the outer
mitochondrial chamber, which includes the
space between the outer and inner mitochondrial membranes and the connecting
intracristae space.
Muscle cells of irregular shape and bizarre ultrastructure were sometimes seen.
The fiber in Fig. 5 contains numerous large
vacuoles, the limiting membranes of which
are in close apposition thereby forming
extended membrane pairs. Patches of disoriented myofibrillar material and several
dense bodies are also seen in this cell.
Additional intracellular alterations included distention of the sarcoplasmic reticulum, vacuolization, excessive lipid accumulations, and myofibrillar breakdown.
In the myotonic animals, junctional areas
with an atypical reduction or absence of
postjunctional folds were observed on both
abnormal and apparently normal "fast'
fibers (Fig. 6). This conclusion of pathologic deviations from the normal state was
based on a large sampling of end plates,
many of which were studied in serial section. Such density of sampling was necessitated by the variability in postjunctional
folding evidenced by normal fibers of this
422 Pachter, Davidowitz, and Breinin
Investigative Ophthalmology
June 1975
Fig. 5. In this irregular shaped fiber, an abundance of large vacuoles (V) are apparent.
Several dense bodies (DB) and disoriented myofibrils (arrow) are also seen. Medial rectus,
xl83000.
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Extraocular muscles of myotonic mouse 423
Fig. 6. Several terminal axons (Ax) are smoothly apposed to the sarcolemma of this fiber,
the myofibrils of which are clearly delineated in both the 1 ( 1 ) and A (A) bands. The axons
and the fiber appear to be otherwise normal. Levator palpebrae, x9,000.
Fig. 7. Normal end plate of a typical morphologically "fast" fiber. There are numerous axomal
terminals (Ax) apparent, one of which (arrow) makes a simple apposition to the muscle
fiber. The other terminals have well developed postsynaptic membranes with complex junctional folding. Superior rectus, x6,000.
type. Extraocular muscle end plates are
characterized by separate small axonal
terminals which vary in number from one
to approximately six within a given plane
of section. The complexity of postsynaptic
folding appears to vary from one to another of such junctional components. Some
axonal terminals may show numerous deep
folds, while other terminals of the same
end plate may be virtually flat within a
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given plane of section (Fig. 7). In sequential sections of a given axonal terminal, essentially flat junctions (Fig. 8, A)
were sometimes seen to develop folding
(Fig. 8, B)y or conversely, the junctional
folding of some terminals was seen to
gradually disappear. Thus, flat terminals in
themselves can not be considered as indicative of abnormality. In the abnormal tissue,
however, a reduction or absence of post-
424 Vachter, Davidowitz, and Breinin
Investigative Ophthalmology
June 1975
Fig. 8. Nearby sections through one axonal terminal of an end plate. (A) The axonal terminal (Ax) is seen to make a simple apposition to the muscle fiber which lacks postjunctional
folding. (B) Same axonal terminal (Ax) deeper in the motor end plate zone. Note the
well developed postsynaptic area with numerous postjunctional folds. Superior rectus, (A)
and (B), xl5,000.
junctional folding was seen with much higher frequency as compared to the normal,
and flat terminals were seen to persist in
their simplicity in serial sectioning.
Another variety of apparently abnormal
end plates in this tissue were those showing a profuse elaboration of the end plate
region with excessive numbers of terminals
(Fig. 9). Occasionally, dense granules were
seen between the axonal terminal and postsynaptic membrane of otherwise apparently normal end plates (Fig. 10).
Discussion
Most of the alterations which were seen
in this study to affect the muscle fibers
themselves, i.e., dilation of the sarcoplasmic
reticulum, fiber splitting, central nuclei,
excessive lipid accumulations, and myofibrillar breakdown were previously described in the limb musculature of this
and other strains carrying the dy2j gene.G> 1:i
Some of these changes were also observed
in human myotonic peripheral and extraocular muscle.4"5' 14~15
ParacrystalHne mitochondrial inclusions
appear not to have been previously described in any of the animal strains inbred
for a neuromuscular disorder. Such inclusions have been observed in a variety of
human muscle diseases,10"17 though not in
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myotonic dystrophy. These distinctive structures are apparently nonspecific, though
they would seem to be the dominant finding in some pathologies.18 In extraocular
muscles, such mitochondrial inclusions were
observed in a case of human muscular
dystrophy and in human chronic progressive ophthalmoplegia.39"""
The bizarre formations of numerous very
large vesicles seen here (Fig. 5) would
seem not to have been previously described
in dystrophic disorders, but have been
observed in muscle fibers after long periods
of denervation,21 and in primary hypokalemic periodic paralysis." It is unclear
whether these are true vacuoles or represent excessive membrane proliferation.
The most prominent alterations affecting
the motor end plate were a reduction in
the number of postjunctional folds, and
an increase in the number of axonal terminals in hyper trophic fibers. The reduction
of postjunctional folding has been observed in dystrophic mice of the Re-129
dy/dy strain,-3 in myasthenia gravis,21 and
in mice paralyzed by local injection of
tetanus toxin.25 Excessive numbers of axonal
terminals on hypertrophic fibers have been
reported in myotonic and other dystrophies.26"-7 Conceivably, such increase in
area of contact between axonal terminals
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Extraocular muscles of myotonic mouse 425
Fig. 9. Enlarged end plate region on hypertrophic fiber containing an atypical abundance
of axonal terminals (Ax). Superior rectus, x9,000.
Fig. 10. Motor end plate showing two axonal terminals (Ax). Dense granules (arrowhead)
are seen interposed between each terminal and sarcolemma of the muscle fiber. Superior
rectus, x39,000.
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Investigative Ophthalmology
June 1975
426 Pachter, Davidowitz, and Breinin
and the muscle surface may be a structural compensatory response to a reduced
efficiency of impulse transmission or a
partial functional denervation induced by
the decreased available area of postjunctional synaptic contact.
The presence of dense granules between
axon and muscle has been reported in
mice paralyzed by tetanus toxin.25 It was
suggested that they might have originated
from lysosomes of muscle fibers which had
become atrophic due to prolonged denervation. In the present case, however, these
granules were associated with junctions on
nonatrophied fibers.
A prior ultrastructural study of a different
strain of the Bar Harbor mouse, carrying
this myotonic (dy 2j ) gene(i emphasized
abnormalities of the end plate in the limb
musculature, the most prominent of which
was a decrease in the number of synaptic
vesicles in the axonal terminals. A comparable reduction in the number of synaptic
vesicles was found by us in the limb musculature of the presently studied strain of
animals.11 This alteration of the axonal
terminal did not seem to be a significant
factor in their extraocular muscles, however. It would seem that alterations of
the synaptic vesicles is not a critical modification of the end plate.
In this study of extraocular muscle, (1)
the reduction of postjunctional folding,
(2) the splitting of fibers, and (3) the
presence of central nuclei were considered
to be abnormal only when they occurred
in relation to "fast" fibers, as defined above.
These three structural characteristics are
an apparently normal aspect of the morphology of the well known "slow" fibers
of these muscles. Insofar as structural
changes which were unequivocally abnormal were not observed in these "slow"
fibers, it would seem that they are the
most resistant of the fiber varieties observed.
The authors wish to acknowledge the skillful
technical assistance of Mrs. Gloria Philips and
Miss Barbara Zimmer.
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