June 1967
Volume 6, Number 3
INVESTIGATIVE OPHTHALMOLOGY
Symposium on extraocular muscles
October 15,1966
Chairman: Irving H. Leopold, M.D.
The structure of vertebrate slow and twitch
muscle fibers
Arthur Hess
Slow muscle -fibers, which undergo a contracture after a nerve stimulus rather than a twitch,
have been shown in various animals to have, in contrast to twitch fibers, the following morphological characteristics: multiple nerve terminals, absence of a regularly occurring transverse
tubular T system, reduced amounts of sarcoplasmic reticulum, irregular disposition and size of
fibrils, zig-zag Z line, no M line, and no postjunctional sarcolemmal folds under the nerve
terminals. The slow fibers of the frog iliofibularis muscle, the chicken anterior latissimus dorsi
muscle, and the cat superior oblique muscle have been studied and compared with the twitch
fibers in these animals to determine if they have any or all of the above characteristics. All
slow muscle fibers have multiple nerve terminals, no well-organized T system (only aberrant T
system elements occur), reduced amounts of sarcoplasmic reticulum, irregidar disposition and
size of fibrils, and a zig-zag Z line. The frog slow muscle fiber does not have an M line; that
of the chicken and cat does. Virtually no postjunctional sarcolemmal folds occur under the
nerve terminals of slow fibers. However, even in the twitch fibers of the chicken and cat, postfunctional folds are irregular, infrequent, and inconspicuous.
S
described in the muscles of frogs,1 chickens,- ^and cats.3 These slow muscle fibers
also differ morphologically from twitch
fibers in both their internal structure and
their innervation. The following are at
least some of the morphological differences
between slow and twitch fibers which have
been said to exist: (1) the fibrils of the
twitch fiber are well separated from each
other, the fibrils of the slow fiber are not;
(2) the sarcoplasmic reticulum of the
twitch fiber is relatively abundant compared to that of the slow; (3) the trans-
low muscle fibers which respond to a
nerve stimulus with a prolonged contracture rather than a rapid twitch and usually
do not exhibit an action potential have been
From the Department of Physiology, University of
Utah College of Medicine, Salt Lake City, Utah.
This investigation was supported by a Public
Health Service research career development
award (NB-22,494) from the Institute of Neurological Diseases and Blindness and by research
grant NB-05752 from the United States Public
Health Service.
217
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218
Hess
Figs. 1-3. For legends see opposite page.
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Investigative Ophthalmology
June 1967
Volume 6
Number 3
verse tubular T system of the twitch fiber
occurs regularly in each sarcomere, the T
system is virtually absent or consists of
only aberrant elements in the slow fiber;
(4) a zig-zag Z line occurs in the slow
fiber and the Z line of the twitch fiber is
relatively straight; (5) the M line or thickening of the filaments in the middle of the
A band is absent in slow fibers; (6) the
twitch fiber is innervated by an individual
robust motor end plate, while the slow
fiber has multiple nerve terminals, delicate
and usually of "en grappe" type; (7) postjunctional sarcolemmal infoldings are conspicuous in twitch fibers and virtually
absent in slow ones.
The slow and twitch fibers of the three
animals, frog, chicken, and cat, which have
been shown physiologically to have slow
fibers, have been examined in the electron
microscope to see if they conform to the
aforementioned morphological characteristics.
Material and methods
The iliofibularis muscle of the frog, the anterior
and posterior latissimus dorsi muscles of adult
chickens of one to two years of age, and the
superior oblique muscle of the cat were fixed in
1 per cent osmium tetroxide in phosphate buffer
or in 2.5 per cent glutaraldehyde in phosphate
buffer. The latter pieces were treated with 1 per
cent osmium tetroxide in distilled water. All pieces
were dehydrated and embedded in Maraglas.
Sections were stained by lead citrate and uranyl
acetate and examined in the electron microscope.
The glutaraldehyde-fixed sections were used for
Structure of slow muscle fibers 219
study of the internal structure of the muscle
fibers, the osmium tetroxide-fixed sections were
used for study of nerve terminals.
Results
Frog. The twitch fiber outside the tonus
bundle of the iliofibularis muscle (Figs. 1
and 4) has been shown to have sarcoplasmic reticulum regularly separating
fibrils of fairly uniform diameter, T system
elements regularly occurring at the level
of the Z line, a straight smooth Z line across
the width of the fibril, an M line running
across the A band, and a long individual
nerve terminal with postjunctional sarcolemmal folds of fairly regular occurrence
and not too long in extent."1'0
The slow fiber in the tonus bundle
(Figs. 2 and 5), comprising about 40 per
cent of the fibers in the tonus bundle, has
scarce sarcoplasmic reticulum which does
not separate the fibrils regularly, no regularly occurring T system (only aberrant T
system elements occur), fibrils of uneven
diameter, a zig-zag Z line, no M line, and
multiple nerve terminals with virtually no
postjunctional sarcolemmal folds.'1"0
There is a third kind of fiber, comprising
about 60 per cent of the fibers in the tonus
bundle, which has an individual nerve
terminal, but not as long as that of the
twitch fiber outside the tonus bundle and
more branched and variable in shape." This
fiber (Fig. 3) has sarcoplasmic reticulum
regularly separating fibrils of fairly uniform diameter, regularly occurring T sys-
Fig. 1. Frog twitch muscle fiber outside the tonus bundle of the iliofibularis muscle. The
separation of fibrils by sarcoplasmic reticulum, the straight Z line (Z), the regularly occurring
T system (arrows), and the M line (M) are seen. Compare with Figs. 2 and 3. (Glutaraldehyde fixation; longitudinal section:; electron micrograph. xl6,000.)
Fig. 2. Frog slow muscle fiber in the tonus bundle of the iliofibularis muscle. The poor separation of fibrils by sparse amounts of sarcoplasmic reticulum, the zig-zag Z line (Z), the virtual
absence of T system elements (only aberrant T system elements occur), and the absence of
the M line are illustrated. Compare with Figs. 1 and 3. (Glutaraldehyde fixation; longitudinal
section; electron micrograph. xl6,000.)
Fig. 3. Frog twitch muscle fiber in the tonus bundle of the iliofibularis muscle. The separation
of fibrils by sarcoplasmic reticulum, the straight Z line (Z), the regularly occurring T system
(arrows), and the M line (M) are seen. Compare with Figs. 1 and 2. (Glutaraldehyde fixation; longitudinal section; electron micrograph, x] 6,000.)
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June 1967
Hess
i i- m
Figs. 4-6. For legends see opposite page.
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Volume 6
Number 3
tern elements at the level of the Z line, a
straight smooth Z line, and an M line.'1'5
Orkand7 showed that this muscle fiber can
indeed twitch. The disposition of the sarcolemmal folds under the individual nerve
terminal of these muscle fibers has not yet
been described. The nerve terminal has
numerous, frequently occurring folds under
it (Fig. 6). The folds are very conspicuous
and extensive and branch and divide as
they push into the muscle fiber. These
fibers, with bizarre-shaped, variable nerve
terminals, also have bizarre, variable postjunctional folds under the endings.
The twitch and slow muscle fibers of the
frog thus appear to conform in their internal structure and in the disposition and
appearance of their nerve terminals with
all the morphological features mentioned
in the introduction.
Chicken. It has been shown physiologically2 that the posterior latissimus dorsi
consists almost completely of twitch fibers
and the anterior latissimus dorsi of slow
fibers; this has been confirmed morphologically.8 The twitch fibers have fibrils
regularly separated from each other and
are innervated by a single robust end
plate; the slow fibers have fibrils of irregular size and distribution and multiple
delicate nerve terminals of "en grappe"
nature.8
The twitch fibers in the electron microscope (Fig. 7) have a regularly occurring
T system which appears at the level of the
I band almost at the junction of A and I
Structure of slow muscle fibers
221
bands. The T system appears in every sarcomere. Sarcoplasmic reticulum is present
between the fibrils. A smooth Z line runs
straight across the fibril. An M line is seen
bisecting every A band in every sarcomere.
The slow fiber (Fig. 8) does not have
a regularly occurring T system. A few
elements are seen (Fig. 9), sometimes at
the level of the A-I junction and sometimes
above or below this level. When these
irregular elements are seen, they are usually not transversely oriented, but run
obliquely or longitudinally. Sarcoplasmic
reticulum is much less in amount than in
twitch fibers. The Z line of the slow fiber
frequently runs zig-zag across the width
of an individual fibril and does not appear
smooth as in the twitch fiber. The M line
is present in eveiy sarcomere in the slow
fiber of the chicken.
The end plate of the twitch fiber (Fig.
10) appears well organized and makes a
prominent elevation on the muscle fiber.
Several units of the ending are usually
seen in any section. Each unit contains accumulations of mitochondria and synaptic
vesicles. Basement membrane material
separates nerve terminal from muscle fiber.
The postjunctional folds under the nerve
tenninal are not conspicuous. They are
very short and infrequent. The sole plate
sarcoplasm is devoid of fibrils, has several
nuclei, and many clusters of ribosomes are
seen.
The motor terminal of the slow fiber
(Fig. 11) is not as well organized and
Fig. 4. Nerve terminal on frog twitch muscle fiber outside the tonus bundle of the iliofibularis
muscle. The arrows indicate some of the postjunctional sarcolemmal infoldings under the
nerve tenninal. The sarcolemmal infolding is moderate in frequency and extent. Compare with
Figs. 5 and 6. (Osmium tetroxide fixation; longitudinal section; electron'micrograph. x32,000.)
Fig. 5. Nerve tenninal on frog slow muscle fiber in the tonus bundle of the iliofibularis muscle.
Postjunctional sarcolemmal infoldings under the nerve tenninal are virtually absent. Compare
with Figs. 4 and 6. (Osmium tetroxide fixation; longitudinal section; electron micrograph.
x32,000.)
Fig. C. Nerve terminal on frog twitch muscle fiber in the tonus bundle of the iliofibularis
muscle. The anows indicate some of the postjunctional sarcolemmal infoldings under the nerve
tenninal. The sarcolemmal infoldings are very frequent and extensive. Compare with Figs.
4 and 5. (Osmium tetroxide fixation; longitudinal section; electron micrograph. x32,000.)
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Hess
Investigative Ophthalmology
June 1967
Fig. 7. Chicken twitch muscle fiber in the posterior latissimus dorsi muscle. The sarcoplasmic
reticulum separating fibrils, the straight Z line (Z), the regularly occurring T system elements
(arrows), and the M line (M) are seen. Compare with Fig. 8. (Glutaraldehyde fixation; longitudinal section; electron micrograph. xl6,000.)
Fig. 8. Chicken slow muscle fiber in the anterior latissimus dorsi muscle. The reduced amounts
o£ sarcoplasmic reticulum, the more irregular Z line (Z), the virtual absence of T system
elements, and the M line (M) are seen. Compare with Fig. 7. (Glutaraldehyde fixation; longitudinal section; electron micrograph. xl6,000.)
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Fig. 9. Chicken slow muscle fiber in the anterior
latissimus dorsi muscle. The M line (M) is seen.
The arrows indicate aberrant T system elements,
which are few in number and can be longitudinally, rather than transversely, oriented in slow
muscle fibers. (Glutaraldehyde fixation; longitudinal section; election micrograph, xj.6,000.)
does not make a conspicuous elevation oil
the muscle fiber. Usually only one or two
units of the nerve ending are seen in any
section. The contents of the nerve terminal,
mitochondria and vesicles, are the same
as in twitch endings, and basement membrane material likewise intervenes between
nerve terminal and muscle fiber. Postjunctional folds under the nerve terminal are
usually absent. The relations of nerve
terminal and muscle fiber appear in general much smoother in slow than in twitch
fibers with no tendency toward the occurrence of postjunctional folds. The area of
sole plate sarcoplasm is less extensive and
the accumulations of nuclei and clusters
of ribosomes less marked.
In summary, the chicken twitch fibers
have a T system, the .slow fibers do not,
although, as is probably the case in all slow
fibers, aberrant T system elements can
occur. The sarcoplasmic reticulum is reduced in amount in slow fibers. Both
Structure of slow muscle fibers
223
twitch and slow fibers have an M line.
Marked postjunctional folds are not extensive under any nerve terminals on chicken
muscle fibers, but smoother relations are
seen in slow fibers than in twitch ones. It
should be stressed that the major differences between nerve terminals on twitch
or slow fibers are that the slow fibers have
multiple terminals, while the twitch fibers
are individually innervated, and the end
plate of the twitch fiber is better organized,
larger and more robust than the more
delicate, filamentous terminal of the slow
fiber.
Mammalian extraocular muscles. Irregular separation of muscle fibrils, a zig-zag
Z line, and multiple nerve terminals have
been found on the slow fibers of the extraocular muscles of guinea pigs,0 monkeys/ 0
cats,8 and man.11
Further study of the cat superior oblique
muscle reveals that the twitch fiber (Figs.
12 and 14) has fibrils regularly separated
from each other by abundant amounts of
sarcoplasmic reticulum, a regularly occurring T system at the junction of A and I
bands, a straight Z line, an M line, and an
individual motor end plate with poorly
developed postjunctional sarcolemmal infoldings. The slow fiber (Figs. 13 and 15),
on the contrary, has scarce amounts of
sarcoplasmic reticulum poorly separating
the fibrils, fibrils of irregular size, no regularly occurring T system with only a few
aberrant T system elements present, a zigzag Z line, an M line, and multiple motor
terminals under which sarcolemmal folds
are virtually absent.
Hence, the cat extraocular twitch fiber
conforms with almost all the features of
twitch fibers elsewhere, except in that the
postjunctional sarcolemmal folds are not
very conspicuous. The slow fiber also has
many of the morphological characteristics
of slow, fibers elsewhere, but it has an M
line.
Discussion
A muscle is a muscle is a muscle. When
pointing out differences between twitch
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Hess
Investigative Ophthalmology
June 1967
Fig. 10. Nerve ending on chicken twitch muscle fiber in the posterior latissimus dorsi muscle.
The arrows indicate some of the places where the sarcolemma tends to invaginate, although
postjunctional sarcolemmal folds are not conspicuous. The muscle fiber membrane under the
nerve terminal is relatively irregular. Compare with Fig. 11. (Osmium tetroxkle fixation; longitudinal section; electron micrograph, x32,000.)
Fig. 11. Nerve ending on chicken slow muscle fiber in the anterior latissimus dorsi muscle.
The muscle fiber membrane under the nerve terminal is smooth. Compare with Fig. 10.
(Osmium tetroxide fixation; longitudinal section; electron micrograph, x32,000.)
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Fig. 12. Cat twitch muscle fiber in the superior oblique muscle. The separation of fibrils by
sarcoplasmic reticulum, the straight Z line (Z), the regularly occurring T system (arrows),
and the M line (M) are seen. Compare with Fig. 13. (Glutaraldehyde fixation; longitudinal
section; electron micrograph. ~16,000.)
Fig. 13. Cat slow muscle fiber in the superior oblique muscle. The poor separation of fibrils
by sparse amounts of sarcoplasmic reticulum, the zig-zag Z line (Z), the virtual absence of T
system elements (only aberrant T system elements occur), and the M line (M)are illustrated.
Compare with Fig. 12. (Glutaraldehyde fixation; longitudinal section; electron micrograph.
~16,000.)
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Fig. 14. Nerve terminal on cat twitch muscle fiber in the superior oblique muscle. The muscle
fiber membrane under the nerve terminal is relatively irregular; postjunctional sarcolemmal
folds are infrequent, irregular and inconspicuous. Compare with Fig. 15. (Osmium tetroxide
fixation; ~ongitudindsection; electron micrograph. ~32,000.)
Fig. 15. Nerve ttmninal on cat slow muscle fiber in the superior oblique muscle. The muscle
fiber membrane under the nerve terminal is relatively smooth; postjunctional sarculemmal
folds are virtually absent. Compiue with Fig. 14. (Osmium tetroxide fixation; longitudinal
)
section; electron micrograph. ~32,000.
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and slow fibers, and saying that twitch
fibers have more sarcoplasmic reticulum
than slow ones and contain a regularly occurring T system in contrast to only aberrant T system elements in slow fibers, it
also serves to illustrate that these two
types of muscle fiber have obvious similarities in that both contain sarcoplasmic reticulum and T system elements. It is the
differences in occurrence and distribution
of these elements that serves to illustrate
the fundamental differences between these
two types of muscle fiber. A twitch fiber
may have more reticulum than another
twitch fiber adjacent to it, but in general,
both will have more reticulum than the
slow fibers. Some slow fibers may have
more aberrant T system elements than
others, even in the same muscle, but the
T system elements of slow fibers do not
occur regularly. The M line is absent in
frog slow muscle fibers, and snake slow
muscle fibers (although this is not mentioned by Hess1- it is apparent from the
photographs) also do not have an M line;
however, the M line is present in slow
muscle fibers of the chicken and cat. This
does not mean that slow muscle fibers of
the chicken and cat are the same morphologically as their twitch fibers. Rather, it
means that the M line cannot be used as
a structural entity in identification of slow
muscle fibers in the chicken and cat, but
that other morphological criteria, as mentioned above, must be used. It also does
not signify that more types of muscle fiber
should be enumerated, but rather that
variation from the typical, which is perhaps to be expected in biology, is present
in the two main types of muscle fiber that
exist.
Similarly, comparison of individual nerve
endings can. lead to a distortion of the
fundamental characteristics which serve
to differentiate twitch from slow fibers. The
slow fiber has delicate endings, usually appearing like a bunch of grapes and called
"en grappe"; the twitch fiber has a robust
end plate. However, in the snake, the individual end plates on twitch fibers look
Structure of sloiu muscle fibers
227
more like a bunch of grapes than the
multiple nerve terminals on slow fibers.13
The multiple terminals on different slow
fibers in the same muscle can differ in
appearance. In guinea pig extraocular
muscles, there are thin fibers along the
periphery of the muscle which are multiply
innervated by terminals that appear like
"blobs" of staining material, while the
multiple terminals on thick fibers in the
interior of the muscle appear as extensive
ramifications "en grappe" in shape.9 The
individual nerve terminal of a frog twitch
fiber does not appear like a motor end
plate, but is an elongate, branched structure equivalent to the more compact end
plate of twitch fibers in other animals.
Multiple terminals on cat muscle fibers do
not appear like a bunch of grapes.3 The
postjunctional sarcolemmal folds under the
nerve terminals on some twitch muscle
fibers are very prominent1-; on others, such
as the chicken and cat, they are not conspicuous. Nevertheless, the chicken and
cat fibers under discussion are still twitch
fibers and have enough of the features of
a twitch fiber to be identified as such
morphologically. None of these peculiarities can negate the fact that slow muscle
fibers have multiple nerve terminals and
twitch muscle fibers are individually innervated, despite the varied shapes of the
nerve terminals themselves or the occurrence or lack thereof of postjunctional
sarcolemmal infoldings. The delineation of
more fiber types in addition to the fundamental twitch and slow categories does not
serve to elucidate the problem of discussing .muscle fibers, but rather to muddle
and confuse the issue. A description of the
way in which certain muscle fibers may
vary in their morphology from the general
scheme would be most helpful in promoting the knowledge of the structure of
muscle fibers.
The absence of a propagated potential
is a fundamental difference between slow
and twitch fibers. Perhaps a slow fiber,
under certain physiological conditions, can
be made to twitch. Perhaps this slow fiber
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Investigative Ophthalmology
June 1967
Hess
contains a certain arrangement of fibrils,
T system elements, and nerve terminals
which allow it to twitch under certain circumstances. But fundamentally slow muscle fibers do not twitch, and it was this
characteristic that drew the attention of
investigators to these muscle fibers. It is
these mucle fibers, which fundamentally
do not twitch and are slow, which can be
differentiated from twitch fibers because
of fundamental morphological differences.
The author wishes to thank Miss P. Y. Cossey
and Mr. L. Bradshaw for capable technical assistance.
REFERENCES
1. Kuffler, S. W., and Vaughan Williams, E.
M.: Small-nerve junctional potentials. The
distribution of small motor nerves to frog
skeletal muscle, and the membrane characteristics of the fibres they innervate, J.
Physiol. 121: 289, 1953.
2. Cinsborg, B. L.: Spontaneous activity in
muscle fibres of the chick, J. Physiol. 150:
707, 1960.
3. Hess, A., and Pilar, C : Slow fibres in the
extraocular muscles of the cat, J. Physiol.
169: 780, 1963.
4. Hess, A.: The structure of extrafusal muscle
fibers in the frog and their innervation studied
by the cholinesterase technique, Am. J. Anat.
107:129, 1960.
5. Peachey, L. D., and Huxley, A. F.: Structural
identification of twitch and slow striated
muscle fibers of the frog, J. Cell Biol. 13:
177, 1962.
6. Page, S. C : A comparison of the fine structures of frog slow and twitch muscle fibres,
J. Cell Biol. 26: 477, 1965.
7. Orkand, R. K.: A further study of electrical
responses in slow and twitch muscle fibres
of the frog, J. Physiol. 167: 181, 1963.
8. Hess, A.: Structural differences of fast and
slow extrafusal muscle fibres and their nerve
endings in chickens, J. Physiol. 157: 221,
1961.
9. Hess, A.: The structure of slow and fast
extrafusal muscle fibers in the extraocular
muscles and their nerve endings in guinea
pigs, J. Cell. & Comp. Physiol. 58: 63, 1961.
10. Hess, A.: Further morphological observations
of "en plaque" and "en grappe" nerve endings on mammalian extrafusal muscle fibers
with the cholinesterase technique, Rev.
Canad. Biol. 21: 241, 1962.
11. Dietert, S. E.: The demonstration of different
types of muscle fibers in human extraocular
muscle by election microscopy and cholinesterase staining, INVEST. OPHTH. 4: 51, 1965.
12. Hess, A.: The sarcoplasmic reticulum, the T
system, and the motor terminals of slow and
twitch muscle fibers in the garter snake, J.
Cell Biol. 26: 467, 1965.
13. Hess, A.: Two kinds of extrafusal muscle
fibers and their nerve endings in the garter
snake, Am. J. Anat. 113: 347, 1963.
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