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Uniformity of structural characteristics throughout the length of skeletal muscle fibers.

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Uniformity of Structural Characteristics Throughout
the Length of Skeletal Muscle Fibers'.'
PATRICIA R. FARRELL AND M. R. FEDDE
Neuromuscular Laboratory, Department of Physiological Sciences,
Kansas State University, Manhattan, Kansas 66502
ABSTRACT
Fresh-frozen, serial cross-sections of the transversus abdominis muscle
of four mature chickens (98 fibers) were examined to determine structural and histochemical characteristics throughout the entire length of skeletal muscle fibers. Fiber
diameter and nicotinamide adenine dinucleotide diaphorase (NAD-D) and myosin
adenosine triphosphatase ( ATPase) activities were used as criteria to classify fibers
as Type I or 11. Measurements were made a t 10 to 22 locations along the length of
the fibers. An unimodal distribution of mean fiber diameters ranging from 48 I( to 86 p
was found. Fibers did not appear larger in the belly of the muscle than near the
ends. Although small fluctuations i n fiber diameter occurred through the length of
a fiber, large and small fibers tended to remain relatively large or small at each
location. NAD-D activity was either consistently high or low throughout the length
of a fiber. Likewise, myosin ATPase activity was either high or low for an entire
fiber. It is concluded that skeletal muscle fibers maintain rather uniform structural
End histochemical characteristics along their entire length.
Fibers in a skeletal muscle generally are
classified as Type I' or Type 11' depending on histochemical reactions and fiber
diameter (Dubowitz and Pearse, '60;
Engel, '62, '65; Drews and Engel, '66).
Type I fibers have high oxidative enzyme activity, low glycolytic enzyme
activity, low myosin adenosine triphosphatase (myosin ATPase) activity, and a
smaller diameter than Type I1 fibers, which
have low oxidative enzyme activity, high
glycolytic enzyme activity, high myosin
ATPase activity, and larger diameters.
Fibers with enzyme activities and diameters between these two classes have been
termed intermediate (Nachmias and
Padykula, '58; Beckett, '62; Gauthier and
Padykula, '66).
The percentages of fibers with various
diameters and with certain histochemical
characteristics have been determined for
several mixed muscles (Nene and Chinoy,
'65; Nene and George, '65; George and
Berger, '66; Padykula and Gauthier, '67).
These values have been based on measurements of cross-sectional samples taken
at certain regions within the muscle. However, before measurements of diameter
ANAT. REC., 164: 219-230.
and cytochemical characteristics of fibers
from a single section or region can be
used to express the proportion of each
fiber type within the muscle as a whole,
it is necessary to be certain that these
characteristics remain the same within
a single fiber regardless of the location
along its length at which the section was
taken. Our study was designed to determine if muscle fibers in the transversus
abdominis of the chicken, many of which
course from tendon to tendon, are
structurally and cytochemically uniform
throughout their length. Characteristics
studied were fiber diameter and certain
enzyme activities.
METHODS
Four transversus abdominis muscles
(Chamberlain, '43), including the aponeurosis of insertion and bony origin,
were removed from four mature, male,
White Leghorn chickens killed by an overdose of sodium pentobarbital. The muscles
Received Dec. 2, '68. Accepted Jan. 30, '69.
1 Contribution no. 50, Neuromuscular Laboratory,
Department of Physiological Sciences, College of
Veterinary Medicine KSAES, KSU, Manhattan.
Supported by USPHS, NIH grant NB-05786.
219
220
PATRICIA R. FARRELL AND M. R. FEDDE
were placed on a saline dampened gauze
in a petri dish over ice approximately
one hour or until a mechanical stimulus
elicited no contraction. A strip of tissue
extending from aponeurosis to pubis was
excised from the central part of the muscle parallel with the muscle fibers. Tissue
strips from birds I and I1 were divided into
five pieces of equal length which were positioned adjacent to each other with the
ventral transverse planes on corkboard
and frozen in 2-methylbutane cooled to
- 125" C in liquid nitrogen. Muscle strips
from birds I11 and IV were frozen intact,
then cut into two pieces, each of which was
mounted on a microtome chuck and surrounded by embedding media (O.C.T.,
- 15 to - 30" C, Ames Lab-Tek, Inc.,
Westmont, Ill.). All tissue pieces were
carefully orientated so cross-sections
would be obtained. The blocks were serially sectioned at 10 v with a cryostat at
- 15" to - 20" C. Two adjacent sections
were picked up on cover slips in each
40 c1 interval (birds I and 11) or 100 c1 interval (birds I11 and IV). One section was
stained with hematoxylin-eosin ( H and E ) ,
while the adjacent section was incubated
for histochemical determination of nicotinamide adenine dinucleotide diaphorase
(NAD-D) activity in birds I, 11, and I11
(Pearse, '60) or myosin ATPase activity
in bird IV (Padykula and Herman, '55).
Fibers were much easier to follow when
the strips of muscle were frozen prior to
blocking and affixing to the cryostat chuck
(birds 111, IV), than when the muscle
was blocked into pieces and then frozen.
Prefreezing resulted in fewer lost sections
when blocks were changed and produced
less chance for fibers to deviate from true
transverse sections.
A clearly discernible area of the muscle
was traced under a microscope through all
the H and E sections. Photographic enlargements of the area were then made at
approximately 400 p intervals along the
length of the muscle, and individual fibers
were identified and numbered on the photographs. Fibers that did not course the entire length of the muscle were not used.
Photographs of NAD-D and myosin ATPase
sections were also made and cells that had
been followed on the H and E photographs
were identified.
Cross-sectional areas of identified fibers
were measured on photographs (370 X
enlargement) with a planimeter at ten
locations throughout the length of the
muscle on 34 fibers in bird I and 27 fibers
in bird I1 while those measurements were
made at 22 locations on 14 fibers in bird
111; and at 20 locations on 23 fibers in
bird IV. The cross-sectional area measurements were then converted to fiber diameter, assuming the fibers to be circular
(Gauthier and Padykula, '66).
A two-way analysis of variance (Snedecor, '56) was used to determine: (1) if
significant differences existed between
mean diameters of the various muscle
fibers; and ( 2 ) if a significant difference
existed between the mean of the diameters at various locations along the muscle.
Fibers on photographs of the NAD-D
preparations were qualitatively scored as
Type I, Type I-intermediate, intermediate,
Type 11-intermediate, or Type I1 on the
basis of the density of NAD-D localizations. Fibers on the photographs of the
myosin ATPase preparations were classified as Type I1 if a predominant dark
appearance prevailed or Type I if very
little reaction was observed.
RESULTS
Many fibers of the transversus abdominis muscle in the chicken coursed from
tendon to tendon. Figure 1 illustrates that
it is comparatively easy to trace an individual fiber over short distances of
muscle by observing the size, shape, and
relative position within a fasciculus. Sections A and D were only 200 c~ apart. The
fasciculi in each photograph are similar
and the two fibers marked (0) and (+)
maintain approximately the same shape,
size, and spatial relation in the muscle.
Fig. 1 Spatial arrangement of fibers over a
short distance in the transversus abdominis
muscle of the chicken. (Bird I. Fresh frozen sections, H and E.) Sections A and B, and C and
I
3 are 4 0 p apart: sections B and C are 1 2 0 p
apart. Fibers marked ( 0 ) and (+) are the same
in each section and were easily identified by
similarity in size and spatial relationships. Scale
011 this and following photomicrographs is 35 p .
STRUCTURE OF MUSCLE FIBERS ALONG THEIR LENGTH
Figure 1
22 1
222
PATRICIA R. FARRELL AND M . R. FEDDE
However, neither the configuration of
the fasciculi nor the spatial arrangement
of the fibers was constant throughout the
length of the muscle. Figure 2 shows
sections taken 6000 c~ apart. It is not evident from the sections that the labeled
fibers are the same in each photograph.
That conclusion could only be reached by
using close serial sections like those in
figure 1. Inspection of the two labeled
fibers in figure 2 leads to the conclusion
that they retain their relative diameters
in each section although the shape and
position of the fibers within a fasciculus
are not constant over long distances.
Relative relationships of three fibers
with different diameters are shown in
figure 3. Although the diameter of a given
fiber fluctuates from place to place, large
ones remained large and small ones remained small throughout the length of
the muscle. Smallest and largest diameters at a given location of the 98 fibers
studied were 30 and 115 u.
To determine whether fiber diameters
were larger in the belly of the muscle
than near the origin or insertion, average
diameter of all fibers at each location
was computed for birds I11 and IV. Although differences were significant (P <
0.05) in average diameter of fibers at
various locations, fibers in the muscle
belly were not consistently larger than
elsewhere (fig. 4).
Differences between mean diameters of
fibers in a muscle were very highly significant (P < 0.005). However, there were
no distinct populations of small, intermediate or large fibers. A histogram of
the mean diameter of 98 fibers from all
birds indicates a normal unimodal population of fibers in the transversus abdominis muscle (fig. 5).
Sections stained for NAD-D activity
(adjacent to the H and E sections presented in fig. 2), are shown in figure 6.
Differences between Type I and I1 fibers
are less marked than in many mammalian muscles. However, the smaller fibers
have more NAD-D activity than the
larger ones and other fibers exist between
the extreme intensities. The larger fiber
( 0 ) had consistently less oxidative en-
zyme activity than the smaller fiber (+)
throughout the muscle.
Sections illustrating myosin ATPase activity in fibers from bird IV are shown in
figure 7. Sections A and D are 16,700 I.I
apart. The fiber labeled (+) had low activity at each location; the fiber labeled
( 0 ) had high activity through its entire
length. Of the 23 fibers studied in this
bird, 18 were Type I1 while five were
Type I. The mean diameter of the Type
I1 fibers (69.5 rt 2.4 P, S.E.) was significantly different ( P < 0.05) from that of
the Type I fibers (62.4 2 2.1 H).
DISCUSSION
Several lines of evidence indirectly suggest that muscle fibers may contain rePions which differ chemically and morphologically. Segmentation of fibers could
occur due to the nature of embryological
development since the long multinucleate
muscle cell probably forms by successive
fusion of individual cells (Konigsberg,
'61 ). Furthermore, pathological changes
accompanying necrobiotic myopathies include an unexplainable segmented necrosis of some fibers (Adams, '64; Pearson,
'65). Also, serial transverse sections of
muscle spindles of the rectus femoris of
the cat and of the lumbricals of the rat
contain structural changes in individual
intrafusal fibers (Merrillees, '60; Barker,
'62).
It has previously been implied that
muscle fibers are morphologically, histochemically and physiologically uniform
along their length (Nachmias and Padykula, '58; Dubowitz and Pearse, '60,
'61, '64; Engel, '65; Drews and Engel,
'66; Engel and Irwin, '67). However, the
only confirming data which have been
presented consist of photomicrographs of
longitudinal sections of muscle fibers
which were only a few hundred microns
Fig. 2 Spatial relationships of fibers over a
long distance in tranversus abdominis muscle of
the chicken. (Bird I. Fresh frozen sections, H and
E.) Sections are 6 0 0 0 ~apart. Fibers marked
( 0 ) and (+) are the same in each section. Although they differ markedly in spatial relationship, they maintain the same relative sizes.
STRUCTURE O F MUSCLE FIBERS ALONG THEIR LENGTH
Figure 2
223
224
PATRICIA R. FARRELL AND M. R. FEDDE
95
90
a
85
L 80
P)
c 75
70
._
0 65
60
55
50
1
2
3
4
5
6
7
8
10
9
11
12 13
14
15
16
17 18
19 20
location
Fig. 3 Diameters of three fibers a t 20 locations throughout the length of the transversus
abdominis muscle. (Bird IV.) Despite fluctuations in fiber diameter each maintains a relative large, intermediate, or small size a t each location.
a.
74
72
L
70
E
66
.-
0 66
64
1
'
~
"
"
'
'
'
"
"
"
"
"
'
Fig. 4 Average diameter of 23 fibers at 20 locations throughout the tranversus abdominis
muscle of the chicken. (Bird IV.) Despite increased variation of diameters i n the belly of
the muscle, no consistent increase in fiber diameter a t any location could be observed.
in length. The present study supports
these suggestions on the basis of a more
thorough examination of the entire fiber
length.
The transversus abdominis of the
chicken is a mixed muscle containing
fibers of various diameters and histochemical characteristics. Our data indicate that
a given fiber is structurally and cytochemically uniform throughout its length. Fiber
diameter remained relatively constant with
large fibers tending to remain large and
small ones tending to remain small. NADD activity was reasonably constant and
myosin-ATPase activity was either high
or low for individual fibers.
The small fluctuations in the diameter
of a fiber along the length of the muscle
may have resulted from failure to obtain
true cross-sections at each location. An
estimate of the reliability of the histological technique used in obtaining true
cross-sections was made by plotting deviation percentages in the diameter of a
fiber at each location from its mean diameter for fibers of different sizes located
close to each other. Similar deviation
percentages from the mean for fibers at
each location would indicate that the
tissue might not be cut in exact crosssection. However, the deviation percentages from the mean diameter were not
STRUCTURE OF MUSCLE FIBERS ALONG THEIR LENGTH
225
pected that the diameter of adjacent fibers
would alter in unison.
Recent investigations regarding the
trophic function of nerve on skeletal
muscle suggest reasons for the structural
and cytochemical uniformity of muscle
fibers throughout their length (Guth, '68).
There is a rapid increase in ACh sensitivity of the muscle membrane following
denervation (Axelsson and Thesleff, '59).
Furthermore, innervated end plates in the
partially denervated frog sartorius muscle
(each fiber of which is dually innervated
by two distinct nerve bundles) were
found to possess an increased sensitivity
to ACh (Miledi, '60; Frank and Inoue,
'66) suggesting that the nerve regulates
ACh sensitivity throughout the entire
length of the muscle fiber.
It has also been shown (Guth et al.,
'66) that reinnervation of a fiber at a
distant point from the original sole plate
restores the high level of cholinesterase
active at that point indicating that the
nerve regulates cholinesterase activity
throughout the length of the entire muscle
fiber. Ions, metabolic energy sources, and
proteins all appear to be regulated by
specific neural influences (Drahota and
Gutmann, '63; Guth and Watson, '67;
Mommaerts, '68).
Some substance, apparently not acetylcholine, appears to be liberated from the
nerve that directs the metabolic and/or
contractile properties of the muscle (Miledi, '60, '63; Gutmann, '67, '68). It has
16
been shown that radioisotopically labeled
$
C1 4 1
leucine and orotic acid injected into the
ventral horn of the spinal cord can travel
down the axon of a motoneuron at the
rate of 2-3 mm/day and appear in newly
synthesized protein and RNA at the nerve
ending (Bray and Austin, '68; Peterson,
Bray and Austin, '68). If such a substance
acts as a controlling factor on the muscle,
i t apparently is capable of influencing
the entire muscle fiber from its point
45 50 55 60 65 70 75 80 85 90
of release since the present study has
Diameter p
shown that fibers with very diverse diFig. 5 Histogram of the mean diameter of
ameters and cytochemical composition
98 individual fibers from the transversus abdom- maintain relative uniformity throughout
inis muscle of four birds. Distinct populations of
their length.
fibers based on fiber size were not apparent.
the same for neighboring fibers of widely
varying diameters at many locations in
the muscle (fig. 8). In many cases, deviation percentage from the mean diameter of one fiber increased while that of
the other decreased. For example, between
location 11 and 12 in bird IV, the small
fiber (open circles) became smaller while
the large fiber (closed circles), only about
70 w away, was becoming larger. It therefore appears that the small variations in
diameter at different locations did not
result from failure to obtain true crosssection of the fibers.
A more plausible explanation for the
observed small fluctuations in fiber diameter is that the sarcomere lengths were
not constant throughout the length of the
muscle. It has been shown that the short
sarcomeres, representing some degree of
a contraction, are thicker than the longer
sarcomeres in the same fiber (Jordan,
'33; Brandt et al., '67) and since the
muscle was not rigidly fixed during freezing, i t is possible that small degrees of
contraction may have occurred at various
places along the length of a fiber. If the
variations in sarcomere length within a
fiber were random, it would not be ex-
::/
IJ
226
PATRICIA R. FARRELL AND M. R. FEDDE
Fig. 6 Sections from the transversus abdominis muscle of the chicken illustrating consistency
of NAD-D activity throughout the fibers. Sections are 6000 ,u apart; ( 0 ) or ( + ) marked fibers are
the same in each section.
STRUCTURE OF MUSCLE FIBERS ALONG THEIR LENGTH
227
Fig. 7 Sections from the transversus abdominis muscle of bird IV illustrating myosin ATPase
activity in fibers throughout their lenzth. A and B, 6000 $L apart; B and C, 5300 p apart; C and D,
5400 ,u apart. The large fiber ( 0 ) showed positive reaction throughout its length while the
showed a comparative negative reaction throughout its length.
small fiber (f)
228
PATRICIA R. FARRELL AND M. R. FEDDE
1
2
3
4
5
6
7
8
9
10
11
12
13 14
15 16 17
18
19 2 0
location
Fig. 8 Deviation percentages of fiber diameters from the mean diameter at the locations
studied. (Bird IV.) Two fibers of different sizes (open circles, mean diameter, 56p; closed
circles, mean fiber diameter, 84 p ) within approximately 70 p of each other are compared.
The fibers deviated from the mean diameter in the same direction in 11 intervals while
they deviated from the mean diameter in the opposite direction in eight intervals, indicating
that small fluctuations in fiber diameter throughout a muscle, as shown in figure 3, did not
result from poor cross-sections.
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