Download A continuum of myofibers in adult rabbit extraocular muscle: force

J Appl Physiol 111: 1178 –1189, 2011.
First published July 21, 2011; doi:10.1152/japplphysiol.00368.2011.
A continuum of myofibers in adult rabbit extraocular muscle: force,
shortening velocity, and patterns of myosin heavy chain colocalization
Linda K. McLoon,1,2 Han na Park,1 Jong-Hee Kim,3 Fatima Pedrosa-Domellöf,4
and LaDora V. Thompson3
Departments of 1Ophthalmology and 2Neuroscience and 3Physical Medicine and Rehabilitation, University of Minnesota,
Minneapolis, Minnesota; and 4Department of Clinical Sciences and Ophthalmology, Umeå University, Umeå, Sweden
Submitted 25 March 2011; accepted in final form 20 July 2011
extraocular muscles; myosin heavy chain isoforms; muscle force
MUSCLE CONTRACTILE CHARACTERISTICS are a function of the array
of fiber types within each muscle. These characteristics depend, in part, on the expression patterns of myosin heavy chain
(MyHC) isoforms within any given muscle. These MyHC
isoforms exist as part of a multigene family (90). Classically,
limb skeletal muscles are considered to be composed of four
main fiber types: three fast fiber types containing MyHC
isoforms IIA, IIX, IIB, and one slow fiber type, containing
MyHCI. While the majority of myofibers within limb skeletal
muscles contains only one MyHC isoform (e.g., slow myofibers in the soleus), others may show significant MyHC polymorphism (79), containing more than one of these MyHC
isoforms. This MyHC coexpression pattern is most common in
Address for reprint requests and other correspondence: L. K. McLoon,
Dept. of Ophthalmology, Univ. of Minnesota, Rm. 374 LRB, 2001 6th St.
SE, Minneapolis, MN 55455 (e-mail: [email protected]).
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physiologically fast muscles, such as diaphragm and plantaris
muscles, where IIA, IIB, IIX, and I can all be coexpressed (15).
The contractile properties of hybrid myofibers reflect the mixture of MyHC isoforms and other fiber type specific proteins,
and these hybrid myofibers are thought to play a role in refining
the control of shortening velocity (53).
The extraocular muscles (EOM) have a unique embryological origin from nonsegmented head mesoderm compared with
skeletal muscles, which are derived from somites (85). The
EOM retain a number of characteristics that are downregulated
in somite-derived skeletal muscle (58), including myofibers
with multineuronal (49, 69) and polyneuronal innervation (22),
expression of N-CAM (60), as well as expression of the
immature isoform of the nicotinic acetylcholine receptor (40).
In contrast to the four MyHC isoforms found in trunk and limb
skeletal muscles, gene and protein expression analyses show
that nine different isoforms exist in EOM, including an EOMspecific isoform (8, 10, 13, 45, 66, 87, 91). The nine, and
possibly more, MyHC isoforms expressed in the EOM are:
MyHCI (MYH7, slow beta-cardiac), MyHCIIA (MYH2), MyHCIIB (MYH4), MyHCIIX/D (MYH1), MyHCembryonic (developmental, MYH3), MyHCperinatal (neonatal, MYH8), MyHCalpha-cardiac (MYH6), MyHCEOM-specific (MYH13),
and MyHCslow-tonic (MYH14/7b) (73). A recent report indicates MYH15 is also expressed in the EOM (73). The differential patterns and proportions of MyHC isoform expression
within the orbital and global layers combine with this diversity
of MyHC isoforms to result in increased overall muscle fiber
complexity (10, 43, 69, 96).
There are distinct variations of MyHC isoform expression
longitudinally in individual EOM, with the most striking differences in the middle region (midbelly) of the EOM compared
with the tendon ends (13, 59, 74). Single EOM myofibers also
coexpress multiple isoforms of the MyHC family, which are
distributed differentially along the length of the myofibers (42,
45, 74), adding to the already complex three-dimensional
pattern of MyHC expression in different regions of the whole
muscle.
There is increasing evidence for coexpression of multiple
MyHC within single myofibers in all mammalian EOM thus far
investigated, from mouse to humans (13, 42, 45, 47, 48). The
EOM can, within short time periods, exhibit a wide array of
movements, from steady fixation, slow and fast saccades,
smooth pursuit, as well as slow vergence, in an infinite number
and wide range of eye and gaze positions. On the basis of these
studies, we hypothesize that the individual myofibers within
EOM represent a continuum, in terms of MyHC isoform
expression patterns, force generation, and shortening velocities. To support this hypothesis, 220 single skinned myofibers
8750-7587/11 Copyright © 2011 the American Physiological Society
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McLoon LK, na Park H, Kim JH, Pedrosa-Domellöf F, Thompson LV. A continuum of myofibers in adult rabbit extraocular muscle:
force, shortening velocity, and patterns of myosin heavy chain colocalization. J Appl Physiol 111: 1178 –1189, 2011. First published July
21, 2011; doi:10.1152/japplphysiol.00368.2011.—Extraocular muscle
(EOM) myofibers do not fit the traditional fiber typing classifications
normally used in noncranial skeletal muscle, in part, due to the
complexity of their individual myofibers. With single skinned myofibers isolated from rectus muscles of normal adult rabbits, force and
shortening velocity were determined for 220 fibers. Each fiber was
examined for myosin heavy chain (MyHC) isoform composition by
densitometric analysis of electrophoresis gels. Rectus muscle serial
sections were examined for coexpression of eight MyHC isoforms. A
continuum was seen in single myofiber shortening velocities as well
as force generation, both in absolute force (g) and specific tension
(kN/m2). Shortening velocity correlated with MyHCIIB, IIA, and I
content, the more abundant MyHC isoforms expressed within individual myofibers. Importantly, single fibers with similar or identical
shortening velocities expressed significantly different ratios of MyHC
isoforms. The vast majority of myofibers in both the orbital and global
layers expressed more than one MyHC isoform, with up to six
isoforms in single fiber segments. MyHC expression varied significantly and unpredictably along the length of single myofibers. Thus
EOM myofibers represent a continuum in their histological and
physiological characteristics. This continuum would facilitate fine
motor control of eye position, speed, and direction of movement in all
positions of gaze and with all types of eye movements—from slow
vergence movements to fast saccades. To fully understand how the
brain controls eye position and movements, it is critical that this
significant EOM myofiber heterogeneity be integrated into hypotheses
of oculomotor control.
SINGLE SKINNED EOM MYOFIBERS
from adult rabbit EOM were analyzed individually to determine force generation and shortening velocity. The MyHC
isoform composition for each single identified myofiber was
determined with gel electrophoresis (SDS-PAGE) followed by
densitometric determination of the relative abundance of each
distinct MyHC isoform. For EOM there was an extremely wide
continuum of both shortening velocity and contractile force
and practically all myofibers analyzed contained more than one
MyHC isoform.
MATERIALS AND METHODS
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phoresis system (Hoefer SE600, GE Healthcare) and run at 250 V for
24 h. Water baths maintained the temperature at 12–15°C. Following
electrophoresis the gels were silver-stained for MyHC identification
(30). MyHC isoform mobility was assessed by comparing each fiber
to standards prepared from EOM and leg samples. Densitometry was
performed on each lane/gel by scanning them with a molecular
multi-imaging system (GS-800, Bio-Rad). The relative expression of
the MyHC isoforms was determined from the optical density using a
densitometry image analysis software program (Quantity One). Data
were graphed based on Vo and MyHC isoform.
A second group of four adult rabbits was used for histological
examination of coexpression patterns of MyHC in serial sections.
Individual rectus muscles were removed after the rabbits were euthanized with an overdose of barbiturate anesthesia. The specimens were
frozen in 2-methylbutane chilled to a slurry on liquid nitrogen. In a
cryostat, 12 ␮m serial frozen sections were prepared and mounted on
glass microslides. Serial sections were immunostained with antibodies
specific for the following MyHC isoforms using standard methods
(59): pan-fast (specific for MyHCIIA and B, 1:40), slow (MyHCI,
1:40), and neonatal (MyHCneo, 1:20; all Vector Labs, Burlingame,
CA) and embryonic (MyHCemb, F1.652; 1:20), EOM-specific
(MyHCeom, 4A6; 1:10), slow tonic (MyHCslowtonic, S46; 1:20),
fast IIA (MyHCIIASC-71; 1:20), fast IIB (MyHCIIB, BF-F3;
straight), and fast IIX (MyHCIIX, 6H1; 1:20) MyHC isoforms
(Hybridoma Bank, University of Iowa, Ames, IA). Sections were
selected for analysis midway between the tendon end and the
midbelly region of the rectus muscles. After incubation in blocking
serum, the sections were incubated in primary antibody for 1 h.
The sections were rinsed and incubated using the reagents of
the Vectastain Elite mouse ABC peroxidase kit (Vector Labs). The
reacted tissue sections were processed for visualization of the
bound secondary antibodies using the heavy metal intensified
diaminobenzidine procedure. Specificity of antibody binding was
verified by immunostaining sections in the absence of primary
antibody. The immunostained serial sections were examined microscopically and photographed for identification of coexpression of
MyHC isoforms in individual myofibers. Reconstructions of individual myofibers positive for neonatal MyHC were performed by tracing
individual myofibers in serial sections using the Bioquant NovaPrime
software (Bioquant, Nashville, TN). Three-dimensional reconstructions were then built using the Topographer software (Bioquant; 17).
Statistical analysis. Data are presented as means ⫾ SE. Pearson’s
correlation coefficients were calculated for the data and, where significant, are indicated with both r and P values. Data were deemed
significant if P ⱕ 0.05. All statistical analyses were performed using
Prism software (Graphpad, San Diego, CA).
RESULTS
Single myofiber diameter, force, and shortening velocity.
Contractility (force and Vo) was determined on 220 myofibers.
Histograms representing the number of myofibers for both
specific tension and Vo are shown in Fig. 1. Notably, individual myofibers from the EOM showed a wide range of contractile values compared with limb skeletal muscle myofibers.
Specific tension (in kN/m2) ranged significantly among the
fibers examined, with a low of 9.99 to a high of 143, over a
14-fold difference. Specific tension (⫾SE) for the 220 individual fibers was 40.24 ⫾ 1.66 kN/m2. Vo in single myofibers
ranged significantly too, with a low of 0.439 fl/s to a high of
19.8 fl/s, almost a 50-fold difference. Mean Vo (⫾SE) was
7.569 ⫾ 0.196 fl/s. Mean myofiber diameter was 53.4 ⫾ 0.623
␮m, with a range from 31.2 to 80.8 ␮m. It should be noted that
there is an ⬃10 –30% swelling of skinned fibers (31); however,
there is minimal change to contractile proteins (23). These are
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New Zealand White rabbits obtained from Bakkom Rabbitry were
housed with Research Animal Resources at the University of Minnesota in an AAALAC approved facility. All research conformed to the
guidelines of the NIH for the Use of Animals in Research and was
approved by the Institutional Animal Care and Use Committee at the
University of Minnesota.
Normal adult rabbits were euthanized with an overdose of barbiturate anesthesia and muscles were prepared for single fiber analysis.
The individual rectus muscles were dissected free and removed,
taking care not to stretch the muscles during removal. The muscles
were placed on ice in relaxing solution at pH 7.0 consisting of (in
mM) 7 EGTA, 0.016 CaCl2, 5.6 MgCl2, 80 KCl, 20 imidazole, 14.5
creatine phosphate, and 4.8 ATP, with a pCa of 9.0. Bundles of
myofibers, ⬃8 by 1 mm, were tied to capillary tubes and stored in
permeabilization buffer for up to 1 wk at ⫺20°C. Permeabilization
buffer contains (in mM) 125 K proprionate, 2 EGTA, 4 ATP, 1 MgCl,
20 imidazole, 0.1% leupeptin, pH 7.0, plus 50% glycerol (vol/vol).
For contractile analysis, individual myofiber segments, ⬃2– 4 mm
long, were randomly isolated from the fiber bundles and placed in an
experimental chamber mounted to an inverted microscope. The chamber allowed the fiber to be transferred to wells containing activating or
relaxing solutions, and the solutions were maintained at 20°C. One
end of the fiber was attached to a force transducer (Cambridge
Technology/Aurora Scientific) and the other end to a length ergometer
using microtweezers (Cambridge Technology/Aurora Scientific). Sarcomere length was visually measured using a microscope and calibrated eyepiece micrometer. Sarcomere length along an isolated
single muscle segment was adjusted to a sarcomere spacing of 2.5
␮m. Each fiber segment was transferred from relaxing solution to
activating solution to determine force generation. Activating solution
was the same as relaxing solution except calcium concentration was
brought to a pCa of 4.5 and KCl to 180 mM. Fiber cross-sectional
areas were determined at three locations along the fiber segment. The
slack test method was used to determine the maximal unloaded
shortening velocity (Vo). Briefly, the myofiber was maximally activated and then rapidly shortened by 7–18% of the fiber length such
that force was reduced to zero. The time between zero force and
initiation of force redevelopment was measured, and this procedure
was repeated at various fiber lengths, graphed, and Vo determined by
the slope of the linear regression line divided by fiber length as
previously described (86). A total of 220 single skinned fibers from 10
rabbits was examined.
After determining force and Vo, each fiber was removed from the
experimental chamber and solubilized in sample buffer containing 6
mg/ml EDTA, 0.06 M Tris, 1% SDS, 2 mg/ml bromphenol blue, 15%
glycerol, and 5% ␤-mercaptoethanol. Aliquots were removed for
separation of single fiber MyHC isoforms using SDS-PAGE and
visualized with silver staining. Stacking gels were composed of 4%
acrylamide:N-N=-methylenebisacrylamide (bis) (50:1), 0.5 M Tris (pH
6.8), 0.1 M EDTA (pH 7.0), 10% SDS, 80% glycerol, 10% ammonium persulfate (APS), and 0.5% tetramethylethylenediamine
(TEMED). Separating gels were composed of 5% acrylamide:bis
(50:1), 1.5 M Tris (pH 8.8), 1.0 M glycine, 10% SDS, 80% glycerol,
10% APS, and 0.5% TEMED. Samples were loaded on an electro-
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Fig. 1. Histograms of contractility for the 220 myofibers. A: specific tension in kN/m2 with a mean of 40.24.
B: shortening velocity in fl/s with a mean of 7.569.
Shortening velocity was determined by the slack
method.
similar to previously published fiber diameters (54), and their
values ranged from 22 to 83 ␮m, with the highest value of
82.55 ␮m in rabbit superior rectus muscle.
Contractile parameters of individual fibers from limb
skeletal muscles are traditionally graphed diameter vs. force
(mg) because the size of the individual fiber predicts the
force-generating capacity. Figure 2 represents the contractile parameters of the 220 myofibers from the EOM plotted
in this traditional manner. When the diameter and force
properties of individual myofibers were plotted, contrary to
what is seen in limb skeletal muscle, there was no correlation between myofiber diameter and force generation (Fig.
2A). When individual myofiber diameter and Vo (fiber
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lengths/s) were graphed, as was expected and similar to limb
skeletal muscle, there was no correlation between these two
parameters (Fig. 2B).
Myofiber force, Vo, and MyHC isoform composition. After
force and Vo were determined for individual fiber segments,
their MyHC isoform composition was determined by electrophoresis. The electrophoresis method used in the initial analyses allowed separation of only five of the nine MyHC isoforms found in adult EOM (Fig. 3). Almost 50% of single
myofiber segments examined expressed three MyHC isoforms
(Table 1), while another 37.8% coexpressed two MyHC isoforms. Only ⬃5% expressed one MyHC based on mobility in
these electrophoresis gels.
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SINGLE SKINNED EOM MYOFIBERS
Fig. 3. Representative silver-stained electrophoresis gel showing separation of
myosin heavy chain (MyHC) isoforms obtained from single myofibers after
force measurements had been obtained. The corresponding Vo, specific tension
(mN/cm2), and percent composition of MyHC isoforms in each fiber are
beneath each gel lane.
A proportion of the 220 myofibers analyzed was graphed in
increasing order of Vo (in fl/s) on the x-axis, the percent
content of five MyHC isoforms (MyHCIIA, IIX, neonatal, IIB,
and I) on the y-axis (Fig. 4A, Table 1), and the individual
MyHC types on the z-axis. Certain trends were immediately
apparent. First, as expected, the fiber segments with the slowest
shortening velocities contained the largest proportion of
MyHCI, and the fiber segments with the fastest shortening
velocities contained a large proportion of MyHCIIB. However,
it was readily apparent that there was a continuum of single
myofiber shortening velocities, and this was associated with a
significant variation in percent expression of the five MyHC
isoforms typically visualized in the silver-stained electrophoresis gels. There was a highly significant and positive correlation between Vo and MyHCIIB (r2 ⫽ 0.82, P ⫽ 0.00001) and
between Vo and MyHCI (r2 ⫽ 0.99, P ⫽ 0.028). There was
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Table 1. Percentage of fibers coexpressing individual MyHC
Number of Myosin Bands
Percent of Total Fibers Analyzed
1
2
3
4
5
6
5.17
37.76
48.98
6.12
1.53
0.51
MyHC, myosin heavy chain.
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Fig. 2. Contractility correlations. A: graph of fiber diameter of single myofibers
plotted against force (n ⫽ 220). B: graph of fiber diameter of single fibers
plotted against shortening velocity (Vo) in fl/s determined by the slack method.
Each colored set of symbols for each indicated series represents representative
values for single fibers (n ⫽ 220).
also a statistically significant correlation between Vo and
expression of MyHCIIA (r2 ⫽ 0.54, P ⫽ 0.00028).
A subset of randomly selected myofibers out of the total 220
analyzed were graphed in increasing order of contractile specific force (in mg) on the x-axis, the percent content of five
MyHC isoforms (MyHCIIA, IIX, neonatal, IIB, and I) on the
y-axis, with individual MyHCs depicted on the z-axis (Fig. 4B).
No clear relationship was seen between force and MyHC
isoform content, and statistical analysis confirmed the lack of
correlation between force and MyHC isoform expression (r2
varied between 0.003 and 0.017; P varied between 0.07 to
0.998). This analysis may be colored by the fact that only five
MyHC isoforms were visualized.
Patterns of MyHC coexpression in the orbital layer. We
further investigated the occurrence of coexpression of MyHC
isoforms in single identified myofibers in serial histological
sections of the orbital layer immunostained for fast, neonatal,
IIB, slow, slow-tonic, IIA, and IIX (Fig. 5) to confirm the
SDS-PAGE data. While the vast majority of the orbital layer
fibers in this region of the muscle express embryonic and IIA
MyHC isoforms, they are differentially positive for other
isoforms. Six fibers are followed in all eight sections to serve
as examples (Fig. 5). Looking at the three fibers indicated in
the most superficial part of the orbital layer (Fig. 5, green, blue,
and red arrows), if they are followed in all eight sections, the
fibers indicated by the green and blue arrows show positive
immunostaining for pan-fast, embryonic, and IIA; slightly
positive for neonatal; and negative for IIB, slow, slow-tonic,
and IIX. The fiber indicated by the red arrow shows positive
immunostaining for slow, slow-tonic, embryonic, and IIA;
slightly positive for neonatal; and negative for the pan-fast,
IIB, and IIX isoforms. In another group of three fibers (Fig. 5,
yellow, purple, and orange arrows), the orange arrow points to
a fiber that immunostains positive for slow, slow-tonic, and
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SINGLE SKINNED EOM MYOFIBERS
embryonic MyHCs and negative for pan-fast, neonatal, IIB,
IIA, and IIX MyHCs. The two fibers indicated by the purple
and orange arrows are both positive for the pan-fast and IIA;
negative for neonatal, IIB, and IIX; and differentially positive
for slow, slow-tonic, and embryonic MyHC isoforms. An
additional two fibers are indicated by the black and maroon
arrows in the slow and slow-tonic photomicrographs. While
both are positive for slow tonic, one is lightly positive for
slow MyHC and the other negative (Fig. 5, black and
maroon arrows). Other fibers with equally complex coexpression patterns can easily be seen in these serially sectioned and immunostained tissues. This complex MyHC
coexpression patterning in single myofibers has been described for orbital layer fibers in the EOM of other mammalian EOM as well as in humans, although not with eight
isoforms simultaneously (10, 44).
Patterns of MyHC coexpression in the global layer. The
coexpression of MyHC isoforms in single identified EOM
myofibers was examined histologically in serial sections of the
global layers (Fig. 6). The majority of the myofibers in this
region midway between the tendon and the midbelly region of
the global layer expressed fast IIB MyHC (Fig. 6). While the
myofibers in the global layer did not show as complex a
coexpression pattern as seen in the orbital layer, most myofibers
expressed more than one MyHC isoform. Three fibers are followed in all nine cross-sections (Fig. 6, green, red, and blue
arrows). The fibers indicated by the green and red arrows are
relatively similar throughout the distances examined here (⬃216
␮m). These fibers are immunopositive for the pan-fast, IIB, and
EOM-specific MyHC isoforms; negative for neonatal, slow,
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DISCUSSION
The traditional description of EOM fiber types stems from
early studies where mitochondrial density and patterns of
innervation were the major criteria used (56, 82). These authors
described two fiber types in the orbital layer: 1) a fast, fatigueresistant singly innervated fiber type with large numbers of
mitochondria and 2) a less common multiply innervated slowtonic fiber type. Four fiber types were described in the global
layer, also based on innervation and concentration of mitochondria: 1) a fast, fatigue-resistant fiber type with large
numbers of mitochondria, 2) a fast fiber type with intermediate
level of contraction speed and fatigue resistance, and 3) a fast
fatigable type with few mitochondria. These are singly innervated. A fourth global layer fiber type is multiply innervated,
has very few mitochondria, with slow tonic contractile properties. As our study and others have shown, when additional
biochemical and physiological characteristics are considered,
this classification scheme becomes inadequate (7, 44, 45).
Single fiber physiology. Analysis of the 220 skinned myofibers in this study demonstrated several important properties of
EOM myofibers that differ from those seen in limb musculature. First, there was no correlation between fiber diameter and
force (in mg). Second, when single fiber shortening velocity
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Fig. 4. Continuum of contractile properties across the MyHC isoforms.
A: graph of shortening velocity plotted against myosin heavy chain isoform
composition based on densitometric analysis of silver-stained electrophoresis gels of a representative sample (48) of individual fiber segments
analyzed. Velocity determined by the slack method. B: graph of force in
milligrams plotted against MyHC isoform composition.
slow-tonic, embryonic, and IIA MyHCs; but one fiber (green
arrow) is lightly stained with the IIX/A antibody while the
other is negative. The fiber indicated by the blue arrow is
positive for slow and slow tonic MyHCs. The orange box (Fig.
6) is another example of the myofiber complexity within the
EOM; these four myofibers can be seen to change their intrafascicular and interfascicular position in this portion of the
muscle. Additionally these four myofibers again demonstrate
coexpression of multiple MyHCs within single fibers and
significantly different patterns of expression among the four
indicated fibers. When the percent of myofibers that coexpress
slow and slow tonic is assessed, in the global layer it varied
from 70 to 96% (mean 77.25 ⫾ 6.65%) and in the orbital layer
it varied from 47 to 59% (mean 53.33 ⫾ 3.48%). It should be
noted that these percentages will vary significantly when the
muscle is examined in the midregion or in the tendon region of
the EOM (56). In three sections immunostained for embryonic
myosin, the change in expression within single fibers along
even a small part of their length can be seen (Fig. 7). The total
distance from the section in Fig. 7A to the section in Fig. 7C is
396 ␮m. All three identified myofibers change their expression
for embryonic MyHC isoform in this distance. While immunohistochemistry is not quantitative, these serial sections demonstrate the complex MyHC expression patterns of individual
myofibers, as well as the differential presence of detectable
protein along the length of single myofibers.
Segmental changes in MyHC in single myofibers. To further
demonstrate the variation in expression of MyHC along individual myofibers, five myofibers were reconstructed from serial sections of an EOM where every other section was immunostained for neonatal MyHC (Fig. 8). The arrow indicates
where the reconstructions were begun, and the entire length of
each randomly selected myofiber was reconstructed. Note that
one myofiber was uniformly positive, one basically negative,
and three other fibers whose expression of neonatal MyHC
substantially changed in different fiber segments.
SINGLE SKINNED EOM MYOFIBERS
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and contractile force are examined, very wide ranges of values
were seen in the fiber segments. Jointly the single fibers
spanned a full spectrum of velocity and force measurements. It
should be noted that when motor nerves in the EOM are
directly stimulated, motor unit and whole muscle forces are not
intrinsically weaker than limb muscles; in fact EOM and limb
muscles can generate the same force and specific tension (26,
32). However, normal eye movements produce only small
amounts of muscle force, and the force produced is less than
predicted from direct motor unit measurements (32, 33). This
loss in muscle force in contracting EOM is hypothesized to be
due to myofiber branching and short myofibers that do not run
Fig. 6. Photomicrograph of serial sections of
global layer of a superior rectus muscle from
a normal adult rabbit immunostained for
each of the following MyHC isoforms:, fast,
neonatal (neo), slow (I) (from Vector) and
type IIB (BF-F3), slow tonic (S46), embryonic (emb, F1.652), type IIA/X (6H1), type
IIA (SC-71), embryonic (F1.652), EOM-specific (4A6) (from Hybridoma Bank). Three
myofibers are followed in all 9 panels identified by blue, red, and green arrows. An
additional 4 fibers are enclosed in an orange
box. The most common fiber type in the
global layer in this region of the muscle is
positive for MyHCIIB, with some light immunostaining for MyHCIIA. Blue arrows
indicate a myofiber immunopositive for slow
and slow-tonic, lightly positive for embryonic, and negative for all other MyHC isoforms stained. Green arrows point to a myofiber positive for pan-fast, MyHCIIB, and
EOM-specific and lightly positive for IIX.
Red arrows indicate a myofiber positive for
pan-fast, IIB, and EOM-specific. Bar represents 20 ␮m.
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Fig. 5. Photomicrograph of serial sections of
orbital layer of a superior rectus muscle from
normal adult rabbit immunostained for each
of the following MyHC isoforms: fast, neonatal, MyHCI (slow) (from Vector) and type
IIB (BF-F3), slow-tonic (S46), embryonic
(F1.652), type IIA (SC-71), and type IIX
(6H1) (from Hybridoma Bank). Six individual myofibers are followed in the 8 serial
sections, each identified with a single colored arrow (green, blue, red, yellow, purple,
and orange). Black and maroon arrows indicate fibers that are differentially positive for
slow and slow-tonic antibody staining. Bar
represents 20 ␮m.
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SINGLE SKINNED EOM MYOFIBERS
Fig. 7. Three cross-sections of an inferior rectus muscle immunostained for
embryonic MyHC. There are 216 ␮m between A and B and 180 ␮m between
B and C. Three fibers are identified in all 3 sections with blue, green, and red
arrows. In each identified fiber the expression of embryonic MyHC changes
along the length. Bar is 100 ␮m.
tendon-to-tendon within individual EOM (32, 33, 36, 81).
EOM have the fastest shortening velocities of any skeletal
muscle (6) and are extremely fatigue resistant (7). Despite the
fact that 85–90% of the myofibers within the EOM are “fast,”
this unusual combination supports the view that they represent
a distinct allotype compared with limb and body skeletal
muscle. This is based on their complex coexpression patterns
of the many molecules responsible for these physiological
properties (39).
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Fig. 8. Reconstruction of 5 myofibers from serial sections immunostained for
neonatal MyHC (Vector). Blue indicates regions that were negative, pink
indicates regions that were stained at an intermediate level, and red indicates
regions that were strongly positive. The green dot represents the section where
the reconstructions were started. The reconstructed myofibers averaged 1.296
to 2.45 mm in length.
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As stated, the shortening velocity of the EOM is faster than
all other muscles that have been examined (6), while additionally demonstrating a higher fusion frequency and greater fatigue resistance (27, 28) than all other fast twitch muscles.
While the extraocular muscles as a whole normally operate at
less than their maximal force potential, they can generate
significant maximal tetanic tension (26, 33). However, the
single myofibers in the present study generally had lower
specific tensions than those from single limb fibers (86).
However, eye movements can use very few of the thousands of
EOM myofibers in any given muscle to effect movement.
Estimates suggest that as few as two motor units appear to be
sufficient to move the eye by one degree, indicating a precise
control of eye position by a small number of myofibers (32).
Indeed, experimental removal of a large portion of the lateral
rectus muscle did not alter force generation or eye movement
accuracy (33). No change in eye movement accuracy was seen
after a loss of 22% of the normal motoneuron pool in monkeys
after chronic electrophysiological recordings in eye movement
studies, suggesting a large amount of myofiber and neuronal
redundancy as well as polyneuronal innervation (22, 57).
Despite attempting to fit their data into the traditional EOM
fiber typing classification scheme based on innervation and
mitochondrial content (82), a number of studies have data
where the range in values in EOM myofibers suggests a
continuum, as well as far exceeding ranges seen in myofibers
derived from limb muscles. For example, in a study of crossbridge kinetics that measured myofiber dynamic stiffness,
EOM single fibers had a greatly increased mechanical kinetic
range compared with limb fibers (52). In a study of Ca2⫹ and
Sr2⫹ activation, EOM myofibers not only had a wider range of
values than limb muscle fibers, but over one-third of the
myofibers exhibited mixed fast- and slow-twitch contractile
properties within single contracting units (54). Twitch contraction time, fusion frequency, and fatigability in motor units of
SINGLE SKINNED EOM MYOFIBERS
J Appl Physiol • VOL
forms were observed at the tapered ends of single myofibers,
but not within the entire fiber length (72). Functionally, can this
longitudinal variation in MyHC isoforms within single fibers
affect muscle contractile characteristics? This type of heterogeneity of MyHC expression in segment lengths within single
fibers occurs in frog muscle and was shown to result in
measurable differences in maximum shortening velocity along
the fiber length (1, 25). When examined physiologically not
only did fiber segments from these frog muscles display different shortening velocities, but often strained in opposite
directions for up to one-third of the contraction cycle. This
supports the hypothesis that these MyHC variations along a
fiber’s length must play a functional role in EOM contractile
function.
Another example of variation along fiber length is EOMspecific MyHC, which was localized to the innervation zone in
both the orbital and global layers, colocalizing with IIB and
less commonly with IIX (13). In another study only the
myofiber ends were shown to express neonatal MyHC (75).
Functionally, in rat single orbital layer EOM myofibers, the
end-plate region expressing fast MyHC displayed fast-twitch
contractile properties, while the fiber ends expressing slow
tonic MyHC showed graded tonic contractions (41, 42).
Individual skeletal muscle myofibers are long multinucleated cells that develop by fusion of many precursor cells. Each
myonucleus regulates protein expression in what is called its
myonuclear domain, the fiber segment in which it is contained
(2, 65). It was elegantly shown that gene regulation in individual myonuclei is independently regulated, and activation of
any given nucleus within a myofiber is dynamic and occurs in
pulses (62). Thus the machinery that controls gene expression
functions to regulate protein content independently within each
fiber segment; this would include MyHC isoforms. Parsing this
continuously changing continuum into six fiber types overly
simplifies the physiology of these complex muscles.
Other support for the continuum hypothesis. In addition to
the considerable complexity in MyHC coexpression patterns in
single myofibers in EOM, other proteins also show coexpression patterns that do not correspond to the “historically”
defined fiber types. EOM, as is true in limb muscle, express
MyHC binding protein C (MBPC). In contrast to limb muscle,
where MBPC-fast is associated with fast-expressing MyHC
and MBPC-slow with slow myofibers, in EOM MBPC-slow is
expressed in all myofibers with a complete absence of MBPCfast expression (44). This is despite the fact that 80 –90% of the
myofibers express a fast MyHC isoform (45). EOM fast-twitch
myofibers express both SERCA1 and SERCA2 (43), again in
contrast to limb skeletal muscle where fast fibers express only
SERCA1 (92). An examination of succinate dehydrogenase
(SDH) and ␣-glycerophosphate dehydrogenase (GPDH) expression patterns in EOM showed that the fast myofibers
formed a continuum relative to coexistence of these two
metabolic pathways (7). When expression of SDH and GPDH
was graphed as scatterplots, there was significant overlap of all
the fast fibers; only the slow tonic fibers formed a distinct
group. Even the first publication parsing the EOM muscle
fibers into fiber types using innervation and mitochondrial
content describe the mitochondrial density as a “spectrum”
(56). Examined in aggregate, the overwhelming evidence leads
to the conclusion that, with the exception of the slow tonic
myofibers, the twitch myofibers form a continuum with signif-
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cat lateral rectus muscles showed a wide range of measured
values (81), and the individual motoneurons and myofibers
showed a continuum along the range of values for each of the
physiological parameters examined (81). In contrast to limb
skeletal muscle, their data show overlapping ranges when
parsed into the “traditional” fiber type groups. It should be
noted that none of these studies examined the MyHC composition in relation to shortening velocity or force.
Colocalization of MyHC isoforms in single myofibers. We
showed that the vast majority of the myofibers within the rabbit
EOM expresses more than one MyHC isoform, and in fact,
serial sections demonstrate that single fibers can coexpress up
to six isoforms simultaneously in the same region of the
myofiber. The coexpression of various isoforms of MyHC in
single myofibers is somewhat common in skeletal muscle (12,
68, 77, 80). In plantaris muscle, for example, over 50% of the
myofibers examined coexpressed at least two fast MyHC
isoforms (12). The same was shown for diaphragm muscle;
polymorphic fibers constituted the majority of myofibers examined (15). A number of studies demonstrated coexpression
of multiple MyHC isoforms in single EOM myofibers in a wide
range of animal species (10, 13, 42, 47, 59, 74 –76, 89, 91, 96)
including humans (45, 48, 89). While none examined the
number of MyHC isoforms evaluated in the present study and
species differences exist, hybrid fibers with multiple MyHC
isoforms are the rule not the exception. EOM muscles appear
to represent the far end of the allotypic continuum, where the
majority of the myofibers are hybrid (48, 93).
A direct correlation has been demonstrated between twitch
contraction time and MyHC composition (11, 50, 67). The
complex spectrum of coexpression of MyHC isoforms in EOM
myofibers allows the continuous spectrum of contraction shortening velocities and force generation found. When limb skeletal muscle fibers were assessed physiologically, the contractile velocities spanned an overlapping continuum ranging from
0.35 to 2.84 fl/s, approximately a 10-fold difference (11). In
contrast, the values for EOM in this study ranged from a low
of 0.439 fl/s to a high of 19.8 fl/s, almost a 50-fold difference.
We clearly showed the presence of single myofibers coexpressing fast and slow MyHC isoforms in rabbit EOM, and this
phenomenon has been seen in limb muscle also. These were
described in diaphragm muscle (15, 51), plantaris and soleus
(12), masseter (14), and several other muscles (84). These
distinct myofibrils intermingle within any given cross-section
(29). In aging muscle, 20% of the myofibers in limb muscle
coexpressed fast and slow MyHC (5). However, in a comprehensive study of the MyHC composition of human EOMs,
there were no fibers coexpressing MyHC slow and fast (45).
Heterogeneity in MyHC composition along single myofiber
length. The reconstructed myofibers expressing neonatal
MyHC in the current study demonstrate the significant heterogeneity along individual myofiber segments. This also suggests that the diversity of MyHC isoforms within single myofibers is probably greater than can be gleaned from fiber
segments with a limited pool of protein, as performed using
skinned fibers. Despite the traditionally described six fiber
types for EOM, as early as 1984 variability of the histochemical composition in single myofibers in EOM was described
(64). Limb skeletal muscles also can have nonuniform MyHC
expression in different regions of single fibers along their
length (76, 77, 83, 95). For example, immature MyHC iso-
1185
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The cranial motoneurons that form the oculomotor, trochlear, and abducens nerves have properties that are different
from those of spinal motoneurons and different from each
other. Comparative studies of spinal vs. extraocular cranial
motoneurons suggest several mechanisms that might explain
these differences. In a study of long-term cultures of EOM
primordia with either spinal or cranial motoneurons, the EOM
survived long term only in the presence of oculomotor neurons
(70). Normal adult extraocular motoneurons continue to express a number of neurotrophic factors and their receptors that
are downregulated in spinal motoneurons, including tyrosine
receptor kinase type 1 (TrkA; 9) and growth factors such as
fibroblast growth factor isoforms (35, 37) and insulin growth
factors I and II (4, 38). Growth factors such as brain-derived
neurotrophic factor and neurotrophin-3 differentially alter extraocular motoneuron firing patterns (19). Extraocular motoneurons also significantly differ from spinal motoneurons in
their differential survival rates in spinal motor degenerative
diseases (63). These intrinsic differences extend to the EOM
themselves. Pax3, the gene responsible for the formation of
somite-derived skeletal muscle, does not play a role in EOM
formation (85); instead formation of the EOM depends on the
gene Pitx2 (21). Thus the variability of MyHC isoforms within
individual EOM myofibers is just one manifestation of a large
array of molecular and genetic differences both at the muscle
and motoneuron levels.
From a functional point of view, there is accumulating
evidence that the motoneurons that control EOM contractile
characteristics also represent a continuum in terms of their
firing rates, eye position sensitivity, and velocity sensitivity
(20). Abducens motoneurons control both initial eye position, by a burst of firing followed by maintenance of lateral
rectus muscle tension by tonic firing during the “slide
phase” for any given duration of eye position. Even in the
presence of a rigorous eye movement fatigue protocol,
where eye velocity kinematics decrease, the EOM compensate and maintain the accuracy of final eye position (71). In
other words, despite a decreased neuronal firing velocity
after rapid and repetitive eye saccades, no evidence of
change in EOM function or eye position was seen. Presumably, because populations of muscle fibers contract after any
given stimulation, the summed EOM dynamics do not
change. The ability of extraocular motoneurons to draw on
a myriad of “fiber types” supports the findings of these
studies. The existence of a myofiber continuum relative to
shortening velocity and force and specific tension generation will hopefully inform future electrophysiological experiments, allowing a more accurate modeling of the eye
plant relative to oculomotor control of eye movements.
Conclusions. The complexity of patterns of coexpression
of MyHC isoforms in EOM results in a wider spectrum of
shortening velocities and contractile forces than is seen in
single fibers from limb skeletal muscle and forms a continuum. Single fibers with the same apparent shortening velocity can have significantly different MyHC isoform ratios.
This complexity is compounded by differential MyHC isoform expression along the length of single EOM myofibers.
This may play a role in increasing the range of power output
of an individual muscle. For example, if initial shortening
velocity is reduced, force development is accelerated when
the fiber is activated (24). How this myosin isoform com-
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icantly overlapping metabolic and functional properties. As the
technology for protein separation improves, we hypothesize
that single fibers will be shown to contain more MyHC isoforms than can currently be demonstrated.
Functional consequences. This complexity must play an
important role in the fine control of EOM contraction velocities, force, and specific tension generation, with the resultant
ability to control eye position with great accuracy, regardless
of movement (e.g., fixation, slow vergence movements, or fast
saccades). How do these complex coexpression patterns of
MyHC affect contractile behavior? This has been partly explained in an interesting series of experiments where activation
characteristics of single fast and slow myofibers tied together
either in series or in parallel were examined (55). When fast
and slow myofibers were attached in parallel, the contraction
was directly related to the known proportion of fast and slow
fiber components. However, when tied in series, the contractile
characteristics did not match the ratio of fast and slow fiber
components, but instead resulted in “mixed” contractile behaviors that generally behaved more similar to fast-twitch fibers.
In other words, the fast MyHC isoforms were “dominant” (55).
This directly mirrors the mixed contractile properties that
would be expected of the majority of EOM myofibers examined in the current study.
Directions of movement controlled by limb skeletal muscles
are generally constrained by their bony attachment sites and the
weight of the moving limb. The EOM do not insert into bone,
but rather into the sclera of the globe. The eye in the bony orbit
is angled at 25° off the midline (94). To maintain eye position
in primary gaze, e.g., straight ahead and fixating on an object
in the far horizon, the EOM must maintain continuous muscle
tension of the medial rectus muscles to keep the direction of
gaze parallel to the direction of the head. The EOM also must
be able to hold the eye in a single position of gaze during
fixation, move the eyes slowly during vergence, as well as
move the eyes quickly when making saccades. A number of the
unusual properties of extraocular motoneurons and associated
muscle fibers, i.e., the motor unit, can be partly explained by
myofibers with a continuum of MyHC isoform expression.
Despite the wide array of movement speeds performed by the
eye, ocular motor units do not have specialized roles (81). This
is presumably the functional correlate of having very small
motor units with diverse MyHC isoforms, allowing different
motor units to produce distinct fiber tensions and shortening
velocities. In fact, a continuum of fusion frequencies within
EOM motor units was demonstrated electrophysiologically
(61). Also, no correlations were seen between the mechanical
parameters of single abducens motoneurons and recruitment
order (81). In a series of studies in cat and monkey, stimulation
rate and resultant muscle tension were nonlinear. Based on
predicted force vs. actual force in whole abducens nerve
stimulations, it was determined that as few as two or three
motor units could move the eye 1° (32, 33). There is also
evidence that unlike adult limb and trunk musculature, the
adult EOM maintain a number of myofibers with polyneuronal
innervation (22). As the pattern of motor nerve stimulation
plays an important role in determination of MyHC isoform
composition (34), single fibers receiving “instructions” from
more than one motoneuron would be predicted to respond with
increased MyHC isoform diversity.
SINGLE SKINNED EOM MYOFIBERS
plexity affects control of eye position and eye movements is
unknown; however, motor unit and whole muscle responses
to whole nerve stimulation also show a continuum of properties. In addition it is known that these properties rapidly
adapt to changes in stretch, innervation, hormone status,
drug treatments, and the like (3, 16 –18, 88). Future studies
are needed to understand how these complex coexpression
patterns of MyHC isoform are controlled. In addition, it is
critical to consider the implications of this heterogeneity
when investigating how the oculomotor control system is
able to accurately and reproducibly move the eye in a wide
range of velocities and gaze directions.
16.
17.
18.
19.
20.
GRANTS
DISCLOSURES
21.
22.
23.
No conflicts of interest, financial or otherwise, are declared by the
authors.
24.
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This work was supported by Grants EY15313 and EY11375 from the
National Eye Institute, the Minnesota Medical Foundation, the Minnesota
Lions and Lionesses, Swedish Research Council (K2010-62x20399-04-02,
FPD), Research to Prevent Blindness Lew Wasserman Mid-Career Development Award (to L. K. McLoon), and an unrestricted grant to the Department
of Ophthalmology from Research to Prevent Blindness.
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