Download Fibrillin and elastin networks in extrafusal tissue and muscle

Fibrillin and Elastin Networks in Extrafusal Tissue and
Muscle Spindles of Bovine Extraocular Muscles
Alfred Maier, Christopher N. McDaniels, and Richard Mayne
Purpose. Bovine extraocular rectus muscles were examined to map the distribution of elastin
and fibrillin in extrafusal tissue and muscle spindles.
Methods. Immunohistochemical techniques and immunolocalization were employed to pinpoint the placement of molecules relative to muscle fibers.
Results. Strands containing elastin and fibrillin surrounded all extrafusal fibers. They also
covered the external surface of intrafusal fibers, more extensively at the equator than at the
pole. Within strands elastin was placed in the center, whereas fibrillin was located in microfibrils on the periphery.
Conclusions. The wide distribution in extrafusal tissue of elastin and fibrillin suggests that they
are factors in determining the mechanical properties of extraocular muscles. Their placement
in proximity to individual intrafusal fibers should affect the viscoelastic properties of these
fibers and, thus, influence the dimensions of the afferent discharge. Invest Ophthalmol Vis
Sci. 1994:35:3103-3110.
Connective tissues are important functional units of
striated muscles. They securely link contractile elements to the muscle tendon and act as conduits for
force transmission. Connective tissue macromolecules
whose distribution in muscle have been investigated
include collagen types I, III, and VI, basal lamina components, and fibronectin.12 Data obtained from previous investigations on connective tissue matrix have
led to an understanding of how muscle fibers are bundled into fascicles and how they are affixed at the myotendinous junction.3 Yet, little information is available
on the distribution in muscle of elastin and its associated microfibrils. One component of these microfibrils is a large (350 kD) protein called fibrillin.45 Recent evidence indicates that Marfan syndrome, a condition involving pathologic changes in elastic fibers, is
associated with a mutation in the fibrillin gene located
on chromosome 15.6'7 The current study employed antibodies against fibrillin and elastin to evaluate the dis-
t'rom the Department of Cell Biology, University of Alabama at Birmingham,
Birmingham, Alabama.
Supported by National Institutes of Health grants DE08228 (RM) and EY09908
(RM).
Submitted for publication October 6, 1993; revised January 3, 1994; accepted
January 5, 1994.
Proprietary interest category: N.
Reprint requests: Alfred Maier, Department oj Cell Biology, University of Alabama
at Birmingham, 1670 University Boulevard, Birmingham, AL 35294-0019.
investigative Ophthalmology & Visual Science, June 1994, Vol. 35, No. 7
Copyright © Association for Research in Vision and Ophthalmology
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tribution of these molecules in extrafusal tissue and
muscle spindles. Extraocular muscles were selected
because of the fine movements they perform in positioning the globe, a task that might require an extracellular matrix containing extensible and rigid components, as exemplified by elastin and collagens, respectively. Muscle spindles were examined because
equatorial regions of intrafusal fibers, or structures
immediately adjacent to them, require resiliency to
produce the dynamic peak of the afferent discharge in
response to phasic changes in muscle length.8 Elastin
could be the vehicle that renders this resiliency.
MATERIALS AND METHODS
Extraocular rectus muscles were obtained from six
healthy male or female domestic cattle of a local meat
processing plant. Twenty muscles were examined. Immediately after death, globes were enucleated, packed
in ice, and taken to the laboratory. No deterioration of
muscle tissue was observed on subsequent microscopic
examination. Muscles were detached from the sclera
and frozen in melting isopentane, and they were held
with with tweezers to approximate their in situ length.
Tissue was frozen under these conditions to avoid
structural artefacts introduced by curling of muscle
fibers. Serial cross-sections were cut (6 to 8 /im) in a
cryostat and allowed to dry at room temperature for
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3104
30 minutes. Using standard immunohistochemical
procedures, individual sections were then incubated
with any one of the primary monoclonal antibodies
against myosin heavy chains (MHC) or connective tissue macromolecules listed in Table 1. Repetitive series
of three or more consecutive sections were processed
to permit tracing of the same connective tissue structures and of extrafusal and intrafusal fibers for appreciable distances through the muscles. Before incubation, sections were fixed for 2 minutes in 2% phosphate-buffered paraformaldehyde and washed repeatedly in phosphate-buffered saline (PBS). Incubation
with the primary antibodies lasted for 60 to 90 minutes
(37°C). After several PBS washes, sections were incubated with the appropriate fluorescein-, rhodamineor HRP-conjugated secondary antibodies for 30 to 45
minutes (37°C). Diaminobenzidine was used as chromogen on sections incubated with HRP-conjugated
second antibodies. Sections were washed several more
times with PBS and coverslipped with 30% glycerol in
PBS (fluorochrome sections) or dehydrated and
mounted in Permount (HRP sections). In some instances, sections used for demonstrating elastin or fibrillin were double labeled with rhodamine-conjugated phalloidin toxin. This compound combines with
actin to facilitate identification of muscle fibers and
pinpointing of the connective tissue matrix location
relative to extrafusal and intrafusal fibers.
10 minutes with 1 % gluteraldehyde and paraformaldehyde in PBS. Sections were then incubated for 30 minutes (4°C) in 0.5% sodium borate in PBS to remove
excess fixative. This was followed by washing in PBS,
incubating for 2 hours in 0.1% BSA in PBS, and rinsing in PBS. Sections were incubated with the primary
antibodies overnight (4°C), washed with PBS, and
then incubated with 5 nm colloidal gold-conjugated
second antibodies (Auroprobe, Amersham, Arlington
Heights, IL) for 4 hours (4°C). The remaining procedure was a standard protocol for embedding in plastic
and sectioning of electron microscopic specimens.
Silver or gold sections were cut with a diamond knife
on an ultramicrotome, stained with uranyl acetate and
lead citrate, and inspected on a JEOL (Peabody, MA)
100X transmission electron microscope.
RESULTS
There were no noticeable differences between data
collected from male and female animals.
Immunohistochemistry
Extrafusal fibers. Individual fiber types were identified to determine if the distribution of elastin or fibrillin differed around different fiber types. About
78% of the fibers reacted strongly with the anti-MHC
MY32 antibody and stained dark after incubation for
mATPase, pH 9.6 preincubation. Both reactivities are
characteristic of fast-twitch muscle. All fibers also exhibited low to moderate amounts of slow MHC, as
seen with antibody CA83, and stained moderately to
strongly for mATPase, pH 4.6 preincubation. Thus,
most fibers were at least to some extent fast/slow hybrids, also known to occur in other mammalian extraocular muscles.1011
In a limited number of series, some sections were
incubated for myosin (m) ATPase, acid, and alkaline
preincubation, according to the method of Guth and
Samaha.9 This was done to identify, in conjunction
with MHC sections, muscle fiber types.
Tissue from several muscles was prepared for colloidal gold immunolocalization. Forty micro-thick sections of frozen blocks containing muscle spindles were
cut, allowed to dry for 30 minutes (RT), and fixed for
TABLE
l. Antibodies Used
Reference or
Manufacturers
Designation
Reactivity
11C1
Fibrillin
1:100
Wright5
E-4013
Elastin
1:100
Sigma Chemical, St. Louis, MO
V-5255
Vi men tin
1:100
Sigma Chemical
ALD58
Slow-tonic MHC
1:5
Shafig27
1975
Laminin
1:100
Chemicon, Temecula, CA
MY32
Neonatal/fast MHC
1:500
2B6
Embryonic MHC
1:100
Sigma Chemical
Harris2"
Gamoke29
CA83
Slow MHC
1:100
Sweeney™
Working Dilution*
* Diluent: 0.05% Triton X-100 and 0.05% bovine serum albumin in phosphate-buffered saline.
MHC = myocin heavy chains.
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Fibrillin and Elastin Networks
3105
Incubation of sections with anti-elastin (Fig. 1A)
and anti-fibrillin (Fig. IB) antibodies produced similar
staining around all extrafusal fiber types; however, instead of a uniform reaction around each muscle fiber,
there was for the most part alteration of thicker and
thinner staining, interspersed with occasional unstained segments. It is unlikely that this pattern resulted from shortened muscle fibers because muscles
were kept near their in situ length as they were frozen.
When disregarding unstained segments, the pattern
resembled in shape and location that seen for basal
lamina macromolecules, such as laminin (Fig. 1C).
Double labeling with phalloidin established that the
anti-elastin and anti-fibrillin staining was external to
musclefibers,close to the basal lamina. Distribution of
fibrillin and elastin extended into the perimysium and
epimysium. Strong immunostaining for both molecules and for vimentin was observed in the walls of
larger blood vessels and around intramuscular nerve
trunks (not shown).
Muscle spindles. Most spindles were located in the
orbital sections of the muscles.12 They displayed the
same general structure as receptors in limb muscles,
consisting of an intrafusal fiber bundle wrapped by an
inner capsule. More peripherally, there was a connective tissue outer capsule that bulged at the equatorial
region to accommodate an extensive periaxial space.
Intrafusal fiber types could be classified with conventional mATPase histochemistry and MHC immunohistochemistry. Nuclear chain fibers presented contractile protein profiles resembling fast fibers; nuclear bag
fibers appeared more like slow fibers. The nuclear
bag, fibers did not react with the antibody against neonatal/fast MHC, whereas the nuclear bag2 fibers
reacted moderately. (Fig. 2A; Table 2).
The outer spindle capsule contained small to moderate amounts of fibrillin and elastin. Fibrils of these
proteins situated within the outer capsule were seemingly continuous with similar structures in the adjacent perimysium (Fig. 2F).
Intrafusal fibers were routinely circled by thin and
thick strands of fibrillin, which in cross-section appeared as dashes or dots, respectively (Fig. 2B, 2D, and
2F). Their location suggested that they were part of
the inner portion of the inner capsule. When traced
through serial sections, it became apparent that these
strands were not of constant diameter nor did they
pursue a straight course. Instead, they changed their
size, shape, and course along the lengths of intrafusal
fibers (Fig. 3A). Elastin was similarly distributed (Figs.
2C and 2G), except that thinner strands were more
numerous. Even though not strictly applicable to each
intrafusal fiber, on average elastin and fibrillin were
more prevalent and occurred more often in distinct
strands at the equator than at the pole (Figs. 2B and
2C versus Figs. 2F arid 2G). None of the three intrafu-
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FIGURE 1. (A) Cross-sectioned extrafusal fibers in bovine extraocular rectus muscles incubated with antibodies against
the indicated molecules. (B, C) Equally enlarged consecutive
sections. Identical points are marked with arrowheads. Bar
= 50 /*m. EF = Extrafusal fiber; Pm = perimysium.
sal fiber types had significantly more elastin and fibrillin associated with it than the other two. There was
strong immunostaining for the intermediate filament
vimentin (Fig. 2E), comparable in location to that for
elastin and fibrillin.
From the network of elastin and fibrillin around
intrafusal fibers, slender extensions projected across
the periaxial space in the direction of the outer cap-
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Investigative Ophthalmology Sc Visual Science, June 1994, Vol. 35, No. 7
FIGURE 2. Cross-sectioned muscle spindles from bovine extraocular recms muscles incubated with antibodies against
the indicated molecules. (B-G)
Small circles mark the positions
where centers of intrafusal
fibers would be. (A, B) Consecutive sections, (D, E) separate
images of one double-labeled
section. Bar = 20 /xm. b, = nuclear bag| fiber; b 2 = nuclear
baga fiber; c = nuclear chain
fiber; liL — elastin; FI = fibrillin; N/F MHC = neonatal/fast
myosin heavy chain; oc = outer
capsule; pm = perimysium;
ps = periaxial space; VI = vimentin.
sule. They did not traverse the space in a fixed plane
but meandered in and out of the field of view. Ultimately, however, they appeared to connect to the
outer capsule (Figs. 4A and 4B). Extensions of fibrillin
were more frequently observed than were similar extensions of elastin.
EM Immunolocalization
Extrafusal fibers. Labeling was present just external to extrafusal fibers and their basal laminas. In support of findings obtained with the light microscope,
immunoreactivity was also observed in the perimysium, epimysium and perineurium; however, labelling
was often less extensive than in the immunohistochemical sections. Elastin label was relatively sparse and
concentrated in the more interior of the elastin-fibrillin complex (Fig. 5A), whereas fibrillin label was abundant on, and principally restricted to, outlying microfibrils (Fig. 5B). There was a greater density of gold
particles in tissue incubated with anti-fibrillin than in
tissue incubated with anti-elastin.
Muscle spindles. Fibrillin label was located along
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the periphery of the inner capsule. It was deposited
around oval patches of elastin (Fig. 5C), typically interconnected by narrower labeled strips (Figs. 3B). No
instances were encountered in which the label clearly
reached the basal lamina of intrafusal fibers. Labeling
for elastin was largely unsuccessful, but elastin fibrils
could always be recognized by their amorphous structure (Figs. 5A and 5C). As in the immunohistochemical
sections, no difference was observed in the distribution and amount of the two molecules around different types of intrafusal fiber.
In accordance with immmunofluorescence data,
thin and curved labeled extensions were seen to project from immunopositive regions immediately adjacent to intrafusal fibers toward the outer capsule (Figs.
4A and 4B). In these extensions, fibrillin was more
strongly represented than elastin.
DISCUSSION
With the immunohistochemical methods employed
here, it has been demonstrated that elastin and elastin-
Fibrillin and Elastin Networks
TABLE 2.
3107
Defining Criteria of Intrafusal Fibers
Reactivity
Anti-MHC Antibodies
mA TPase
Nuclear Fibers
pH4.6
pH9.6
MY32
2B6
CAS3
ALD5S
Bag,
Chain
associated microfibrils513'14 form a network that
spreads throughout bovine extraocular rectus muscles. Fibrillin as identified with antibody 11C15 was
found to be concentrated on microfibrils that
surround cores of elastin. This spatial relationship is
established during elastogenesis. Collections of microfibrils are the initial step in assembling elastin fibrils.
As elastin is laid down and fibrils grow, they acquire a
peripheral lattice of microfibrils15 that persists in mature tissue.
EFN
FIGURE 3. (A) Three-dimensional reconstruction from serial
fluorescing cross sections of one ring of elastin-fibrillin
surrounding one intrafusal fiber near the equatorial region.
Black areas represent regions where strong staining predominates and most thicker strands are located. Light areas denote regions where staining is weak or absent and thinner
strands prevail. To preserve clarity, the intrafusal fiber has
not been drawn. (B) Schematic drawing illustrating in crosssection the spatial relations of intrafusal fiber (IF), basal lamina (BL), inner capsule (IC, stipple), and the elastin-fibrillin
network (EFN). Arrows point to equivalent regions in A
and B.
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In the muscles examined here, elastin and fibrillin
formed cylindrical sleeves that clothed all extrafusal
muscle fibers. They approximated in shape and location sleeves of endomysium and basal lamina. Electron
microscopic immunolocalization indicated that the fibrillin-elastin network was positioned external to the
basal lamina within the endomysium, but it probably
had no extensive direct linkage with the basal lamina.
There is evidence that at the dermal-epidermal junction, microfibrils do attach to basal lamina.1617
Force transmission from contractile elements to
connective tissues is not limited to the distal portions
of muscle fibers that connect at myotendinous junctions but also occurs at lateral surfaces of muscle fibers
via the endomysium, perhaps in association with costameres.3 Even though it is not known how elastin and
fibrillin interconnect with the basal lamina arid the endomysial layer, both are potential links in the chain of
force transmission from muscle fiber to tendon. Differences in conformational states of cross-linking exist
in elastin during length changes,18 and closed and
open conformations have been postulated for fibrillin.10 Because of these properties, both molecules
could dampen excessive force transfer.
Gold immunolabeling was more readily accomplished with anti-fibrillin than with anti-elastin. When
present, label was confined to the periphery of the
elastin fibril, neither entering its center nor extending
to surrounding microfibrils. The lesser success with
anti-elastin may be a penetration problem related to
numerous cross-linkages.13 Moreover, fixation and
subsequent preparation for electron microscopy may
have damaged the epitope. Despite low levels of label,
elastin could be always unequivocally identified at the
ultrastructural level because of its constant association
with microfibrils.
Cooper and Gladden20 found in histologic sections of mammalian spindles from nonextraocular
muscles more elastic fibers around nuclear bag fibers
than around nuclear chain fibers. Preferential location of elastin around nuclear bag] fibers supports the
notion that the rising and declining limbs of the dynamic phase of the afferent signal21 are imparted by
the viscoelastic properties of this intrafusal fiber,
aided by an external extensible connective tissue skele-
Investigative Ophthalmology &: Visual Science, June 1994, Vol. 35, No. 7
3108
PS
was a greater reactivity for fibrillin and elastin at the
equator than at the pole. This observation is consistent
with the thinning and eventual termination of the inner capsule in the polar region.22 The functional implication relating to this architecture is that after releasing them from a stretch, the greatest recoil potential for intrafusal fibers is available at the sensory
region of the equator. As a corollary, release from
stretch would remove tension from the primary affer-
• 1 •
EL
\ .
ps
75 nrji7 5 nm
A
FIGURE 4. Panels on right show electron microscopic immunolocalization of fibrillin (5 nm gold) in the periaxial
space (ps) and the outer spindle capsule (oc). lmmunofluorescing sections to the left illustrate in each case the appearance at the light microscope level. Small circles mark the
position where centers of intrafusal fibers would be. (A)
Thin strands offibrillin(arrowheads) extend from the inner
capsule or fromfibrillin—elastincomplexes around intrafusal fibers (arrows) across the periaxial space. (B) Ultimately,
the strands connect to the outer capsule. Frequently, the
strands persue a wavy course, thus passing in and out of the
plane of view (arrowheads in periaxial space, right panel).
Bar for fluorescing sections = 20 jtm. pm = Perimysium.
ton. It is not clear how the elastic fibers of Cooper and
Gladden20 relate to the distribution of immunohistochemically identified elastin and fibrillin. At any rate,
we found no significant differences in the distribution
of elastin and fibrillin around nuclear bag and nuclear
chain fibers. Hence, the dynamic peak of the afferent
discharge, if present in extraocular spindles, quite
likely is not largely or solely caused by mechanical
properties inherent in nuclear bag, fibers and their
immediate connective tissue coverings but is formed
by contributions from all fibers and their adnexa.
A condition that applied to most intrafusal fibers
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FIGURE 5. Electron microscopic immunolocalization (5 nm
gold) of elastin (EL; A) andfibrillin(FI; B) around extrafusal
fibers, and offibrillinaround intrafusal fibers (C). Label for
elastin is confined to the periphery of an elastin-fibrillin
strand and rarely reaches its core (x). Fibrillin label is concentrated in microfibrils (arrows) surrounding the elastin
core. Inset in (C) is a fluorescing section that shows a ring of
fibrillin surrounding a single intrafusal fiber! A small circle
marks the location where the center of the intrafusal fiber
would be. A double arrow points to a single elastin-fibrillin
strand as it appears at the light microscope and electron
microscope levels. Elastin-fibrillin complexes connect with
neighboring like complexes via thinner lamellae of fibrillin
and elastin. Bar in inset of panel C = 10 /xm. Bar in (A) also
applies to (B). IC = Inner capsule.
Fibrillin and Elastin Networks
3109
ent and change its discharge from the higher level during the stretch to a basal rate.8 The strong staining in
the inner capsule for vimentin, an intermediate filament occurring in fibroblasts, indicates the presence
of a population of fibroblasts or specialized inner capsule cells.23 The coincidence in location of cells and
microfibrils suggests that fibrillin and elastin are products of these cells.
Although the periaxial space in extraocular spindles of some species is diminutive,24 in the bovine muscles examined here it is voluminous. At the equator of
these spindles, the intrafusal fiber bundle is separated
from the outer capsule by as much as 100 /xm. To be an
effective sensor of muscle length, it would appear that
intrafusal fibers and the associated sensory endings
EFN
need to maintain a reasonable parallel alignment with
extrafusal fibers. Hyaluronate within the periaxial
space25 could orient the transducing elements to some
extent. Anchorage of the intrafusal fiber bundle may
be also accomplished via connections from the inner
to the outer capsules, involving modified inner or
outer capsule cells, or fibroblasts.23'26 Our data suggest that struts containing fibrillin and elastin also play
a role in suspending intrafusal fibers within the periaxial space (Fig. 6). There are no large amounts of
fibrillin or elastin within the outer capsule, and it has
yet to be determined how fibrillin bonds to other capsular matrix. The undulating nature of the struts indicates that the bracing is not rigid but allows for some
movement in each plane. This arrangement could
compensate for changing conditions, permitting the
intrafusal fiber bundle to slide back and forth in relation to the outer capsule when the gross muscle is
lengthened in response to an applied pull, or when
lateral pressure is applied.
Key Words
extraocular muscles, muscle spindles, elastin-fibrillin network, electron microscopic immunolocalization, mechanical
properties
Acknowledgments
The authors thank Drs. Brigitte Gambke and Radovan Zak
for gifts of the 2B6 and CA83 antibodies, respectively. The
ALD58 antibody was obtained from the Developmental
Studies Hybridoma Bank maintained by the Department of
Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, and the
Department of Biology, University of Iowa, Iowa City, Iowa,
under contract N01-HD-2-3144 from the National Institute of Child Health and Human Development.
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FIGURE 6. Schematic representation of salient features of the
elastin-fibrillin network (EFN) in bovine extraocular rectus
muscle spindles. Only one-half [equator (E) to pole (P)] of
the spindle is drawn. Afferents, efferents, and the inner capsule are not shown. Rings of elastin-fibrillin individually
surround intrafusal fibers (IF) in close association with the
inner capsule. Extensions project from the rings across the
hyaluronate-filled periaxial space (arrow) to adjoin the internal surface of the outer capsule (OC). The system of elastinfibrillin strands is most extensive at the equator. It is less
prominent at the polar region, where the periaxial space is
small and the outer capsule and the intrafusal fiber bundle
are near each other.
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