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Myosin heavy chain expression within the tapered ends of skeletal muscle fibers.

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THE ANATOMICAL RECORD 242:462-470 (1995)
Myosin Heavy Chain Expression Within the Tapered Ends of
Skeletal Muscle Fibers
BENJAMIN W.C. ROSSER, DONNA M. WALDBILLIG, STACEY D. LOVO,
JACALYN D. ARMSTRONG, AND EVERETT BANDMAN
Department of Anatomy and Cell Biology, University of Saskatchewan College of Medicine,
Saskatoon, Saskatchewan, Canada (B.W.C.R., D.M.W., S.D.L., J.D.A.); Department of
Food Sciences and Technology, University of California, Davis, California (E.B.)
ABSTRACT
Background: The pectoralis muscle of the chicken contains
fast-twitch glycolytic fibers, which during development undergo a transformation in their myosin heavy chain (MyHC) content from embryonic to
a neonatal to an adult isoform (Bandman et al., 1990). Little, however, is
known of MyHC expression within the ends of these or other muscle fibers.
Here we test the hypothesis that the tapered ends of mature skeletal muscle
fibers contain a less mature MyHC isoform than that typically found
throughout their lengths.
Methods: We apply an ammoniacal silver histological stain for endomysium and monoclonal antibodies against neonatal and adult MyHCs of
chicken pectoralis to transverse serial sections of pectoralis from five mature chickens. The “lesser fiber diameters” of populations of fibers from
each bird are also measured.
Results: Most (-81.8%) of the small (<12 pm) and none of the larger (>20
pm) diameter fibers contain the neonatal MyHC. Following these smaller
fibers through serial sections, we show that they are the tapered ends of the
larger fibers. Whereas neonatal MyHC is restricted to the tapered fiber
ends, adult MyHC is present throughout the entire lengths of all fibers. We
also demonstrate acetylcholinesterase (AChE) activity at some of these fiber ends.
Conclusions: We postulate that longitudinal growth of myofibrils in adult
muscle is characterized by the sequential expression of MyHC isoforms
similar to that observed in rapidly growing muscle and that the presence of
the neurotransmitter hydrolase AChE at the tapered fiber ends may be
related to the retention of neonatal MyHC. o 1995 Wiley-Liss, Inc.
Key words: Muscle, Myosin, Fiber, End, Chicken, Pectoralis
The heavy chain of the myosin molecule contributes
Longitudinal growth of skeletal muscle is facilitated
more to myofibrillar mass than any other protein in by the addition of new sarcomeres to the ends of the
skeletal muscle (Yates and Greaser, 1983; Stryer, existing myofibrils, as shown by models of stretch1988). Myosin heavy chains (MyHCs) are encoded by a activated muscle growth (Williams and Goldspink,
diverse multigene family (Nguyen et al., 1982; Robbins 1971, 1973; Ziv et al., 1984). Chronically stretched
et al., 1986) and consist of a large number of related muscles can lengthen by the addition of nascent fibers
isoforms (Bandman, 1985). MyHCs are indicators of derived from satellite cells to the ends of existing
muscle fiber specialization, and distinct MyHC iso- fibers, and it has been suggested that these nascent
forms are found in different muscle fiber types (Pette fibers might repeat the MyHC transitions observed in
and Staron, 1988; Williams and Dhoot, 1992; Staron other fibers during development (Kennedy et al., 1988,
and Johnson, 1993), in different muscles (Crow and 1989; Dix and Eisenberg, 1990). However, i t is not
Stockdale, 1986; Bandman and Bennett, 1988), and in known whether the ends of fibers normally express a
the same muscle fiber at different periods of its normal
development (Lyons et al., 1983; Bandman et al., 1990).
Developing muscle fibers are capable of co-expressing
more than one MyHC isoform (Taylor and Bandman,
1989; Gauthier, 1990; Gordon and Lowey, 19921, as are
Received August 4, 1994; accepted March 7, 1995.
transitional fibers (Pette and Staron, 1988; Schiaffino
Address reprint requests to Dr. B.W.C. Rosser, University of
et al., 1990) and adult fibers under certain experimen- Saskatchewan
College of Medicine, Department of Anatomy and Cell
tal paradigms (Pette and Vrbova, 1992; Russell et al., Biology, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Can1992).
ada.
0 1995 WILEY-LISS, INC.
MYOSIN EXPRESSION WITHIN MUSCLE FIBER ENDS
different MyHC than that found throughout their bodies.
In this study, we test the hypothesis that the tapered
ends of mature skeletal muscle fibers contain a less
mature MyHC isoform than that typically found
throughout their lengths. The model used is the pectoralis muscle of the adult chicken. We show that the
smaller diameter fibers in transverse sections of the
pectoralis are in fact the tapered ends of the more commonly occurring larger diameter fibers. We also demonstrate within these tapered fiber ends the presence of
a MyHC isoform characteristic of early development
and that acetylcholinesterase activity is located on
some of these ends.
MATERIALS AND METHODS
Experimental Model
The pectoralis of birds is a comparatively massive
muscle extending from the sternum to the humerus
(Raikow, 1985),providing most of the energy necessary
to power the downstroke of the wing during flight (Dial
et al., 1988; Dial, 1992). Its fiber type complement is
generally correlated with a species mode of flight, or
flightlessness, although phylogenetic lineage also influences fiber type (George and Berger, 1966; Rosser
and George, 1986; Rosser et al., 1994). The pectoralis
muscle of the chicken, a nonvolant species, is fairly
unusual in that all but the deepest regions of the muscle consist entirely of fast-twitch glycolytic fibers (Gauthier et al., 1982; Matsuda et al., 1983). Distinct MyHC
isoforms appear sequentially within each of the fibers
of the superficial regions of the chicken pectoralis during embryonic, neonatal, and adult stages of development (Shear et al., 1988; Bandman et al., 1990; Gauthier and Orfanos, 1993). The pectoralis muscle of the
chicken and other birds consists of serially arranged
fibers that overlap one another t o a considerable extent
with their long tapered ends (Trotter et al., 1992;
Gaunt and Gans, 1993).This arrangement differs from
that found in the muscles of most mammals in which
fibers either run the entire length of the muscle or, if
arranged serially, do not overlap to the extent that
avian fibers do (Gaunt and Gans, 1990,1992).A transverse section through the belly of a muscle in which the
fibers are arranged serially, such as the chicken pectoralis, will reveal populations of very small diameter
fibers that are in fact the tapered ends of larger fibers
(Swatland, 1981, 1983).
The long tapering fiber ends in this study terminate
intramuscularly. It has not been widely appreciated,
until relatively recently, that the ends of muscle fibers
might commonly do so (Gaunt and Gans, 1990, 1992).
As the majority of earlier researchers believed that
most muscle fibers generally ran from the origin to the
insertion of a muscle, most earlier works were confined
to those ends located at or near musculotendinous junctions. In this study, we naturally discuss our findings
in light of some of these earlier works. We are not
aware of any publications demonstrating biochemical
differences between ends located intramuscularly and
those found at the musculotendinous junction. It is,
however, possible that some differences might exist.
Therefore, in order to avoid any future confusion,
throughout this study we refer to those ends located
intramuscularly as “tapered.”
463
Tissue Preparation
Five adult white leghorn chickens (Gallus domesticus), each 14 months old, were killed by cervical dislo-
cation. Blocks of muscle (each -0.6 x 0.6 x 3 cm) were
excised from the most superficial areas of the proximal
one-half of the belly of the right pectoralis muscle of
each bird, at a point located approximately midway
between the cranial and caudal edges of the muscle.
Blocks were immediately coated with Tissue-Tek
O.C.T. compound (Miles, Elkhart, IN), then quick frozen in 2-methylbutane cooled to -160°C by liquid nitrogen (Dubowitz, 19851, and stored at -80°C. From
tissue blocks representative of each bird, sections of 8
km thickness were cut in a cryostat maintained at
-20°C. An extensive series of serial sections was obtained from each pectoralis muscle studied. Microscope
slides were coated with gelatin-chrome alum (Alder,
19781, and two sections were picked up on each slide.
Myosin Heavy Chain lsoforms and Primary Antibodies
Embryonic, neonatal and adult MyHCs of the
chicken pectoralis muscle can be distinguished by immunocytochemical techniques utilizing monoclonal antibodies, each antibody generated in mouse against a
specific MyHC isoform of the chicken pectoralis (Bandman et al., 1990). MyHCs of adult fibers are identified
by their reactivity with the primary antibody AB8. The
MyHCs of neonatal fibers are labelled by 2E9 and the
MyHCs of embryonic fibers by a combination of their
reactions with several antibodies.
lmmunocytochemical and Histological Techniques
Immunocytochemicaltechniques, described by Shear
et al., 1988, were used to demonstrate MyHC isoforms.
Briefly, sections from each series were first blocked in
a solution consisting of 5% horse serum, 1%bovine
serum albumin, and 5 mM ethylenediaminetetraacetic
acid (EDTA) in phosphate-buffered saline (PBS; 0.02 M
sodium phosphate buffer, 0.15 M sodium chloride, pH
7.2). Sections were then incubated for 75 minutes at
room temperature with an appropriate primary antibody; either AB8 used at a dilution of 1:5,000 in blocking solution, or 2E9 a t 1:500. Sections were then rinsed
twice, each rinse for 5 minutes, with PBS. Binding of
the primary antibodies was visualized by incubating
the sections for 30 minutes at room temperature with a
fluoresceinated horse antimouse IgG secondary antibody (Vector Laboratories, Burlingame, CA) used at a
dilution of 1:64 in PBS. Sections were rinsed twice, 5
minutes each rinse in PBS, and subsequently fixed for
3 minutes in 4% buffered formalin. Sections were again
rinsed twice, 5 minutes each rinse in PBS, and then
mounted in citifluor mountant (Marivac, Halifax,
Nova Scotia).
The ammoniacal silver histological technique, as described by Swatland (19821, facilitates a more precise
measurement of muscle fiber diameter by producing a
blackening of the reticular fibers within the endomysium surrounding each muscle fiber (Swatland, 1983).
Briefly, sections serial to those used for immunochemistry were dipped in 95 ml 0.5% potassium permanganate acidified with 5 ml 3% sulfuric acid and then
rinsed in distilled water. Sections were subsequently
dipped into 1%oxalic acid, rinsed, dipped into 2.5%
464
B.W.C. ROSSER ET AL.
ferric ammonium sulphate, rinsed, dipped into an am- to estimate the size and frequency of the fibers or ends
moniacal silver hydroxide solution (see Swatland, 1982 containing the neonatal MyHC. In addition, from four
for preparation), and then rinsed. Each of the preceding of the birds, a t least 20 fibers or tapered fiber ends that
steps took 5-10 seconds. Sections were then rinsed, reacted positively with 2E9 were followed from the
fixed in a solution of 4% paraformaldehyde, rinsed, de- middle third through to the beginning andlor end of the
series of histological and immunocytochemical slides.
hydrated, cleared, and mounted.
Slides bearing serial sections were processed in the This enabled us to ascertain whether they were in fact
following sequence: 2E9, AB8, silver, 2E9, AB8, silver, small nascent fibers or, rather, the tapered ends of the
2E9, AB8, silver, etc. Individual fibers, their diameters much larger fibers.
narrowing toward their tapered ends (see Swatland,
1981) were followed in these transverse serial sections
RESULTS
for 1-2 mm distance. We searched for 2E9 labelled fiOn average, in the five birds studied, 9.84
1.86
bers in the middle third of each of our series of sections,
tracing fibers outward to the beginning and the end of (standard error) % of all fibers were labelled by the 2E9
each series. Sections were examined and photographed antibody against the neonatal MyHC. The mean “lesser
using a Zeiss microscope equipped for epifluorescence. fiber diameter” of all muscle fibers was 35.28 2 3.23 ( 5
Acetylcholinesterase (AChE) activity was demon- standard deviation) pm, but the mean diameter of the
strated on representative sections following the fibers reacting with 2E9 was only 6.50 2.25 pm (Table
method of Karnovsky and Roots (1964) as adopted by 1). Approximately 16.5% of all fibers had a “lesser fiber
Stoward and Pearse (1991). Immediately prior to pro- diameter” of <20 pm (Fig. l ) , and only fibers <20 pm
cessing slides for AChE activity, however, neonatal in diameter reacted with 2E9 (Fig. 2). Although not all
MyHCs in the sections were labelled by immunocy- fibers <20 pm in diameter were labelled with 2E9,
tochemical techniques using the 2E9 antibody as out- -81.8% of those with a diameter <12 pm were labelled
lined in the preceding. AChE activity was then dem- (Fig. 2). This percentage, however, dropped to -30% of
onstrated on these same sections by placing them for Y2 those fibers ranging in diameter from 12-16 pm and to
of those fibers ranging from 16-20 pm diameter
hour, at 37”C, into a n incubation medium containing 4%
0.17 mM acetylthiocholine iodide, 5 mM sodium cit- (Fig. 2). Overall, approximately 56% of all fibers under
rate, 3 mM CuSO,, and 0.5 mM potassium ferricyanide 20 microns diameter were labelled with 2E9. In each
in a 0.065 M, pH 5.9, maleate buffer. Slides were then fiber in which we localized the neonatal MyHC using
rinsed in PBS and mounted in citifluor. In this way we the 2E9 antibody (Fig. 31, the adult MyHC was cowere able to double-label our slides for both reactivity localized using AB8 (Fig. 4). All fibers in all sections
to the 2E9 antibody and AChE activity. Control slides contained the adult MyHC.
The 2E9 antibody (Fig. 3) appeared to label more
were processed a s in the preceding, except that butylthiocholine iodide (0.17 mM) was substituted for ace- intensely than the AB8 antibody (Fig. 4). This was
tylthiocholine iodide or eserine sulfate (0.10 mM) was most probably a result of differences in the dilutions we
added to the incubation medium, to verify that we had employed; 2E9 was diluted 1 5 0 0 and AB8 1:5,000. Aldetected AChE rather than nonspecific esterase activ- though 2E9 has been routinely used a t a dilution of
1:2,500 in developmental studies (Bandman and Benity.
nett, 1988; Shear et al., 19881, we found that our secQuantitative Measurements
tions were poorly labelled at this higher dilution.
Serial sections of the fibers reacting with 2E9 demA Wild-Leitz Ortholux microscope, with a n attached
Hitachi videocamera connected to a n FT-100 video- onstrated that these smaller diameter fibers were in
board (Imaging Technology, Vancouver, B.C., Canada) fact the tapered ends of the much larger adult fibers.
mounted inside a DN-4000 graphics workstation This was true for 71 of 94 2E9 labelled fibers, which we
(Apollo Computer, Calgary, Alta., Canada) was used to followed through our more extensive series of transmeasure the “lesser fiber diameters” of representative verse serial sections. One such fiber is shown in Figpopulations of muscle fibers on the silver stained slides ures 5-13. At a “lesser fiber diameter” of -10 pm, the
from each chicken studied. “Lesser fiber diameter” is a neonatal MyHC was present (Fig. 5). Using the ammomeasurement used to overcome distortion that can niacal silver stain for endomysium, we followed this
occur when a muscle fiber is cut slightly obliquely fiber through the serial sections (Figs. 6-8). The
rather than perfectly transversely and is defined as the “lesser fiber diameter” continued to increase to -17
maximum diameter across the lesser aspect of a fiber pm (Fig. 8) some 440 pm distance from the section in
(Dubowitz, 1985). Approximately 200 individual fibers which we first observed the fiber (Fig. 5). At -650 pm
per chicken were measured in contiguous fascicles, and from the first section, it had a “lesser fiber diameter” of
the percentage of fibers of different diameters within -22 pm and no longer contained the neonatal MyHC
each bird was calculated. This information enabled us (Fig. 9). The same fiber was then followed through silto construct a profile of the fiber population within the ver stained sections for a n additional 632 pm (Figs.
pectoralis of each chicken.
10-131, where it continued to increase in size until it
The percentage of the fibers or tapered fiber ends attained a “lesser fiber diameter” of -32 pm. Thus one
reacting with 2E9 and the “lesser fiber diameters” of small fiber containing the neonatal MyHC is shown to
these and other randomly selected nonreacting fibers be the tapered end of a n average size, more commonly
or fiber ends of comparable diameter were determined occurring larger fiber, which does not contain this
using a calibrated eyepiece with the Zeiss microscope. MyHC. This pattern was typical of 71 of the 94 small
At least 29 reacting fibers or tapered fiber ends per 2E9 labelled fibers we followed.
animal were measured in this way. This permitted us
Twenty-three of these 94 small 2E9 labelled fibers,
*
*
MYOSIN EXPRESSION WITHIN MUSCLE FIBER ENDS
465
TABLE 1. “Lesser fiber diameters” (pm) of muscle
fibers in chicken pectoralis’
Fibers labelled
by antineonatal
MyHC antibody 2E9
4.71 (21.73)
[371
[ 1.88-16.651
4.02 (21.85)
[441
[1.32-9.751
9.66 (k3.14)
L591
[4.50-16.801
6.71 (23.57)
[291
[2.10-13.051
7.43 (k3.54)
L301
13.15-17.001
6.50 (22.25)
Chicken
no.
All
All fibers within
representative
fascicles
38.49 (215.14)
[2111
f3.37-70.311
33.50 (212.94)
PO41
[3.59-60.191
30.92 (212.47)
[2161
14.79-58.131
35.15 (211.61)
[I851
L4.28-59.441
38.32 (213.43)
[2111
13.76-69.221
35.28 (k3.23)
w”
80
T
“‘Lesser fiber diameter” follows Dubowitz (1985). Values are expressed in microns as mean (t standard deviation). Numbers in
square brackets are ‘In” and “minimum-maximum.”
401
-I
L
0)
D
.
I
CL
c)
5:
P)
z
10
0
Lesser Fiber Diameter (pm)
Fig. 1. Range of “lesser fiber diameters” of fibers observed in
chicken pectoralis muscle. Whereas -55% of all fibers measured were
30-50 pm in diameter, only 16.5% of the fibers were <20 p m in
diameter. Each bar is mean (tstandard error) of data from five chickens.
however, did not appear to merge into the larger fibers.
Instead, they retained their comparatively smaller diameters and 2E9 reactivity throughout their lengths to
the first and/or last section in a series of sections. These
fibers were, in all probability, the tapered ends of fibers
Lesser Fiber Diameter (vm)
Fig. 2. Range of “lesser fiber diameters” of fibers in chicken pectoralis muscle labelled by the primary antibody 2E9 against the neonatal myosin heavy chain. No fiber >20 pm in diameter is labelled by
2E9. Approximately 56% of all fibers <20 pm in diameter are labelled, and -82% of all fibers under 12 pm are labelled. Each bar is
mean (? standard error) of data from five chickens. Note: All fibers,
regardless of diameter, were labelled by the primary antibody AB8
against the adult myosin heavy chain.
whose main bodies were located beyond the distance
encompassed by our serial sections. We did not observe
any small caliber fibers that were labelled throughout
their lengths by 2E9 and that appeared both to begin
and to terminate within our series of sections. Furthermore, fiber ends seemed to be fairly evenly distributed
throughout the fascicles of the muscle. They were not
clustered together as one would expect of the intrafusal
fibers of avian muscle spindles (Maier, 19921, nor were
they grouped outside of the fascicles as has been reported for the nascent fibers of avian stretched muscle
(Kennedy et al., 1988; McCormick and Schultz, 1992).
Overall, our observations indicate that the neonatal
MyHC was contained only within the tapered ends of
muscle fibers and that nascent fibers did not appear to
be present within those regions of the pectoralis muscle
that we studied.
Acetylcholinesterase (AChE) activity was localized
on a small minority of the muscle fibers. Although no
attempt was made to quantify the proportion of fibers
or tapered fiber ends demonstrating AChE activity, in
each pectoralis several of the ends that labelled with
2E9 (Fig. 14)also had AChE activity (Fig. 15). In some
of the fibers, a small amount of reaction product appeared to have diffused into the sarcoplasm (Fig. 15).
This was most probably due to our incubation pH of 5.9,
as at a pH greater than 5.0 a small amount of diffusion
is possible (Karnovsky and Roots, 1964). The cholines-
466
B.W.C. ROSSER ET AL.
Fig. 3. Transverse section of chicken pectoralis muscle. Immunofluorescent microscopy demonstrating affinity of the primary antibody
2E9 to the neonatal myosin heavy chain. Arrowheads indicate three
small labelled muscle fibers. Bar = 50 ym.
Fig. 4 Transverse section serial to that in Figure 3, demonstrating
affinity of the primary antibody AB8 to the adult myosin heavy chain.
All fibers are labelled by the antibody. Arrowheads indicate that the
same three small fibers containing the neonatal MyHC in Figure 3
also contain the adult MyHC. Bar = 50 Fm.
terase activity that we demonstrated was eliminated
on control slides when butylthiocholine iodide was substituted for acetylthiocholine iodide or when eserine
sulfate was added to the incubation medium.
DISCUSSION
Approximately one-sixth of all muscle fibers in the
chicken pectoralis are comparatively small (<20 pm)
in diameter, and the majority of these small fibers contain the neonatal MyHC. Larger diameter fibers (>20
pm) do not contain the neonatal MyHC. The smaller
the fiber diameter, the greater the chance that the neonatal MyHC will be present. These smaller diameter
fibers are not nascent fibers but the tapered ends or
terminal tips of the much larger normal adult fibers.
Neonatal MyHC is restricted to these tapered fiber
ends. Adult MyHC co-localizes with the neonatal
MyHC, and all fibers contain the adult MyHC throughout their entire length. Observations indicate that tapered fiber ends containing a neonatal MyHC have also
been identified in the pectoralis muscle of mature pigeons and of ducks (Rosser, unpub. data) and, interestingly, at the ends of fibers at myotendinous junctions
within the hind limb muscles of mature mice (Condon,
pers. comm.).
The forces generated by muscle contraction are
transmitted from the ends of muscle fibers to the collagenous tissue of tendon, periosteum and/or other
muscle fibers (Trotter, 1993). A variety of structural
proteins localized at the ends serve this purpose (Baker
et al., 1994; Tidball, 1991, 1992, 1994). The normally
complex morphology of the myotendinous junction can
be greatly altered by myopathies (Desaki, 1992; Law
and Tidball, 1993, Law et al., 1994) and aging (Trotter
et al., 1987).
Longitudinal growth also occurs at the ends of skeletal muscle fibers through the addition of new sarcomeres to the tips of existing myofibrils (Williams and
Goldspink, 1971, 1973; Ziv et al., 1984). Chronic
stretching of muscle induces the synthesis of a different
MyHC at the muscle and/or muscle fiber end(s).
Williams and colleagues (1986) concluded that adult
slow MyHCs were added to the ends of stretched fast
muscle fibers. However, the ATPase techniques that
these researchers then employed cannot readily differentiate embryonic or neonatal from the adult fiber
types (see Hurov et al., 1992). Nevertheless, more recent molecular work from their laboratory has strongly
suggested that a slow MyHC might indeed be synthesized at the ends of fast muscle fibers that had been
chronically stretched and/or electrically stimulated
(Goldspink at al., 1991), although stimulated fast muscle often converts to slow (Pette and Vrbova, 1992) and
the possibility of isoforms representative of earlier development stages was not addressed. It has been demonstrated that chronically stretched muscles lengthen
by the addition of nascent fibers derived from satellite
cells to the ends of existing fibers, and that these nascent fibers repeat the MyHC transitions observed during the development of the adult fibers (Kennedy et al.,
1988, 1989; Dix and Eisenberg, 1990); slow MyHC isoforms characteristic of early development were present
in nascent fibers at the ends of chicken slow and rabbit
fast muscle. These models of chronic stretch, however,
may be more representative of muscle fiber regeneration than postnatal growth as recent experiments using intermittent stretch have shown that hyperplasia
augments hypertrophy only under the more severe and
unnatural regimens of chronic stretch (Antonio and
Gonyea, 1993a,b).
Intrinsic or genetic factors are responsible for the
initial appearance of MyHCs within an embryonic
muscle fiber (Fredette and Landmesser, 1991; Condon
et al., 1992). However, innervation is essential to promote and maintain subsequent MyHC transformations
that can occur within a muscle fiber as it matures from
embryonic to neonatal to adult stages (Crow and Stockdale, 1986; Miller and Stockdale, 1986; Cerny and
Bandman, 1987; Narusawa et al., 1987), and type of
motoneuron input can be directly correlated with
MyHC isoform (Pette and Vrbova, 1985; Salviata et al.,
1986; Navarrete and Vrbova, 1993). During development, each embryonic muscle fiber initially receives
input from many motoneurons. This polyneuronal innervation gradually disappears, so that by the early
MYOSIN EXPRESSION WITHIN MUSCLE FIBER ENDS
Fig. 5. Transverse section of chicken pectoralis muscle. Immunofluorescent microscopy demonstrating affinity of the 2E9 antibody to the
neonatal myosin heavy chain. Labelled are two small fibers, one indicated by an arrowhead and the other by a small arrow. Bar = 20
Pm.
Fig. 6. Transverse serial section cut 24 pm from that in Figure 5, the
endomysium stained by the ammoniacal silver histological technique
and viewed by light microscopy. Same two small fibers indicated in
Figure 5 are shown. Bar = 20 pm.
Figs. 7 and 8. Transverse serial sections cut, respectively, 264 and
448 pm from that in Figure 5. In each, arrowhead indicates same fiber
as in Figures 5 and 6. Small fiber indicated by small arrow in previous
figures is absent. Staining and magnification as in Figure 6.
neonatal period each twitch fiber has only one motor
endplate and receives input from just one motoneuron
(Bennett and Pettigrew, 1974; Brown et al., 1976; Atsumi, 1977; Hesselmans et al., 1993).
Polyneuronal innervation and/or neurotransmitters
467
Fig. 9. Transverse serial section cut 648 pm from that in Figure 5.
Antibody 2E9 and magnification as in Figure 5. Same fiber indicated
by arrowhead in Figures 5-8 is now larger in diameter and not labelled by the 2E9 antibody against the neonatal myosin heavy chain.
Figs. 10-1 3. Transverse serial sections cut, respectively, 664, 856,
1,032, and 1,280 pm from that in Figure 5. In each, the arrowhead
indicates the same fiber as in Figures 5-9. This fiber continues to
increase in diameter in Figures 10-13. Staining and magnification as
in Figure 6. As demonstrated by these transverse serial sections (Figs.
5-13), the small fiber designated by the arrowhead in Figure 5 is in
reality the tapered end of the much larger fiber shown in Figure 13.
Only the tapered end of this fiber contains the neonatal myosin heavy
chain (Figs. 5 and 9).
may persist at the ends of some muscle fibers. Multiple
motor endplates are normally located a t the ends of
those fibers that possess the capacity for rapid regeneration in the tail muscles of urodele amphibians
(Thouveny et al., 1991). In fast-twitch fibers of the
468
B.W.C. ROSSER ET AL.
termined functions (Massoulie et al., 1993). Butylcholinesterase (BChE) is found in a variety of tissues
throughout the body, and although its physiologic function(s) remains obscure, its presence indicates nonspecific cholinesterase activity (Massoulie et al., 1993).
The lack of BChE activity in our sections indicates that
there was no (or very little) nonspecific cholinesterase
activity at the ends of the fibers. Eserine, which inhibits acetylcholinesterase activity a t motor endplates
(Stryer, 1988), inhibited all staining for AChE activity
in our sections. This inhibition also suggests that the
function of the AChE that we located a t the fiber ends
was to hydrolyze acetylcholine.
The neonatal MyHC that we observe a t the tapered
ends of fibers in adult chicken pectoralis might be related to a motoneuron input or sarcolemmal depolarization different from that found along the lengths of
these fibers. Accessory nerve terminalb) or neurotransmitter located at the ends of a fiber (as discussed
above) might be capable of affecting MyHC expression
a t the ends by either interfering with action potentials
generated by the main motor endplate or by generating
local depolarizations of the sarcolemma. Such a n effect
on fiber phenotype would undoubtedly extend some distance beyond the actual restricted location of the accessory endplatek) or neurotransmitter on the fiber.
Conversely, voltage dependent sodium (Na) channels
are also essential for sarcolemmal depolarization and
muscle fiber contraction (Brazil and Fontana, 1993;
Kallen et al., 1994). An extremely low density of Na
channels has been found a t the ends of muscle fibers in
the r a t and snake, and it has been suggested that sarcolemmal depolarization is likely to be compromised at
the ends of these fibers (Caldwell and Milton, 1988).
Experimental models of growth and regeneration inFig. 14. Transverse section of chicken pectoralis muscle. Immunofluorescent microscopy demonstrating a f k i t y of the 2E9 antibody to
dicate that muscle fibers must recapitulate their develthe neonatal myosin heavy chain. A labelled tapered fiber end is
opmental program to some extent before new areas of
indicated by the arrowhead. Bar = 20 pm.
adult myosin can be formed (Cerny and Bandman,
1987; Stewart et al., 1989; Dix and Eisenberg, 1990).
Fig. 15. Same section as shown in Figure 14, here stained for acetylcholinesterase (AChE) activity and viewed by light microscopy. Our findings suggest that the tapered ends of normal
Same fiber end indicated by arrowhead in Figure 14 has AChE activ- adult fibers remain in a ready state for longitudinal
ity on its surface. Bar = 20 pm.
growth, regeneration, and repair. We extrapolate from
our results to postulate that the longitudinal growth of
myofibrils in adult muscle is characterized by the sechicken, in addition to the single, large, motor endplate quential expression of MyHC isoforms similar to that
typically seen near the center of each fiber, small mul- observed in rapidly growing muscle.
tiple motor endplates were observed 30-40 pm from
ACKNOWLEDGMENTS
the musculotendinous junction (Shear, 1981). The ultrastructure of these small endplates was the same as
Chickens for this study were obtained from the Dethat normally exhibited by active motor endplates, and partment of Animal and Poultry Science, University of
postsynaptic acetylcholinesterase (AChE) activity was Saskatchewan, through the courtesy of Dr. H.L.
also demonstrated at the ends of these muscle fibers Classen and Mr. Robert Gonda. We also express our
(Shear, 1981). AChE activity has been demonstrated at appreciation to Mr. Victor Loewen of Waldheim,
the ends of muscle fibers near the musculotendinous Saskatchewan, and Mr. Wes Freisen of Gruenthal,
junctions of a variety of vertebrate species (Nishikawa, Saskatchewan, for their generosity in supplying chick1981; Sketelj et al., 1991; Trotter, 19931, and acetylcho- ens on which our preliminary observations were made.
line receptors have also been located a t the ends of Access to the DN-4000 graphics workstation was
fast-twitch fibers near musculotendinous junctions in kindly granted by Dr. S. Lozanoff of the Department
frog (Miledi et al., 1984). In the chicken pectoralis, we of Anatomy and Cell Biology, University of Saskatchlocalize AChE activity at the tapered ends of some of ewan. Dr. K. Condon of the Department of Cell Biology
the fibers containing the neonatal MyHC. AChE is and Neuroscience, University of Texas, was kind
found in muscles and the nervous system, and its pri- enough to permit us to share his unpublished observamary role is to terminate the action of the neuromus- tions on muscle fiber ends in hind limb muscles of mice.
cular transmitter acetylcholine a t the neuromuscular We also thank Dr. B.H.J. Juurlink of the Department
junction, although it may have additional still unde- of Anatomy and Cell Biology, University of Saskatch-
MYOSIN EXPRESSION WITHIN MUSCLE FIBER ENDS
ewan, for his many helpful suggestions and stimulating discussions. This study was supported by an operating grant awarded to B.W.C.R. by the Saskatchewan
Health Research Board and by funds from the Scientific Teaching and Research Fund of the College of
Medicine, University of Saskatchewan.
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