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Morphological histochemical and myosin isoform analysis of the diaphragm of adult horses Equus caballus.

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THE ANATOMICAL RECORD 238:317-325 (1994)
Morphological, Histochemical, and Myosin lsoform Analysis of the
Diaphragm of Adult Horses, Equus caballus
Department of Anatomy, College of Veterinary medicine, Cornell University,
Zthaca, New York
The horse provides an interesting model for study of the
structure and function of the mammalian diaphragm. Multiple regions of
diaphragm from seven adult horses were prepared for histochemistry, immunocytochemistry, myosin heavy chain electrophoresis, and native myosin electrophoresis. Two additional adults were dissected to demonstrate
myofiber and central tendon morphology and stained for acetylcholinesterase to demonstrate motor endplates. All regions of the adult diaphragm
were histochemically characterized by a preponderance of type I fibers
with some type IIa fibers. Type IIb fibers were absent in all adult specimens. Myosin heavy chain electrophoresis supported the histochemical
study: two isoform bands were present on SDS gels that comigrated at the
same rate as rat type I and IIa myosin heavy chain isoforms. No isoform
was determined to comigrate with rat type IIb heavy chain isoforms. Native
myosin isoform analysis revealed two isoforms that comigrated with rat
FM-4 and FM-3 (FM = fast myosin) and two isoforms that comigrated with
rat SM-1 and SM-2 (SM= slow myosin) isoforms. In some samples, a third
slow native myosin isoform was observed that comigrated at the same rate
as the SM-3 of the equine biceps brachii muscle. This doublet (or “triplet”)
of slow isoforms is unique to some horse muscles compared with other
adult animals studied. It is not known if these multiple slow native myosin
isoforms confer some functional advantage to the equine muscles. The
adult equine diaphragm also differs in its morphology by having a large
central tendon compared to that in other mammals, and is predominantly
slow in fiber type and myosin isoform composition. o 1994 Wiley-Liss, Inc.
Key words: Horse, Myosin, Muscle, Fiber type
As one of the primary respiratory muscles, the diaphragm is required to maintain contractions to facilitate ventilation for the life of the animal. It also must
increase the rate and strength of these contractions in
response to stress and increased oxygen demand. The
diaphragm has been studied intensively, but primarily
in such small animals such as rat, cat, and dog
(LaFramboise et al., 1991; Gordon et al., 1989; Green et
al., 1989; Farkas and Rochester, 1988; LeSouef, et al.,
1988; Green et al., 1984; Nystrom, 1968; Gauthier and
Padykula, 1966; Sieck e t al., 1983). In light of the fact
that horses are a s much as tenfold larger than a dog,
and have markedly different abdominal anatomy (in
terms of size and position of the abdominal viscera)
which may affect diaphragmatic function, the equine
diaphragm presents challenges to the traditional concept of diaphragm morphology. Therefore, in this
study, we describe the diaphragm in adult horses and
test a general hypothesis that the large body size
should result in a proportionately large population of
slow-twitch and fatigue resistant fibers, thus differing
from the diaphragms of small to medium sized mammals. We based this hypothesis on several reports that
have noted a trend toward a higher percentage of slow
twitch fibers with increasing body size and decreasing
respiratory rate (Gauthier and Padykula, 1966; Green
et al., 1984), although this relationship is not linear
(Farkas and Rochester, 1988). Recently, Rome et al.
(the maximum speed of
(1990) confirmed that V,,
shortening of muscle fibers) of horse soleus muscle fifor muscle fibers from
bers was lower than V,,
smaller mammals (rats and rabbits). In addition, these
in equine soleus fibers
authors demonstrated that V,,
had a wide range that correlated with differences in
myosin heavy chains and light chains associated with
three identified fiber types (I, IIa, and IIb).
We incorporated several techniques in our study of
the adult diaphragm. Gross anatomical studies were
used to determine the sampling sites useful in a histochemical study. The results support the hypothesis
Received December 29, 1992; accepted September 28, 1993.
Address reprint requests to John W. Hermanson, Department of
Anatomy, College of Veterinary Medicine, Cornell University, Ithaca,
NY 14853-6401.
TABLE 1. Adult horses used in this studs
Age (yr)
Weight (kg)
'Heavy chain electrophoresis.
3Native myosin electrophoresis.
4Hiatal region not sampled.
that histochemistry provides a useful indicator of muscle function. However, because the application of standard histochemical techniques to non-traditional species can provide confusing results (Pette and Staron,
1990), our interpretations are supported with electrophoretic separation of myosin heavy chain and native
myosin isoforms. Myosin heavy chain isoforms correlate well with functional parameters such as speed of
shortening (Reiser et al., 1985). Multiple myosin heavy
chain isoforms have been described in the diaphragm of
several species such as the laboratory rat (LaFramboise et al., 1991; Termin et al., 1989) and include a
fiber type composed of a novel myosin heavy chain isoform now labelled IIx (originally called "IId" by the
Termin group). The type IIx myosin heavy chain isoform has been shown to have a n intermediate speed of
shortening compared to type I (slow) and type IIb muscle fibers (Schiaffino et al., 1989) and is believed to be
associated with repetitively recruited muscles.
Morphological studies were performed on fresh muscle specimens obtained from adult horses (Equus caballus) in the postmortem room of the New York State
College of Veterinary Medicine (Table 1).Two entire
diaphragms were removed from adult horses within 30
minutes of death by barbiturate euthanasia. We used a
measuring tape to determine muscle fiber bundle
lengths in situ, before dissecting the muscle free. One
of these diaphragms was immediately fixed to a rigid
surface so that it could be studied further at its in situ
length. This specimen was photographed so that surface area measurements could be made. Both specimens were then stained for acetylcholinesterase by the
technique of Ypey (1978) to demonstrate the motor
endplate distribution patterns within the muscles.
Samples for histochemical and electrophoretic analysis were taken from the right costal diaphragm a t the
approximate level of the costochondral junction of the
fifteenth rib, the right crus of the diaphragm near its
origin on the transverse processes of the lumbar vertebrae, and from the left crus of the diaphragm immediately dorsal to the esophageal hiatus. These regions
will be referred to as costal, crural, and hiatal, respectively, in this study. Costal and crural samples were
obtained from seven animals, and hiatal samples were
obtained from five of these animals.
Histochemistry was performed following the protocol
of Hermanson and Hurley (1990). Samples for histochemistry were taken as 1 to 2 cm3 blocks of tissue
and mounted on cork blocks using 5% gum tragacanth.
These samples were subsequently frozen in isopentane
cooled in liquid nitrogen at -160"C, sealed in plastic
bags and stored a t -80°C. Transverse serial sections
(10 Fm thickness) were obtained on a cryostat microtome at -20°C and mounted on glass slides for staining. Sections for each muscle were stained for myofibrillar ATPase following preincubation at pH 10.3 for
10 minutes a t 37°C in a NaCl glycine buffer solution or
pH ranging from 4.35 to 4.55 in increments of 0.05 for
5 minutes at room temperature in a 0.2M barbital acetate solution. Initial experiments used a wider range
of acidic pH from 4.2 to 4.6. Additional sections of each
sample were stained with alpha-glycerophosphate dehydrogenase (GPD) to determine glycolytic potential
(modified from Wattenberg and Leong, 1960) or with
nicotinamide adenine dinucleotide tetrazolium reductase (NADH) to determine oxidative capacity (Novikoff
et al., 1961). The GPD samples were incubated in 0.2M
phosphate buffer containing 24 mM GPD, 1.2 mM nitro
blue tetrazolium, and 2.3 mM menadione for 1hour at
37"C, following the protocol of Hermanson and Hurley
(1990) but with the increased GPD concentration and
longer incubation. Fibers were classified type I or type
IIa according to the method of Brooke and Kaiser
To verify the histochemical results, additional sections of each sample were stained with antibodies to
fast and slow myosin. Samples were mounted on glass
slides and stored at -20°C. The samples were allowed
to warm to room temperature for 10 minutes and then
preincubated in a 2% solution of normal goat serum in
a 0.1 M phosphate buffered saline (PBS) for 30 minutes
in a humid chamber at 4°C. Following preincubation,
excess goat serum was shaken off the slide and a n antifast myosin or a n anti-slow myosin primary antibody
was applied and allowed to incubate overnight in a
humid chamber at 4°C. Fast myosin reacted with
Sigma MY-32 (Sigma Chemical, ST. Louis, MO) and
slow myosin reacted with S-58 (provided by Dr Frank
Stockdale, Stanford University Medical School). The
following day, slides were dipped in 0.05 M PBS with
0.085% NaCl and then rinsed in the same solution for
10 minutes. Samples were then reacted with a biotinylated rabbit anti-mouse secondary antibody for 10 minutes, a streptavidin peroxidase enzyme conjugate for 5
minutes, and a n AEC substrate chromagen solution for
15 minutes in the dark. Between each of these steps the
samples were rinsed for 10 minutes in the PBS/NaCl
solution. After staining, the slides were soaked for 3
minutes in distilled water and coverslipped using a
glycerol-vinyl alcohol mounting medium.
Black and white photographs (magnifications were
100 x ) obtained using a n Olympus BH-2 photomicroscope were used to determine fiber type percentages
and fiber diameters. For each sample, photographs
were taken from identical regions of each of the histochemically and immunocytochemically stained samples described earlier for fiber type determination.
Seven to ten additional fields of the acid preincubated
ATPase stain showing the best contrast between fiber
types, typically pH 4.45, were taken for fiber type
counts and diameter measurements. For each sample
at least five microscopic fields were counted to provide
a minimum of 1,000 fibers to determine fiber type percentages. Thirty-six fibers of each type were measured
for least diameter (to the nearest 0.05 mm) using dial
calipers. At least five microscopic fields per tissue were
sampled in this measurement process. Fiber type diameters and percentages were compared using a n
ANOVA followed by a Tukey's comparison test of multiple means (Box et al., 1978). Significance was correlated with P 5 0.05.
Myosin heavy chain electrophoresis was performed
following the protocol of LaFramboise et al. (1990). Myosin was extracted from minced muscle samples (20100 mg) on ice for 30-40 minutes in four volumes of a
high salt buffer (Butler-Browne and Whalen, 1984) (pH
6.5). Extracts were then centrifuged at 13,000 rpm for
30 minutes a t 2°C in a Hill Scientific mv 13 microfuge.
The supernatants were then utilized for both native
myosin and heavy chain electrophoresis. Supernatant
for heavy chain electrophoresis was diluted in 9 volumes of buffer (37% [w/v] EDTA and 0.01% [v/v] 2-mercaptoethanol), vortexed and allowed to precipitate
overnight a t 4°C. Centrifugation was repeated and the
resulting pellet dissolved in 0.5 M sodium chloride and
10 mM NaPO, and then denatured by immersion in
boiling water for 2 minutes. Samples were then diluted
1:lOO in a gel sample buffer (62.5 mM Tris/HCl, 2%
[w/v] SDS [sodium dodecyl sulfate], 10% [v/v] glycerol,
and 0.001% [w/v] bromophenol blue; pH 6.8). Electrophoresis was performed in a 4.8% separating gel with
30% (v/v) glycerol and a 3% stacking gel with no glycerol. Ten to 15 p,l aliquots of diluted myosin were electrophoresed for 22 hours a t 120 V and 14°C. Separating
gels were silver stained according to the protocol of
Oakley et al. (1980). The stained gels were photographed on a light table.
Native myosin extraction and electrophoresis was
performed as described by Butler-Browne and Whalen
(1984) and LaFramboise et al. (1990). Native myosin
was obtained in the same manner a s for heavy chain
electrophoresis. However, following the second centrifugation, a fraction of the supernates was recovered and
diluted in 99 volumes of buffer (40 mM sodium pyrophosphate, 50% glycerol, 0.1% [ d v ] 2-mercaptoethanol,
pH 8.5 at 4°C). Gels (6 cm length x 0.5cm diameter)
were cast in glass tubes 1 day prior to electrophoresis
and refrigerated overnight (final gel concentrations
3.88% [w/v] acrylamide, 0.12% [w/v] bisacrylamide, 20
mM sodium pyrophosphate, and 10% [v/v] glycerol; pH
8.6 at 4°C). Electrophoresis was carried out on volumes
of extract (10-15 p,l) in running buffer (20 mM sodium
pyrophosphate, 2 mM magnesium chloride, 10% [v/vl
glycerol, and 0.01% [v/v] 2-mercaptoethanol; pH 8.65 a t
4°C) using a Model 214 Pharmacia vertical gel apparatus. Electrophoresis was performed in a refrigerated
room (2"C), at 100 V and 0°C for 18-20 hours. Gels
were stained in 0.025% (wiv) Coomassie blue, 7% (v/v)
acetic acid, and 2% (v/v) ethanol and destained in 5%
(v/v) methanol and 9% (v/v) acetic acid before storage
in 7.5% ( v h ) acetic acid.
Morphological Studies
The costal part of the diaphragm attaches to the ribs
and costal cartilages on a line passing from the xiphoid
process of the sternum, across the costochondral junction of the tenth rib to a point about midshaft on the
eighteenth rib. It then turns cranially, reaching the
seventeenth rib at the caudal border of its vertebral
attachment. There is a thick layer of connective tissue
covering the thoracic surface of the entire diaphragm.
Virtually all of the muscular part of the costal diaphragm lies directly against the body wall with its fibers running roughly parallel with the craniocaudal
axis of the body (Fig. 1). The two crura of the diaphragm attach to the bodies and transverse processes
of the first two lumbar vertebrae and are attached to
the costal diaphragm by a large central tendon (Fig. 2).
The surface area of this central tendon, measured from
one preparation, was 34% of the entire surface area of
the diaphragm when stretched flat to its in situ length.
Acetylcholinesterase staining of two diaphragms revealed 6 to 9 staggered bands of endplates from costal
to tendinous insertions in the costal diaphragm. These
bands were separated by intervals of about 20 mm.
Both crura of the diaphragm showed three bands of
endplates evenly spaced from vertebral to tendinous
insertions and separated by intervals of about 16 mm.
Histochemical Studies
The adult diaphragms contained type I and IIa fiber
types with type I predominating (Figs. 3,4). Costal and
crural areas were similar, containing 66 and 71% type
I fibers, respectively (N = 7). The hiatal region contained 84% type I fibers (N = 5), significantly more
type I fibers than either costal or crural regions (P <
0.01). The type I fibers were larger than the type IIa
fibers in all three regions (P < .05) and size of both
fiber types was constant across all three regions sampled (Table 2).
Myosin lsoform Electrophoresis
All regions of the adult diaphragm demonstrated two
isoforms subsequent to heavy chain electrophoresis
(Fig. 5) which migrated at the same rate as the type I
and IIa isoforms in rat costal diaphragm described by
LaFramboise et al. (1991). Native myosin electrophoresis of the adult costal, crural, and hiatal regions
showed two or three slow myosin and two or three fast
native myosin isoforms (Fig. 6). The slow myosin isoforms resolved correlated with the SM-1, SM-2, and
SM-3 isoforms of equine biceps brachii (Hermanson et
al., 1991). When only two slow isoforms were present,
they were the SM-1 and SM-2 isoforms. The SM-3 isoform was present in one costal, three crural, and three
hiatal regions in a total of three horses (animals 6, 8,
and 9 in Table 1). The fast isoforms correlated with
FM-4 and FM-3 and in six cases FM-2 of the rat diaphragm and horse biceps brachii. The FM-2 isoform
was present in 4 costal diaphragms (animals 2, 3, 4,
and 5) and three crural samples (animals 2, 3, and 4).
The equine diaphragm demonstrates two interesting
morphological characteristics: multiple, irregularly arranged motor endplate bands are particularly evident
in the costal region, and a large proportion of surface
area is comprised of the central tendon. The endplate
banding pattern is similar to the dog and cat as described by Gordon et al. (1989). This pattern suggests
Fig. 1. Lateral view of the thoracic and abdominal regions of a horse
showing the position of the diaphragm. Craniad is to the left. Muscle
fiber orientation is apparent after stripping off a thick sheet of overlying connective tissue (one block was retained to show the differing
orientation of the connective tissue fibers relative to the underlying
myofibers). The crural portion of the diaphragm is obscured in this
multiple fibers in series between the costal and tendinous insertions of the muscle. Gordon et al. (1989) described a second endplate banding pattern in the rat
and rabbit in which a single continuous endplate band
was seen about midway between the costal and tendinous insertions. The horse diaphragm had a higher
proportion of its surface area made up of central tendon
than the rat, cat, dog, and rabbit described by Gordon
et al. (1989). Interestingly, the rabbit most closely resembled the horse in this characteristic (27% of diaphragm surface area in the rabbit compared to 34% in
the horse.) The cat, by comparison had a much smaller
central tendon (10%). This suggests that size of the
central tendon is not simply a function of the size of the
animal. This large central tendon and the orientation
of the costal diaphragmatic muscle mass along the
craniocaudal axis and directly apposed to the body wall
suggest that at least this part of the diaphragm acts to
pull the central tendinous part directly caudally. This
is somewhat different than the classic view of the diaphragm as a dome which is flattened during contraction. In addition, the connective tissue sheet overlying
the thoracic surface of the diaphragm is important by
imparting significant elastic recoil to the diaphragm
(Griffiths et al., 1992).
Both fiber type staining and myosin heavy chain
electrophoresis of the equine diaphragm revealed a
simple two fiber type design which included predominantly type I fibers with fatigue-resistant and oxidative histochemical characteristics. Our histochemical
study showed a higher proportion of type I presumed
slow fibers than did Zobunzija et al. (1989). However,
these authors based their fiber typing on staining intensity for GPD rather than myosin ATPase, and had
5-10% unclassified fibers in each region sampled. In
our study, type IIA fibers stained more darkly than
type I fibers for GPD, but the resolution was not always
good. It was interesting that the horses had no type IIB
fibers in the diaphragm despite the presence of these
fibers in other equine muscles (Ryan et al., 1992).
Other animals which demonstrate type IIB fibers in
limb muscles have also demonstrated them in the diaphragm (Reid et al. 1987; Gordon et al., 1989; Green et
al., 1984) The type I predominance of the equine dia-
Muscle shown
Motor endplates shown only
CVC Caudal vena cava and
its tributaries
PVC Plica vena cava
Sample sites:
Costal sample
Crural sample
Hiatal sample
Fig. 2. Cranial view of the thoracic surface of an adult horse diaphragm positioned flat with muscle fibers at their in situ length. Two
insets on the muscle show the motor endplate distributions in costal
and crural regions of the diaphragm. Sample sites are highlighted in
the costal, crural, and hiatal region (the latter region transmits the
esophagus through the diaphragm).
Fig. 3.Two histochemical fiber types predominate in the adult diaphragm of the horse. These serial sections were obtained from the
costal diaphragm. Fibers were stained for myosin ATPase after preincubation at pH 4.4 (A) or pH 10.3 (B). Note reversal of staining
properties for type I and I1 fibers in the two pH conditions. Metabolic
properties such as high oxidative potential or glycolytic potential
were demonstrated by dark stains following reaction with NADH (C)
and GPD (D) protocols. Correlation with the ATPase reactions was
provided by antibodies reacting darkly in the presence of slow myosin
heavy chains (E) or fast myosins (F).Scale bar equals 100 km.
Fig. 4. Following acidic preincubation (pH 4.4), sections stained for
myosin ATPase showed the distribution of type I fibers (darkly
stained) for the costal (A), crural (B), and hiatal (C) regions of the
adult diaphragm of horses. Scale bar = 100 pm.
phragm is consistent with the hypothesized requirements of fatigue resistance and continual activity
required for respiration in any mammal and is particularly important for a n endurance athlete such as the
horse (Peters, 1989). In addition, the horse has relatively more type I fibers than did the smaller mammals
(rat: Green et al., 1989; cats: Sieck et al., 1983; dogs:
Reid et al., 1987).
The hiatal region contained a significantly higher
proportion of type I fibers than either the costal or surrounding crural regions in all adult horses studied.
This specialization is consistent with t h a t reported for
the cat (Gordon et al., 1989) and dog (Reid e t al., 1987).
Reid et al. have proposed that this region may provide
a sphincter function for the esophagus. Vomiting is
rare in the adult horse, and when seen, is related to
very high gastric pressures. Often, vomiting is associated with the terminal stages of gastric dilatation
(Smith, 1989). The histochemical features of the hiatal
region of the diaphragm suggest i t may contribute to
this very strong sphincter function and inhibit emesis.
Native myosin electrophoresis of all three regions of
the adult diaphragm showed between one and three
slow myosin isoforms (SM) which migrated at the same
rates as SM-1, SM-2, and SM-3 isoforms of equine biceps brachii muscle (Hermanson e t al., 1991). Identification of this slow triplet isoform pattern has been restricted thus far to the horse where it was observed
consistently in the primarily slow lateral head of the
biceps brachii but not in the faster medial head. In the
equine diaphragm, only the SM-1 isoform was present
in all regions of all animals. The seemingly inconsistent expression of the slow triplet isoform pattern
raises anew a number of interesting questions concerning the origin, expression, transition, and significance
of native myosin isoforms. Hermanson et al. (1991)
suggested that the slow myosin triplet represented the
retention of fetal or neonatal isoforms in the adult. In
consideration of this, a companion manuscript considers the morphology and ontogeny of the diaphragm in
foals (Cobb et al., 1994). The question remains, however, why is the slow triplet seen in the diaphragm of
some adults, and in some regions of individual diaphragms, while i t does not appear in others? The most
plausible explanation is that the SM-2 and especially
the SM-3 isoforms are present at low concentrations in
the equine diaphragm and are, therefore, a t the limit of
resolution for this technique. Another possibility is
that retention of neonatal or fetal isoforms is subject to
individual variation. A combination of these two explanations may be involved.
Bramble and Carrier (1983) showed that respiration
and locomotion were directly coupled in a number of
species, including the horse. Later cineradiographic
and EMG work by Bramble (1989) led to the proposal of
the existence of a “visceral piston” effect in which the
oscillations of the abdominal viscera, most importantly
the liver, caused by the movement of the animal’s body,
played a key role in increasing and decreasing the volume of the thoracic cavity. Movement of the liver cranially upon placement of the forefeet causes cranial
displacement of the diaphragm and effects expiration.
Caudal movement of the liver and diaphragm increase
thoracic volume and effect inspiration. In this model of
respiration during locomotion, the diaphragm would
act to check the forward motion of this piston rather
than the classic view of the diaphragm actively contracting to increase the volume of the thoracic cavity.
The horse differs in several important ways from the
dog which was the animal studied in Bramble’s cineradiographic work. The liver in the horse is a much
TABLE 2. Fiber types and fiber diameters (pm) in adult horses*
Horse No.
% type I
Dia. type I
Dia. type IIa
% type I
Dia. type I
Dia. type IIa
% type I
Dia. type I
Dia. tvDe IIa
Mean (S.E.)
66 (3)
53 (1)
46 (1)
71 (4)
56 (1)
46 (2)
84 (1)
56 (3)
45 (1)
*All type IIa mean diameters were significantly smaller (P 5 0.05) than comparable
mean diameters for type I fibers within each of the three regions.
Fig. 5. 6% SDS-PAGE of myosin heavy chains (MHCs) from the
costal diaphragm of a rat, and the crural, costal and hiatal regions of
an adult horse. The four MHCs labelled in the rat sample are shown
for comparison with the horse samples. Only two MHC isoforms, comigrating with rat type I and IIa MHCs, are present in the adult horse
Fig. 6 . A comparison of 4 6 PAGE of native myosin isoforms for the
costal diaphragm (COS) of a n adult rat, adult horse (10years old);
crural diaphragm (CRUR) and hiatal diaphragm of adult horse (7 and
6 years old, respectively). While rat diaphragm exhibits only one slow
native myosin isoform, the adult horse samples exhibit a t least two
slow native myosin isoforms.
smaller portion of the abdominal mass, and has a relatively smaller area of contact with the diaphragm.
The largest part of the abdominal cavity in horses is
taken up by the cecum and large (ascending) colon.
These structures constitute a massive, freely moveable
mass (restrained only by their mesenteric attachments
to the dorsal body wall) which is in direct contact with
a large portion of the diaphragm. In the dog, the crural
diaphragm was the main modulator of the “visceral
piston.” The morphology of the horse diaphragm, in
which the fibers of the costal diaphragm are arranged
so as to pull the abdominal viscera caudally in a “sling”
formed by the diaphragmatic central tendon, places the
costal diaphragm in an excellent position to perform
that function' In addition, the predominance Of type I
fibers in the diaphragm would provide a largely fatigue
resistant muscular control over these visceral movements. Arguments against the visceral piston model
were recently presented by younget al. (1992), underlining the importance of understanding visceral movements during locomotion and the need for further
conc~usion,this study provides data highlighting
the m o q h o 1 0 0 7
histochemical fiber
myosin isoform composition, and presumed fatigue resistance of adult diaphragms. In contrast to other studies of mammalian diaphragms, the horse has a relatively higher proportion of fatigue resistant type I
fibers. As such, these data support a n hypothesis that
type 1 fibers are prominent in large-bodied mammals.
Based on the observations of Rome et al. (1990) that
show a tight range of V,, for type 1 fibers in another
this high number Of
I fibers is
suited to continuously effect the inspiratory component
of ventilation throughout life. Perhaps the coupling of
ventilation and ~ocomotion,as suggested by the visceral piston model, reduces the need for including a
large Percentage of fast twitch fatigable fibers in the
composition of the diaphragm.
This study was supported by the H.M. Zweig Memorial Fund and benefitted from an NIAID training
grant. Drs. Dorothy Ainsworth, William LaFramboise,
Ron Meyers, and James Ryan read and commented on
draft Of the manuscript' The manuscript was
improved by the comments of two anonymous reviewers. Michael Simmons prepared the figures. Marketa
Aschenbrenner provided excellent technical assistance
for several phases of the study. The assistance of ~
Daaod and Jacqueline Petrie was invaluable. Ruthie
Loeffler patiently worked on word processing of the
manuscript. Finally, this work could not have been
completed without the encouragement
and assistance
and pathologists at the New
College of Veterinary Medicine.
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adults, myosin, equus, caballus, diaphragm, isoforms, analysis, horse, morphological, histochemical
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