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THE ANATOMICAL RECORD 2461195-207 (1996)
Correlation Between Myofibrillar ATPase Activity and Myosin
Heavy Chain Composition in Equine Skeletal Muscle and the
Influence of Training
JOSlbLUIS L. RIVERO, ROBERT J. TALMADGE, AND V. REGGIE EDGERTON
Department of Comparative Anatomy and Pathological Anatomy, Faculty of Veterinary
Science, University of Cordoba, Spain (J.-L.L.R.); and Department of Physiological Science
and Brain Research Institute (R.J.T., V.RX.1, University of California,
Los Angeles, California
ABSTRACT
Background: The histochemical myofibrillar ATPase
(mATPase) method is used routinely for identification of equine skeletal
muscle fiber types, but important problems have been observed with the
subdivision of fast fiber population when using this method. To verify the
use of this qualitative method, a number of equine muscle biopsies were
analyzed with a combination of histochemical, immunohistochemical, electrophoretic, and morphometric techniques. The influence of training on
these interrelations was also evaluated.
Methods: Five young (2-3 years old) thoroughbred horses were intensively trained for 8 months on a high-speed treadmill. Biopsies were taken
from the gluteus medius muscle at the beginning, after 4 months, and at the
end of the training program. Serial sections of the samples were stained by
mATPase histochemistry and immunohistochemistry by using a number of
monoclonal antibodies specific to selected myosin heavy chain (MyHC)isoforms. The histochemical and immunohistochemical categorization of a
large number of fibers (N = 2,078) was compared fiber by fiber. The MyHC
content of homogenates of the same biopsies were quantified by densitometry of a sensitive gel electrophoretic technique and compared with histochemical and immunohistochemical fiber types.
Results: A large proportion of fibers examined (-20%) were misclassified
by traditional mATPase histochemistry. Many fibers histochemically identified as type IIB displayed both type IIa and type IIb MyHC isoforms, and
nearly all type IIAB fibers in mATPase contained only the type IIa MyHC
isoform by immunohistochemistry. Correlation analyses suggested a weak
relation between the histochemically assessed relative cross-sectional area
occupied by the three major fiber types (I, IIA, and IIB) and the electrophoretically assessed MyHC content, whereas a stronger relation was
found between immunohistochemically defined fiber types and electrophoretic data. The four fiber type populations delineated according to
MyHC content (1, IIA, IIAB, and IIB) had sizes and oxidative capacities
significantly different from each other. No adaptation of any parameter
measured to training was found. Training had no significant effect on the
number of fibers misclassified by mATPase histochemistry.
ConcZusions:These data demonstrate a significant limitation in mATPase
histochemistry for assessing fibers containing fast MyHC isoforms. The use
of monoclonal antibodies against specific MyHC isoforms seems to be a
more sensitive and less subjective method. Q 1% Wiley-Liss, Inc.
Key words: Horse, Guteus medius muscle, Histochemistry, Immunohistochemistry, Electrophoresis, Muscle fiber types
Over the past 2o years, enzyme-histochemica1 methhave provided much information about the plasticity of equine skeletal muscle in relation to growth,
training, and performance potential (for review, see
Ods
0
1996 WILEY-LISS, INC.
Received October 25, 1995; accepted February 27, 1996,
Address reprint requests to Jose-Luis L. Rivero, Ph.D., Department
of Veterinary Anatomy, Faculty of Veterinary Science, University of
Cordoba, Medina Azahara 9, 14005 Cordoba, Spain.
196
J.-L.L. RIVER0 ET AL.
Snow and Valberg, 1994). In horses, as in other mammals, muscle fibers have been routinely categorized
into three major types, designated I, IIA, and IIB, and
the minor IIC, based on the myofibrillar actomyosin
adenosine triphosphatase (mATPase) histochemical reaction proposed by Brooke and Kaiser (1970). In addition to these four fiber types, some fibers with staining
characteristics intermediate between types IIA and IIB
(type IIAB; Ingjer, 1979) have frequently been observed in horses (White and Snow, 1985; Rivero et al.,
1993). Although the use of mATPase methods t o identify slow (type I) and fast (type 11)contracting fibers is
highly reproducible, the further subdivision of type I1
fibers is controversial. The subjective evaluation, the
multiplicity of factors affecting reaction patterns, and,
mainly, molecular diversities of mammalian skeletal
muscle fibers have all been adduced in criticism of this
qualitative method (for review, see Pette and Staron,
1990).
Using immunohistochemical methods, the histochemical staining intensity of the mATPase reaction in
a given muscle fiber may be determined by its myosin
heavy chain (MyHC)content (Billeter et al., 1981;Pierobon-Bormioli et al., 1981).Moreover, now there is sufficient evidence that the MyHC protein expressed in a
single muscle fiber is correlated to its contractile properties (Reiser et al., 1985; Bottinelli et al., 1994a). To
date, four major MyHC isoforms have been identified
in adult skeletal muscle in a number of mammalian
species: the p-, slow, or type I MyHC and the three fast
IIa, IIx or IId, and IIb- MyHCs (Bar and Pette, 1988;
Schiaffino et al., 1989; Gorza, 1990; Aigner et al.,
1993).In contrast, in many other species including primates and carnivores, only two fast MyHCs have been
identified; whereas one of these two fast MyHCs has
been unequivocally identified as type IIa-MyHC, there
are serious doubts regarding the identity of the second
fast MyHC isoform. Because it is mainly present in
histochemical type IIB muscle fibers, it has been
termed type IIb-MyHC. However, two recent studies
using molecular biological techniques (Smerdu et al.,
1994; Ennion et al., 1995) have reported the identification of one human skeletal MyHC gene as fast IIx,
whose transcripts are more abundant in histochemical
type IIB fibers. Similarly, in a companion study, we
have identified one slow and two fast (IIa and IIx or IIb)
MyHC isoforms in the equine skeletal muscle by using
monoclonal antibodies and gel electrophoresis techniques. The differential distribution of these MyHCs
defines three main fiber types containing a single
MyHC isoform (I, IIA, and IIX or IIB) and a number of
intermediate hybrid fiber populations containing both
I and IIa MyHCs (type C fibers) and type IIa and IIx or
IIb MyHCs (type IIAX or IIAB fibers).
Although a direct correlation between the histochemical reactivity for mATPase and the MyHC content of a given fiber has been established in rabbit
single fibers (Staron and Pette, 19861, the disadvantages of the mATPase technique have recently been
highlighted in humans, where gel electrophoresis of
MyHC in single fibers was used in conjunction with
mATPase staining (Klitgaard et al., 1990a,b; Staron,
1991; Staron and Hikida, 1992; Andersen et al.,
1994a,b). These studies suggest that many fibers histochemically classified as type I or type IIB contain to
some degree the fast IIa MyHC, particularly in trained
muscles. Moreover, the coexpression of multiple MyHC
isoforms within a single fiber also occurs under normal
conditions (Biral et al., 1988).In summary, because the
dominant MyHC isoform determines the histochemical
mATPase reaction of a fiber (Danielli-Beto et al.,
1986), this method does not reveal subtle alterations in
the expression of MyHCs in muscle fibers and, therefore, may not adequately characterize muscle fiber distribution in control or in trained horses.
No previous studies in horses have made qualitative
and quantitative comparisons between myofiber distribution and MyHC content in equine skeletal muscle by
combining histochemical, immunohistochemical, and
electrophoretic techniques. This relation would lend
validity to the use of routine histochemical techniques
in distinguishing muscle fiber types in horse muscle.
The main purposes of the present investigation were
(1)t o ascertain the degree of association between the
MyHC content of muscle fibers and histochemical muscle fiber type distribution by combining classical qualitative mATPase histochemistry and immunohistochemical and electrophoretic analyses of MyHC; and
(2) to estimate the effect of training on the relations
among these variables. The study also provides quantitative information regarding fiber size and oxidative
capacity of the various muscle fiber types in horses.
MATERIAL AND METHODS
Horses
Six clinically healthy thoroughbred racehorses (five
mares and one gelding) were used in this study. Two
horses were 3 years old and the other four were 2 years
old a t the beginning of the study. They were all of
comparable size and body weight (range = 440-465
kg). All horses were kept under identical environmental conditions and fed the same diet throughout the
experiment. The horses were housed individually in
3-X-3-mboxes a t a temperature ranging from 5 to
25°C. All exercise workouts were made at a temperature ranging between 16 and 26°C. In general, the animals were fed daily with 4.5 kg of oats and 5 kg of
grass-mix hay, and water and minerals were provided
ad libitum. The days on which the horses were exercised, the horses consumed one more kilogram of oats.
Training Program
Five of the horses were included in a training program aimed at investigating the influence of intensity
and duration of exercise on several physiological variables. The sixth horse was used as a control to evaluate
changes associated with age. All the exercise workouts
and standardized tests were done on a high-speed
treadmill (Mustang 2200@, Kagra SA Fahrwangen,
Switzerland). The experimental period was extended
for about 8 months. After a 1-month acclimatization
period, all five trained horses carried out six phases of
exercise, varying in intensity and duration. Each phase
consisted of 11 exercise workouts (once a day every
second day), so each phase was extended for 21 days.
Between two consecutive phases, horses were allowed
to rest for 1 week. During this week, a standardized
exercise test (SET) was performed for each horse to
determine VLA2.5and VLA4or speeds that run over a
defined period of time and produce a concentration of
MYOSIN ATPase AND MYOSIN HEAVY CHAIN IN HORSES WITH TRAINING
lactate in blood of 2.5 and 4 mmol/l, respectively (Lindner et al., 1992). This test was the parameter on which
the intensity of exercise made in each phase of training
was based. Of the six phases of training, three were
made at an intensity of work of -VLA2.5and the other
three in VLAI.In both sets of phases, the duration of
each exercise session was 5, 15, and 25 min.
SET and Estimation of ,V
,,
and V
,,
The SET consisted of several gallop workouts of 5
min duration each. Between two consecutive gallops, a
resting period of 60 sec was allowed. The velocity in the
first step was 6 d s e c and was increased by 0.5 d s e c in
each consecutive gallop. The test was finished when
the blood lactate concentration of a given horse was
close to or above 4 mmol/l. Before the test and after
warm up, the horses walked for 5 min at 1.5 d s e c and
trotted for 5 min at 4 d s e c .
Immediately after each gallop in the SET, blood samples were collected from pectoral skin (Lindner et al.,
1992) to determine blood lactate concentration. Analysis of lactate was made with a EPOS 5060 lactate
analyzer (Eppendorff-Netheler-Hinz GmbH) using an
enzymatic test kit (Boeringer Mannheim hr 1178 750).
To compare blood lactate concentrations from horse to
horse, the individual blood lactate concentration-running speed relation were calculated by plotting lactate
concentrate vs. running speed and drawing the curve of
each horse by linear interpolation.
197
Serial sections were reacted with seven different
monoclonal antibodies specific to rat MyHC isoforms.
Individual cross sections were labeled for fast, slow,
SC-71, N2.261, BF-35, BF-G6, and BF-B6 monoclonal
antibodies. The source and specificity of these monoclonal antibodies and the immunohistochemical procedure employed are described in the companion paper
(Rivero et al., 1996).
Quantitative Tissue Analysis
After histochemical and immunohistochemical
staining of the tissue sections, a region of the cross
sections containing 100-135 fibers per muscle biopsy
was randomly selected for further analysis. These fibers were numbered and classified at random with histochemical and immunohistochemical methods. Myofibers were classified into types I, IIC, IIA, IIAB, and
IIB (Brooke and Kaiser, 1970; Ingjer 1979) according to
the mATPase staining characteristics a t the different
levels of preincubation acidity. The same fibers were
allotted to types I, I + IIA, IIA, IIA + IIB (or IIA +
11x1and IIB (or 11x1according to the MyHC content as
revealed by reactivity against monoclonal antibodies
(see Rivero et al., 1996). To compare histochemical vs.
immunohistochemical data, these five fiber types were
also renamed types I, IIC, IIA, IIAB, and IIB, respectively. The fiber type distribution of each muscle biopsy
was established by counting and typing the relative
frequency of the various fiber types in each sample.
The cross-sectional areas (CSAs), and optical densities
Muscle Biopsies and Tissue Preparation
(ODs) for SDH activity of the same fibers were deterMuscle biopsies (75-150 mg) were obtained from the mined by using a computer-enhanced image processing
right gluteus medius muscle of each horse according to system as described in detail elsewhere (Martin et al.,
Lindholm and Piehl(1974). Biopsies were taken before, 1985). Tissue sections were digitized within the first 2
4 months after training, and a t the end of the 8-month days after staining as gray-level (range of gray levels
training program. Control horse biopsies were taken a t was 0-255) images. The gray level for each picture
the same time periods. Attempts were made to extract element was converted to an OD by using a calibration
tissue from approximately the same relative location of curve, which was linear up to 1.0 OD unit. Succinate
the muscle by using the prebiopsy scar and depth dehydrogenase activity was interpolated from a single
markings on the needle. All the muscle biopsies were endpoint measurement after a 30-min incubation, and
removed a t 2-cm sampling depth. After collection, mus- the values were reported as final OD. For each muscle
cle samples were frozen, stored, and transported as de- biopsy, CSA and SDH activity were averaged for each
scribed elsewhere (Rivero et al., 1996).
fiber type.
Frozen biopsy samples were thawed to -20°C in a
In addition, the relative CSA that a fiber type occucryostat and serially sectioned for histochemistry (10- pied in a muscle sample was calculated by dividing the
pm-thick sections placed on cover slips), immunohis- product of the fiber type percentage and the mean CSA
tochemistry (10-pm-thick sections placed on gelatin of the fiber type by the sum of these products for all
coated slides), and gel electrophoresis of MyHC (20- fiber types (Sullivan and Armstrong, 1978).
pm-thick sections placed in precooled microcentrifuge
Myosin Heavy Chain Electrophoresis
tubes).
MyHC analysis was performed on the biopsy samples
Histochemistry and lmrnunohistochernistry
using the 8% sodium dodecyl sulfate-polyacrylamide
Serial cross sections were stained qualitatively for electrophoretic (SDS-PAGE) protocol of Talmadge and
the demonstration of mATPase activity after alkaline Roy (1993) and detailed in the companion paper (Riv(pH 8.75) and acid (pH 4.2 and pH 4.5) preincubation ero et al., 1996). Three MyHC isoforms were clearly
by using a modification (Nwoye et al., 1982) of the separated in the gels and identified as types I, IIa, and
Brooke and Kaiser (1970) method. Serial sections were IIb. The gels were scanned with an Alpha Innotech
stained for quantitative succinate dehydrogenase IS-1,000 videoscanning densitometric system, and a
(SDH) activity (Blanco et al., 1988). Briefly, tissue sec- quantification of MyHC isoforms was obtained in reltions were incubated in a medium containing 48 mM ative terms for each sample.
succinate, 1.5 mM NBT, 5 mM EDTA, 1mM l-methoAlthough five fiber types were distinguished by both
syphenazine methosulfate (mPMS; Dojindo Laborato- histochemical and immunohistochemical methods, the
ries, Japan), 0.75 mM sodium azide, and 100 mM hybrid fiber types (IIC and IIAB) made up a minor
sodium phosphate buffer (pH 7.60) for 30 min (qualita- portion of the entire biopsy. To correlate quantitative
tive procedure) a t room temperature.
information from MyHC electrophoresis vs. histochem-
198
J.-L.L. RIVER0 ET AL.
ical and immunohistochemical data, the type IIC fibers
(always fewer than 1%) were split so that one-half of
these were combined with type I and one half with type
IIA fibers. Similarly, one-half of the type IIAB fiber
population was combined with type IIA fibers and the
other half with type IIB fibers. These revised data for
fiber type distribution (which gives a more accurate
representation of the three major fiber types) were then
used in combination with the CSA data to obtain the
relative CSA of the three major fiber types.
Statistical Analyses
The statistical package STAT 5.2 SYS developed for
Macintosh was used for all statistical analyses. Descriptive statistics were used to derive means (and
standard deviations) for all variables. One-way analysis of variance (ANOVA) with repeated measurements
was used to determine whether significant effects of
training existed. In the presence of a significant F ratio, post hoc comparisons of means were provided by
Tukey’s t procedure. Correlation analysis was performed to compare the histochemistry, immunohistochemistry, and electrophoretic information. In addition, coefficients of variation between histochemical
and immunohistochemical data were calculated according to the formula
c v = V E-XZ F i
where CV is the coefficient of variation, d is the difference between duplicate determinations, n is the number of repeated analyses, and 5i is the mean value. Finally, because there were significant variations in both
fiber CSA and SDH between muscle biopsy specimens,
the CSA and SDH activity of each fiber was standardized to Z scores based on the following algorithm:
-x
z = -xi
,
S
where Xiis a n individual CSA or SDH measure, X is
the mean for all fibers within the biopsy, and s is the
standard deviation (Sieck et al., 1986). Differences
among fiber types for the CSA and SDH activity were
then examined by a one-way ANOVA for all data standardized to Z scores. Statistical significance was accepted at P < 0.05.
RESULTS
Fiber Typing
In all of the 18 muscle cross sections examined, histochemical and immunohistochemical analyses of muscle revealed a classical mosaic pattern, and the shape
and size of fibers appeared normal. Based on histochemical analysis (mATPase reaction), the muscle fibers could be divided into five categories: I, IIC, IIA,
IIAB, and IIB (Fig. 1B-D). A continuum in the staining
intensity for mATPase after acid preincubation (pH
4.5) was observed between the type IIA and type IIB
fiber population (Fig. 1D). All of these intermediate
fibers were identified as type IIAB.
Five different fiber populations were also demonstrated immunohistochemically by using specific
monoclonal antibodies (Fig. 1F-L). Fibers that reacted
exclusively with the slow MyHC antibody were termed
I. Fibers th a t were unreactive with the slow MyHC
antibody and reacted with fast, SC-71, N2.261, BF.35,
and BF-G6 MyHC antibodies were identified as IIA,
and those fibers that did not react with all monoclonal
antibodies except the fast MyHC antibody were identified as IIB. Two subgroups of fibers coexpressing two
different MyHCs were also identified, and these were
designated a s IIC (fibers coexpressing types I and IIa
MyHCs) and IIAB (fibers coexpressing both fast
MyHCs). No fibers were labeled with the BF-B6 monoclonal antibody, indicating th a t no embryonic or neonatal MyHCs were present in the muscle biopsies examined. A more detailed description of these five
immunohistochemical fiber types has been shown in
the previous study (River0 et al., 1996).
Combined Histochemical and
lmmunohistochemical Analyses
Combined histochemical and immunohistochemical
analyses demonstrated a certain degree of correlation,
albeit not unequivocal, between mATPase staining intensities (pH 4.2, 4.5, and 8.75) and MyHC content.
Type I fibers were histochemically uniform and reacted
with the slow, N2.261, BF-35, and BF-G6 MyHC mAbs
(e.g., fibers labeled 1 in Fig. 1). However, the fasttwitch fiber population was histochemically and immunohistochemically heterogeneous. Most fibers histochemically classified a s IIA contained only IIa MyHC
(e.g., fiber labeled 3 in Fig. 1). However, about 2% of
the IIA fibers were composed of both fast IIa and IIb
MyHCs (and thus named IIAB immunohistochemically; not shown in Fig. 1). Another 1%of the IIA
mATPase fibers contained only type IIb MyHC (IIB
fibers immunohistochemically; not shown). Similarly,
a high percentage of fibers histochemically classified as
IIB contained a mixture of IIa and IIb MyHCs (-24% of
IIB mATPase fibers) and were classified as type IIAB
by immunohistochemistry (e.g., fiber labeled 5 in Fig.
1). A small amount of fibers histochemically identified
as IIB contained only the IIa MyHC (-2%; fibers not
shown). Those fibers histochemically identified as type
IIAB frequently contained both IIa and 1% MyHCs, but
a high proportion were composed exclusively of IIa
MyHC (-61%), so they were immunohistochemically
classified as IIA (e.g., fiber labeled 4 in Fig. 1).A few
fibers histochemically classified as type IIAB contained
only IIb MyHC (-4%; not shown).
Finally, there was no optimal correlation between
mATPase activity and MyHC content for the C-fiber
population. Some fibers stained more like IIC fibers by
histochemistry contained only the slow-MyHC (not
shown); conversely, a few fibers coexpressing both the
slow and the fast IIa MyHCs were typed a s I for
mATPase (e.g., fiber labeled 2 in Fig. 1). Overall, 18.5%
of the muscle fibers (N = 2,078) were classified differently by using qualitative mATPase activity and the
MyHC content as shown immunohistochemically.
Effects of Training
No significant changes in either body weight or performance was observed over the training period. V,
values were similar a t all three times in which muscle
samples were collected: pretraining (7.33 * 0.35 d s e c ,
n = 5 horses), middle of training (7.76 * 0.57 d s e c ) ,
and posttraining (7.76 2 0.39 d s e c , P > 0.05).
MYOSIN ATPase AND MYOSIN HEAVY CHAIN IN HORSES WITH TRAINING
199
Fig. 1. Identification of fiber types by enzyme histochemistry and
immunohistochemistry. Serial sections of horse gluteus medius muscle were processed for the demonstration of myosin ATPase and SUCcinate dehydrogenase (SDH) activities and stained with monoclonal
antibodies against MyHC isoforms. A. Scheme showing the pattern of
reactivity of the six most common fiber types identified by enzyme
histochemistry and immunohistochemistry. E D : Myosin ATPase after preincubations at pH 8.75 (B), pH 4.2 (C), and pH 4.5 (D).
E: Qualitative SDH activity. F-L Immunohistochemical staining
with monoclonal antibodies fast (F), slow ( G ) ,SC-71 (HI, N2.261 (I),
BF-35 (J),BF-G6 (K), and BF-B6 (L). The fibers labeled 1-6 correspond to those fibers illustrated in A. Bar = 100 Fm.
Almost no significant changes (P > 0.05) in fiber
type composition, mean CSA, relative CSA, or SDH
activity of muscle fiber types were recorded as a consequence of training (Table 1).A decrease in the percentage of histochemically identified type IIAB fibers
during the second half of the training program was
the only significant (P < 0.001) change recorded after training. Nevertheless, a tendency for both hypertrophy and an increase of the oxidative capacity of
all fiber types was suggested (Table 1).No changes in
percentages, CSA, relative CSA, and SDH activity
were observed for any of the four histochemical and
immunohistochemical fiber types in the control horse
(Table 1). Training had no significant effect on the
number of muscle fibers misclassified by qualitative mATPase activity in correspondence with their
MyHC content as revealed by immunohistochemistry
(Table 2).
200
J.-L.L. RIVER0 ET AL.
TABLE 1. Percentage, cross-sectional area (CSA), relative CSA, and SDH activity of the various fiber types
identifed immunohistochemically and by qualitative myosin ATPase of five horses (training) and one control
horse (control) dona the experimental Deriod'
Month
Training (N = 5 )
Fiber composition (%)
I
IIA
IIAB
IIB
CSA (pm')
I
IIA
IIAB
IIB
Relative area (%)
I
IIA
IIAB
IIB
SDH activity (OD)
I
IIA
IIAB
IIB
Control (N = 1)
Fiber composition (%)
I
IIA
IIAB
IIB
CSA (pm')
I
IIA
IIAB
IIB
Relative area (%)
I
IIA
IIAB
IIB
SDH activity (OD)
I
0
Immunohistochemistry
4
0
Histochemistry
4
8
13.2 (4.2)
42 (7)
9 (3.8)
35.8 (4.8)
8.6 (6.1)
43.2 (7.2)
13.4 (4.2)
34.8 (5.4)
13.8 (11.4)
40.8 (13.7)
11.8 (3.7)
33.6 (11.5)
13.2 (4.2)
34.4 (8.5)
10.2 (2.2)
42.2 (6.4)
8.6 (6.1)
34.6 (5.3)
15 (4.2)
41.8 (4)
13.8 (11.4)
34.2 (10.2)
6.2 (1.5)*
45.8 (8.2)
1,349 (268)
1,995 (223)
2,605 (607)
3,676 (1,059)
1,922 (615)
2,180 (719)
2,765 (922)
4,386 (1,176)
1,848 (354)
2,339 (440)
2,953 (450)
3,954 (758)
1,349 (268)
1,918 (292)
2,069 (226)
3,575 (988)
1,922 (615)
2,003 (608)
3,309 (1,197)
4,115 (1,131)
1848 (354)
2232 (547)
2719 (230)
3817 (651)
7 (3.1)
33 (4.2)
9.6 (4.3)
50.4 (7.1)
5.2 (4)
31.4 (6.5)
12.6 (4.2)
50.8 (6.3)
9 (7.1)
33.4 (13.3)
12.2 (3.8)
45.4 (13.5)
7 (3.1)
26.2 (6.6)
8.6 (0.9)
58.2 (7.2)
5.2 (4)
22.8 (6.8)
16.2 (6.9)
55.8 (2.7)
9 (7.1)
25.6 (7.8)
6 (1.6)
59.4 (8.2)
1.23 (0.35)
1.06 (0.27)
0.92 (0.24)
0.69 (0.22)
1.31 (0.1)
1.17 (0.07)
1.05 (0.1)
0.72 (0.08)
1.32 (0.08)
1.15 (0.06)
1.03 (0.07)
0.81 (0.06)
1.23 (0.35)
1.08 (0.28)
0.98 (0.26)
0.73 (0.23)
1.31 (0.1)
1.19 (0.06)
1.08 (0.08)
0.77 (0.09)
1.32 (0.08)
1.18 (0.06)
1.07 (0.05)
0.85 (0.05)
8
12
38
15
35
7
42
16
35
5
35
27
33
12
32
8
48
7
34
12
47
5
27
23
45
990
1,960
2,158
3,614
923
1,707
2,247
2,750
846
846
1,631
2,547
990
1,918
2,335
3,190
923
2,669
2,719
2,709
846
1,218
1,608
2,340
5
30
13
52
3
34
17
46
2
19
27
52
5
25
7
63
3
27
10
60
2
18
21
59
1.15
0.99
0.83
0.64
1.16
1.08
0.96
0.67
1.17
1.17
0.89
0.62
1.15
1
0.95
0.68
1.16
1.1
1.03
0.72
1.17
1.03
0.92
0.69
'Values are mean (SD). N = number of horses.
'Significantly different fmm second muscle biopsy (P < 0.001).No significant (P > 0.05) variations along the experimental period were found
for the other muscle parameters.
Quantitative Histochemical and Immunohistochemical
Interrelationshipsof Fast-Twitch fibers
showed a histochemical reaction between IIA and IIB
(type IIAB) fibers was similar in average to those fibers
The mean percentage of type I and type I1 fibers cal- identified immunohistochemically as fibers containing
culated on serial sections stained histochemically and both IIa and IIb MyHCs. Statistical comparisons of the
immunohistochemically were identical. However, im- fiber composition, mean CSA, relative CSA, and SDH
portant variations existed between the various sub- activity of the histochemically and immunohistochemtypes of fast-twitch fibers. A comparison of quantita- ically identified fast-twitch fibers for all muscle bioptive immunohistochemical vs. histochemical data of sies revealed significant correlations and relatively low
the three fast-twitch muscle fibers is summarized in covariation coefficients between duplicate determinaTable 3. As shown, the number of pure IIA fibers im- tions for IIA and IIB fibers. However, correlation coefmunohistochemically identified as larger than ex- ficients were not significant (P > 0.05) for the hybrid
pected based on the histochemical analysis (P < 0.001). IIAB fiber population. Similarly, coefficients of variaIn contrast, the proportion of pure IIB fibers was lower tion between immunohistochemical and histochemical
by histochemical analysis in comparison with the information for these fibers were dramatically higher.
MyHC content (P < 0.001). The number of fibers th a t Measurements of fiber size and SDH activity of fast
201
MYOSIN ATPase AND MYOSIN HEAVY CHAIN I N HORSES WITH TRAINING
TABLE 2. Number and percent of muscle fibers misclassified based on qualitative
mATPase histochemistry and MyHC immunohistochemistry in all 18
muscle bioasies
Month
Histochemistry
Immunohistochemistry
N
IIA
IIB
IIAB
IIAB
IIB
IIA
IIC
N
=
iiAB
IIAB
0
4
684
698
8
IIB
3
49
46
3
IIA
10
IIB
I
2
3
1
3
0
IIA
Percent
8
696
Total
5
16
173
160
45
42
3
4
11
3
17
0
0
3
4
1
3
1
59
65
65
5
49
2,078
4
number of fibers examined.
fibers were not significantly different between the two
methods, but the mean CSA was lower for each of the
three fast fibers histochemically identified.
Electrophoretic Data
Electrophoresis of MyHCs separated three bands in
all the 18 muscle biopsies examined from the training
group and the control horse (Fig. 2). These three bands
were in the order of mobility I > IIb > IIa. Training did
not cause significant changes (P > 0.05) in the relative
proportion of the three MyHC isoforms of the gluteus
medius muscle (Table 4). However, the percentage of
type I MyHC showed a clear tendency to increase and
that of type IIB MyHC to decrease in the biopsy samples after 8 months of training (P < 0.1 in both). No
significant changes in the MyHC composition of the
gluteus medius muscle were observed after 4 and 8
months in the control horse (Table 4).
Electrophoretic, Immunohistochemical, and
Histochemical Interrelation
To compare the MyHC content with the fiber type
composition properly, the relative CSA of the three major fiber types (I, IIA, and IIB) was determined (Table
4). The mean percentage of type I fibers derived electrophoretically, immunohistochemically, and histochemically was similar (P > 0.05). Once again, the percentage of relative CSA occupied by IIA fibers
immunohistochemically classified was higher than
that based on the histochemical analysis (P< 0.001 in
all the samples). Conversely, the relative CSA area
occupied by IIB MyHC fibers was lower than that of IIB
mATPase fibers (P< 0.01). The percentage of fast IIa
and IIb MyHC isoforms based on gel electrophoresis
was intermediate between those of the relative CSA
occupied by these fibers identified by immunohistochemistry and those identified by qualitative
mATPase, but this difference was not significant (P>
0.05; Table 4).
The derived relative CSA of the immunohistochemically and histochemically major fiber types (see Materials and Methods) were again compared with the
MyHC content measured in each biopsy sample by gel
electrophoresis (Table 5). Comparisons over the experimental period for all the 18 muscle biopsies examined
revealed low but significant (P < 0.05) correlations between the percentage fiber-type CSA and MyHC content measured from gels, the exception being the rela-
tion between the relative CSA of histochemically
identified IIA fibers and the IIa MyHC (r = 0.07, P >
0.05). When all fiber types and MyHC were combined,
the correlation coefficients ranged from 0.94 to 0.96.
Differences in Fiber Size and SDH Activity Among
Fiber Types
Figure 3 shows the proportional distributions of Z
scores of the mean CSA and SDH activity for the four
fiber types immunohistochemically identified from the
18 muscle biopsy specimens examined in the present
study (N = 2,078 fibers). Unimodal distributions of
CSA for the total populations of type I, IIA, and IIAB
fibers were generally evident for most of the fiber
types, but a generally wider distribution was observed
for type IIB fibers. Despite the overlap in the distributions of CSA among the four fiber types, significant
differences among fiber types were observed (P <
0.0001). Thus, the fiber size was in the order of IIB >
IIAB > IIA I. Moreover, in addition to being larger,
fast type IIB fibers also showed a significantly greater
range of CSAs than the other fiber types (Fig. 3A).
Similarly, distributions of relative SDH activities
were unimodal for the total fiber populations. Despite
the overlap in the distribution of SDH activities among
fiber types, significant variations were recorded (P <
0.0001) for the overall population among fiber types,
with the type I fibers being more oxidative than fast
fibers. Within the fast fibers, the order of oxidative
capacity was type IIA > type IIAB > type IIB.
In the total population of muscle fibers, there was a
significant (P < 0.001) negative correlation (r =
-0.53) between CSA and SDH activity. This negative
correlation between CSA and SDH was evident within
each population of fiber classifications except for type I:
I, r = -0.08, P > 0.05; IIA, r = -0.25, P < 0.001; IIAB,
r = -0.27, P < 0.001; and IIB, r = -0.35, P < 0.001.
DISCUSSION
The interrelations of histochemical, immunohistochemical, electrophoretic, and morphometric analyses
of muscle biopsies from trained and untrained horses
are described in the present study. As in humans
(Staron, 1991; Staron and Hikida, 1992), there is a high
level of predictability between mATPase activity and
MyHC content among most, but not all, fibers in
equine skeletal muscle. Comparisons between the histochemical and immunohistochemical data show that
202
J.-L.L. RIVER0 ET AL.
TABLE 3. Mean values and correlation and variation coefficient (CV)between
immunohistochemical and histochemical data of the various fast-twitch fiber types for
training and control horses (N = 18 biopsies)
Immunohistochemistry
Fiber composition (%)
IIA
IIAB
IIB
CSA (pm2)
IIA
IIAB
IIB
Relative CSA (%)
IIA
IIAB
IIB
SDH activity (OD)
IIA
IIAB
IIB
Histochemistry
33.8
10.4
43.8
41.4
11.9
34.7
2,060
2,647
3,833
1,976
2,564
3,654
32.5
11.9
48.9
24.6
10.7
58.3
1.12
0.98
0.73
1.13
1.03
0.77
r
CV
0.86
0.5"
0.73
26.2
50.5
29.6
0.95
0.56*
0.99
10.3
30.2
6.84
0.79
0.39*
0.69
24.6
64.5
23.2
0.96
0.93
0.99
4.31
8.33
7.23
*Correlation values not significant (P > 0.05).
1A
B
4A
B
C
2A
B
C
3A
B
C
5 A
B
C
6A
B
C
IIAIIBI -
C
Fig. 2. Eight-percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel of
myosin heavy chain (MyHC) of the training group (horses 1-5) and the control horse (horse 6). A-C are
the three muscle biopsies removed from each horse. The MyHC isoforms are identified as types I (I),IIA
(Ira), and IIB (IIb).
-18% of the fibers were misclassified by traditional
mATPase histochemistry. Each MyHC isoform can
be associated rather consistently with a specific
mATPase-based muscle fiber type (Staron and Pette,
1986), except when two or more MyHCs coexist within
the same fiber. A large proportion of the fast fibers in
the equine gluteus medius muscle coexpress the two
fast MyHC isoforms in varying ratios. Likewise, a few
203
MYOSIN ATPase AND MYOSIN HEAVY CHAIN I N HORSES WITH TRAINING
TABLE 4. Percentage of MyHC based on gel electrophoresis and percentage of the
relative cross-sectional area occupied by the three major fiber types identified by
immunohistochemistryand qualitative myosin ATPase of five horses (training)and one
control horse (control)'
Month
Training
Electrophoresis (N)
I
IIA
IIB
Immunohistochemistry
I
IIA
IIB
Histochemistry
I
IIA
IIB
Control
Electrophoresis (N)
I
IIA
IIB
Immunohistochemistry
I
IIA
IIB
Histochemistry
I
IIA
IIB
All horses
Electrophoresis (N)
I
IIA
IIB
Immunohistochemistry
I
IIA
IIB
Histochemistry
I
IIA
IIR
All samples
0
4
8
5
6.2 (2.5)
33.8 (4.6)
60.0 (5.9)
5
5.4 (2.0)
35.4 (2.1)
59.0 (2.7)
5
11.8 (6.1)
34.6 (2.9)
53.4 (5.1)
15
7.8 (4.9)
34.6 (3.5)
57.6 (5.6)
7.0 (2.8)
37.8 (3.9)
55.2 (5.9)
5.2 (3.6)
37.7 (5.9)
57.1 (4.5)
9.0 (6.3)
39.5 (12.4)
51.5 (11.0)
7.1 (4.7)
38.3 (3.5)
54.6 (7.8)
7.0 (2.8)
30.5 (5.8)
62.5 (6.5)
5.2 (3.6)
30.9 (4.3)
63.9 (3.3)
9.0 (6.3)
28.6 (7.2)
62.4 (7.2)
7.1 (4.7)
30.0 (6.0)**
62.9 (6.0)**
1
8.0
30.0
62.0
1
4.0
27.0
69.0
1
6.0
28
66.0
3
6.0 (2.0)
28.0 (1.4)
65.7 (2.9)
5.0
36.5
58.5
3.0
42.5
52.5
2
32.5
65.5
3.3 (1.2)
38.2 (4.1)
58.5 (5.3)
5.0
28.5
66.5
3.0
32.0
65.0
2.0
28.5
67.5
3.3 (1.2)
30.4 (1.6)*
66.3 (1.0)*
6
6.5 (2.3)
33.2 (4.4)
60.3 (5.4)
6
5.2 (1.9)
34.0 (3.7)
60.8 (4.5)
6
10.8 (6.0)
33.3 (3.9)
55.9 (6.6)
18
7.5 (4.6)
33.5 (4.0)
59.0 (6.0)
6.7 (2.6)
37.6 (3.6)
55.7 (5.5)
4.8 (3.4)
38.5 (5.7)
56.7 (4.4)
7.8 (6.3)
38.3 (11.6)
53.9 (11.3)
6.5 (4.6)
38.2 (7.7)
55.3 (7.8)
6.7 (2.6)
30.2 (5.5)
63.1 (6.2)
4.8 (3.4)
31.1 (4.0)
64.1 (3.1)
7.8 (6.34)
28.6 (6.5)
63.6 (6.9)
6.5 (4.6)
30.0 (5.5)***
63.5 (5.6)**
'Values are mean (SD);
N = number of horses or samples. Training had no significant effect (P > 0.05) on muscle
characteristics analyzed.
*P < 0.05, **P< 0.01, and ***P < 0.001 compared with the same fiber type immunohistochemically classified.
TABLE 5. Correlation analysis between percentages of fiber type area and
myosin heavy chain for training and control horses (N = 18 biopsies)'
Immunohistochemistry vs. histochemistry
Immunohistochemistry vs. electrophoresis
Histochemistry vs. electrophoresis
I
1.00
0.57
0.57
IIA
0.82
0.51
0.07*
IIB
0.80
0.48
0.43
All
0.95
0.94
0.96
'All values given are correlation coefficients.
*Only correlation value not significant (P> 0.05).
fibers (designated IIC) coexpress the I and IIa MyHC.
Fibers coexpressing two or more MyHC isoforms may
react histochemically according to the dominant isoform (Danieli-Betto et al., 1986; Klitgaard et al.,
199Oa). The finding that many fibers histochemically
classified as type IIB coexpressed IIa and IIb MyHCs
supports this assumption. Furthermore, some fibers
histochemically identified as type I coexpressed type I
and type IIa MyHCs based on immunohistochemistry .
However, nearly all fibers histochemically classified as
type IIAB contained only IIa MyHC.
The observation that a large number of histochemically determined IIB fibers actually contain a varying
amount of type IIa MyHC is in agreement with previous studies on human single fibers (Staron, 1991;
Staron and Hikida, 1992; Andersen et al., 1994a,b). In
the two latter studies, the researchers reported that
nearly all histochemical type IIB fibers of sprinters
J.-L.L. RIVER0 ET AL.
204
B. SDH Activity
A. Cross-Sectional
Type I fibers (N=235)
. . . .
35
I
Type ZIA fibers ( N = 8 6 1 )
""T
&
35T-
~
Type IIAB fibers ( N = 2 6 2 )
. .
. .
0
1
Type ZIB fibers (N=721)
30
T
35
1
Z Score
Fig. 3. Histogram of frequencies showing population distributions of
cross-sectional area (A) and SDH activities (B)(standardized to 2
scores) for fibers containing types I (I), IIa (IIA), IIa + IIb (IIAB), and
only IIb MyHCs when identified by immunohistochemistry. All fibers
examined (N = 2,078) are computed; all fiber types were statistically
different (P < 0.0001).
MYOSIN ATPase AND MYOSIN HEAVY CHAIN IN HORSES WITH TRAINING
(Andersen et al., 1994a) and soccer players (Andersen
et al., 199413) coexpressed both IIa and IIb MyHC isoforms. The explanation for the finding that practically
all histochemically typed IIAB fibers contained only
IIa MyHC is unclear. The mATPase histochemical
staining may be affected by factors other than the relative content of MyHC. For example, the myosin light
chain isoform composition of a fiber can, independently
of the MyHC, affect the maximal velocity of shortening
of that fiber (Bottinelli et al., 1994b).Thus, the myosin
light chain may also influence the myofibrillar ATPase
staining characteristics of a fiber because ATPase activity and shortening velocity are highly correlated
(Barany, 1967).
However, in a recent study (Sant’Ana Pereira et al.,
1995), the combined quantitative histochemical, immunohistochemical, electrophoretic, and immunoblotting analyses of single human skeletal muscle fibers
reported that the mATPase staining patterns usually
correlated with the expression of distinct MyHC isoforms. A continuum exists from pure IIA to pure IIB in
the staining density for mATPase after preincubation
at pH 4.5-4.6 (White and Snow, 1985; Rivero et al.,
1993). The results of a previous study of the mATPase
activity of equine muscle fiber types by quantitative
histochemistry (White and Snow, 1985) clearly showed
a continuous range within the type I1 fibers, with two
or three overlapping peaks. Because this continuum of
fiber types is difficult to quantify by using a qualitative
technique such as mATPase histochemistry , this
method of classification of muscle fibers into four major
types (five if the type IIC is included) may have major
limitations in objectively differentiating among the
three fast subtypes (IIA, IIAB, and IIB) based on MyHC
content. An alternative explanation is that histochemical type IIAB fibers might contain a third distinct fast
MyHC isoform, which was not revealed by the monoclonal antibodies tested in the present study. This possibility was not suggested, however, by the monoclonal
antibodies used or by the SDS-PAGE assayed in the
present study. Our observation of some fibers histochemically identified as type I, but coexpressing both
I and IIa MyHC, is similar to that reported for human
muscle (Klitgaard et al., 1990a). Klitgaard et al.
(1990a) reported that in the 95%of fibers in endurance
athletes that contained both I and IIa MyHCs the major fraction was the type I MyHC, and this perhaps
explains the histochemical staining profile as type I
fibers.
Another striking observation of the present study
was that training had no significant effect on the correlation between mATPase activity and MyHC content
of muscle fibers. In some studies, it has been argued
that the “misclassification” of many fibers by mATPase
histochemistry seems to be especially pronounced in
trained individuals because of the high number of fibers coexpressing multiple MyHCs (Klitgaard et al.,
1990a; Staron, 1991). Although this assumption would
be supported by the larger number of fibers coexpressing types I and IIa MyHCs in endurance athletes than
in sedentary men reported by Klitgaard et al. (1990a),
it is not confirmed by either the present or previous
results in humans (Klitgaard et al., 1990b;Andersen et
al., 1994a,b). Our results suggest that the coexpression
of the two fast MyHCs within the same fiber is not
205
necessarily a sign of fiber type transformation because
these fibers did not increase in number with training.
The assumption is also supported by the results of two
previous studies in humans, where the percentage of
fibers with coexisting IIa and IIb MyHCs decreased as
a result of training (Klitgaard et al., 1990b; Andersen
et al., 1994a).
The correlations between the relative CSA of the
three major fiber types, as determined by immunohistochemistry and histochemistry, and the percentage
MyHC content derived by SDS-PAGE coincide with the
results reported by Fry et al. (1994) in humans. These
low correlations do not give credence to the use of mATPase histochemistry for assessing fiber type composition, as was concluded in that study. When the five
immunohistochemically and histochemically delineated equine fiber types were compressed into three
major types (I,IIA, and IIB), the percentage of the CSA
occupied by type IIA fibers histochemically determined
was underestimated and that of type IIB fibers was
overestimated in comparison with the relative CSA occupied by the same fibers classified by immunohistochemistry. The stronger correlations found between
immunohistochemical vs. electrophoretic data when
compared with histochemistry vs. electrophoresis (Table 5) give more validity to the use of monoclonal antibodies against selected MyHCs as a means of fiber
typing in horses.
Surprisingly, no significant changes in muscle fiber
type characteristics were observed in the current study
after 4 and 8 months of training. In horses as in humans and other mammals, the most common adaptation to training is an increase of the type IIA to type IIB
histochemical fiber ratio and an increase of both the
oxidative capacity and capillary supply of muscle fibers
(for review, see Snow and Valberg, 1994). In the current study, a higher, albeit not significant, SDH activity of all fiber types as a result of training was also
recorded. The data of the present study for the mean
CSA were similar in range to those reported for the
gluteus medius muscle in young thoroughbred racehorses (Lindholm et al., 1983; Serrano et al., 1996).The
effects of training on muscle fiber size in horses have
been inconsistent but may depend on the nature, intensity, and duration of the exercise involved (Snow
and Valberg, 1994). An important observation of the
present study is that even after 8 months of intensive
training no significant modifications in fiber size occurred. In other studies on horses, little change or a
general or specific type increase was found in fiber size
after different training programs (see Snow and Valberg, 1994). As the intensity and duration (both daily
and in total) of exercise involved in the current study
was greater and longer than in other previous studies
in horses (Snow and Valberg, 19941, the limited training-related changes in muscle fiber type characteristics
recorded in the present study is difficult to explain.
However, the limited changes may be related to the
relative superficial sampling site of the gluteus medius
muscle from which the samples were taken. In a recent
study (Rivero et al., 19951, the adaptation of the gluteus medius muscle fibers to training was not homogeneous in superficial and deep sampling regions of this
muscle. Because these training-linked modifications
were more marked in the deep region of the muscle
206
J-L.L. RIV‘ERO ET AL.
than in the superficial one, different functional demands must be placed on different depths of the gluteus medius muscle. This lack of muscular response to
training may also be explained in part by the fact that
no real metabolic adaptation occurred at the systemic
level in response t o training. For example, the performance capacity did not improve in any of the horses
throughout the training program, as shown by the constant values of ITLAI.
The most common approach to classifying horse skeletal muscle fibers has been dividing them into three
main fiber types (I, IIA, and IIB) based on myofibrillar
ATPase histochemistry (Snow and Valberg, 1994).
Type I and IIA fibers have a high oxidative capacity,
whereas type IIB fibers have a heterogeneous oxidative potential (high or low). Histochemical analysis of
these three fiber types also reveals important differences in sizes, the order generally being I < IIA < IIB.
The current study confirms these concepts. However,
our results, based on the MyHC content of muscle fibers (immunohistochemistry), support the separation
of equine skeletal myofibers into four major types (I,
IIA, IIAB, and IIB) because the hybrid IIAB fibers appear to be a biologically important type in horses
(- 10-15%).
In conclusion, histochemical, immunohistochemical,
and electrophoretic techniques applied to equine skeletal muscle establish a relation between qualitative
myofibrillar mATPase activity and MyHC content.
However, a high percentage of fibers of the equine gluteus medius muscle typed according to the traditional
mATPase histochemistry does not always correspond
to a classification based on MyHC isoforms. Because
the immunohistochemical techniques used are specific
to MyHC isoforms, these results suggest that the qualitative mATPase histochemical technique is an indication of not only the MyHC isoform but also other characteristics of the myofibrillar protein. The use of
monoclonal antibodies against specific MyHC isoforms
seems to be a more sensitive, more reproducible, and
less subjective method for categorizing muscle fibers.
Finally, because a significant metabolic adaptation to
training did not occur in our horses, the results from
the present study are inconclusive to suggest that
the misclassification of a large number of fibers by
mATPase histochemistry may be magnified in trained
individuals.
ACKNOWLEDGMENTS
This study was completed while Jos6-Luis L. Rivero was working at the Department of Physiological Sciences, University of California at Los Angeles;
his work was supported by grants and scholarships
from the Spanish D.G.C.Y.T. (PR94-202) and the University of Cordoba, Spain. We thank Dr. S. Schiaffino
(University of Padova, Italy) for the generous gift of
MAbs. The MAb developed by Dr. H. Blau (N2.261)
was obtained from the Developmental Studies Hybridoma Bank, maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins
University School of Medicine, Baltimore, MD, and
the Department of Biology, University of Iowa, Iowa
City, IA, under contract N01-HD-2-3144 from the
NICHD.
LITERATURE CITED
Aigner, S., B. Gohlsch, N. HBmalainen, R.S. Staron, A. Uber, U.
Wehrle, and D. Pette 1993 Fast myosin heavy chain diversity in
skeletal muscles of the rabbit: Heavy chain IId, not IIb, predominates. Eur. J. Biochem., 211,367-372.
Andersen, J.L., H. Klitgaard, and B. Saltin 1994aMyosin heavy chain
isoforms in single fibres from m. vastus lateralis of sprinters:
Influence of training. Acta Physiol. Scand., 151:135-142.
Andersen, J.L., H. Klitgaard, J. Bangsbo, and B. Saltin 1994b Myosin
heavy chain isoforms in single fibres from m. vastus lateralis of
soccer players: Effects of strength-training. Acta Physiol. Scand.,
150:2 1-26.
Bar, A,, and D. Pette 1988 Three fast myosin heavy chein in adult rat
skeletal muscle. FEBS Lett., 235:153-155.
Barany, M. 1967 ATPase activity of myosin correlated with speed of
muscle shortening. J . Gen. Physiol., 50t197-216.
Billeter. R.. C.W. Heizmann. H. Howard. and E. Jennv 1981 Analvsis
of myosin light and heavy chain t&es in single human skelktal
muscle fibres. Eur. J. Biochem., 116:389-395.
Biral, D., R. Betto, D.D. Betto, and G. Salviati 1988 Myosin heavy
chain composition of single fibers from normal human muscle.
Biochem. J.,250:307-308.
Blanco, C.E., G.C. Sieek, and V.R. Edgerton 1988 Quantitative histochemical determination of succinic dehydrogenase activity in
skeletal muscle fibres. Histochem. J., 20.230-243.
Bottinelli, R., R. Betto, S. SchiafTino, and C. Reggiani 1994a Maximum shortening velocity and coexistence of myosin heavy chain
isoforms in single skinned fast fibres of rat skeletal muscle. J.
Muscle Res. Cell Motil., 15:413-419.
Bottinelli, R., R. Betto, S. Schiaffino, and C. Reggiani 1994b Unloaded
shortening velocity and myosin heavy chain and alkali light
chain isoform composition in rat skeletal muscle fibres. J . Physiol., 478:341-349.
Brooke, M.M., and K.K. Kaiser 1970 Muscle fiber types:
. _ How many
and what kind? Arch. Neurol., 23r369-379.
Danieli-Betto, D., E. Zerbato, and R. Betto 1986 Type 1, 2A and 2B
myosin heavy chain electrophoretic analysis of rat muscle fibers.
Biochem. Biophys. Res. Comm., I38:981-987.
Ennion, S., J. Sant’Ana Pereira, A.J. Sargeant, A. Young, and G.
Goldspink 1995 Characterization of human skeletal muscle fibres
according to the myosin heavy chains they express. J. Muscle Res.
Cell Motil., 16:35-43.
Fry, A.C., C.A. Allemeier, and R.S. Staron 1994 Correlation between
percentage fiber type area and myosin heavy chain content in
human skeletal muscle. Eur. J. Appl. Physiol., 68:246-251.
Gorza, L. 1990 Identification of a novel type 2 fiber population in
mammalian skeletal muscle bv combined use of histochemical
myosin ATPase and anti-myosin monoclonal antibodies. J. Histochem. Cytochem., 38:257-265.
Ingjer,
F. 1979 Effects of endurance training on muscle fibre ATPase
activity, capillary supply and mitochondria1 content in man. J.
Physiol. (Lond.) 2941419-432.
Klitgaard, H., 0. Bergman, R. Betto, G. Salviati, S. Schiafho, R.
Clausen, and B. Saltin 1990a Co-existence of myosin heavy chain
I and IIa isoforms in human skeletal muscle fibres with endurance training. Pliigers Archiv., 41 6:470-472.
Klitgaard, H., M. Zhou, and E.A. Richter 1990b Myosin heavy chain
composition of single fibres from m. biceps brachii of male body
builders. Acta Physiol. Scand., 140:175-180.
Lindner, A., P. von Wittke, M. Schmald, J. Kusserov, and H. Sommer
1992 Maximal lactate concentrations in horses after exercise of
different duration and intensity. J. Equine Vet. Sci., 12t30-33.
Lindholm, A., and K. Piehl 1974 Fibre composition, enzyme activity
and concentration of metabolites and electrolytes in muscle of
standardbred horses. Acta Vet. Scand., 155’87-309.
Lindholm, A,, B. EssBn-Gustavsson, D. McMiken, S. Persson, and J.R.
Thornton 1983 Muscle histochemistry and biochemistry of thoroughbred horses during growth and training. In: Equine Exercise
Physiology. D.H. Snow, S.G.B. Persson, and R.J. Rose, eds.
Granta Editions, Cambridge, UK, pp. 211-217.
Martin, T.P., A.C. Vailas, J.B. Durivage, V.R. Edgerton, and K.R.
Castleman 1985 Quantitative histochemical determination of
muscle enzymes: Biochemical verification. J. Histochem. Cytochem., 33t1053-1059.
Nwoye, L., W.F.H.M. Mommaerts, D.R. Simpson, K. Serayderian, and
M. Marusich 1982 Evidence for a direct action of thyroid hormone
in specifying muscle properties. Am. J. Physiol., 242:R401-R408.
Pette, C., and S. Staron 1990 Cellular and molecular diversities of
mammalian skeletal muscle fibers. Rev. Physiol. Biochem. Pharmacol., 116:1-76.
MYOSIN ATPase AND MYOSIN HEAVY CHAIN IN HORSES WITH TRAINING
Pierobon-Bormioli,S.,S. Sartore, L. Dalla Libera, M. Vitadello, and S.
Schiafho 1981 Fast isomyosins and fiber types in mammalian
skeletal muscle. J. Histochem. Cytochem., 29t1179-1188.
Rivero, J.-L.L., A.L. Serrano, P. Henckel, and E. Agiiera 1993 Muscle
fiber type composition and fiber size in successfully and unsuccessfully endurance-raced horses. J. Appl. Physiol., 75:17581766.
Rivero, J.-L.L., M.C. Ruz, A.L. Serrano, and A.M. Diz 1995 Effects of
a 3-month endurance training programme on skeletal muscle histochemistry in Andalusian, Arabian and Anglo-Arabian horses.
Equine Vet., 27.51-59.
Rivero, J.-L.L., R.J. Talmadge, and V.R. Edgerton 1996 Myosin heavy
chain isoforms in adult equine skeletal muscle: an immunohistochemical and electrophoretic study. Anat. Rec., 246r185-194.
Reiser, P.J., R.L. Moss, G.G. Giulian, and M.L. Greaser 1985 Shortening velocity in single fibres from adult rabbit soleus muscle is
correlated with myosin heavy chain composition. J . Biol. Chem.,
260r9077-9080.
Sant'Ana Pereira, J.,A. Wessels, L. Nijtmans, A.F.M. Moorman, and
A.J. Sargeant 1995 New method for the accurate characterization
of single human skeletal muscle fibres demonstrates a relation
between mATPase and MyHC expression in pure and hybrid fibre
types. J. Muscle Res. Cell Motil., 16r21-34.
Schiaffino, S., L. Gorza, S. Sartore, L. Saggin, S. Ausoni, M. Vianello,
K. Gundersen, and T. Lemo 1989 Three myosin heavy chain isoforms in type 2 skeletal muscle fibers. J. Muscle Res. Cell. Motil.,
1Ot197-205.
Serrano, A.L., J.L. Petrie, J-L.L. Rivero, and J.W. Hermanson 1996
Myosin isoforms and muscle fiber characteristics in equine gluteus medius muscle. Anat. Rec., 244:444-451.
Sieck, G.C., R.D. Sacks, C.E. Blanco, and V.R. Edgerton 1986 SDH
207
activity and cross-sectional area of muscle fibers in cat diaphragm. J. Appl. Physiol., 60r1284-1292.
Smerdu, V., I. Karsh-Mizrachi, M. Campione, L. Leinwand, and S.
Schiaffino 1994 Type IIx myosin heavy chain transcripts are expressed in type IIb fibers of human skeletal muscle. Am. J. Physiol., 267:(Cell Physiol, 36)C1723-C1728.
Snow, D.H., and Valberg, S.J. 1994 Muscle anatomy, physiology and
adaptations to exercise and training. In: The Athletic Horse. D.R.
Hodgson and R.J. Rose, eds. WB Saunders, Philadelphia, pp.
145-179.
Staron, R.S. 1991 Correlation between myofibrillar ATPase activity
and myosin heavy chain composition in single human muscle
fibers. Histochemistry, 9621-24.
Staron, R.S., and R.S. Hikida 1992 Histochemical, biochemical, and
ultrastructural analyses of single human muscle fibers, with special reference to the C-fiber population. J . Histochem. Cytochem.,
40563-568.
Staron, R.S., and D. Pette 1986 Correlation between myofibrillar
ATPASE activity and myosin heavy chain composition in rabbit
muscle fibres. Histochemistry, 86.19-23.
Sullivan, T.E., and R.B. Armstrong 1978 Rat locomotory muscle fiber
activity during trotting and galloping. J . Appl. Physiol., 44:358363.
Talmadge, R.J., and R.R. Roy 1993 Electrophoretic separation of rat
skeletal muscle myosin heavy-chain isoforms. J. Appl. Physiol.,
75t2337-2340.
White, M.G., and D.H. Snow 1985 Quantitative histochemistry of myosin ATPase activity after acid preincubation, and succinate dehydrogenase activity in equine skeletal muscle. Acta Histochem.
Cytochem., 18:483-493.
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