Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

Abstract

Twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background were investigated and classified into three groups according to their physical activity and sport discipline: untrained students (group A), national and sub-national level endurance athletes (group B, 7.8+/-2.9 years of specialised training) and sprint-power athletes (group C, 12.8+/-8.7 years of specialised training). Muscle biopsies of vastus lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin, and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC) composition. Significant differences (P<0.05) regarding composition of muscle fibre types and myosin heavy chains were found only between groups A (41.7+/-1.6% of MyHCI, 40.8+/-4.0% of MyHCIIA and 17.5+/-4.0% of MyHCIIX) and B (64.3+/-0.8% of MyHCI, 34.0+/-1.4% of MyHCIIA and 1.7+/-1.4% of MyHCIIX) and groups A and C (59.6+/-1.6% of MyHCI, 37.2+/-1.3% of MyHCIIA and 3.2+/-1.3% of MyHCIIX). Unexpectedly, endurance athletes (group B) such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C) such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance. Furthermore, the relative amount of the fastest MyHCIIX isoform in vastus lateralis muscle was significantly lower in the athletes from group C than in students (group A). We conclude that the myosin profile in the athletes belonging to group C was unfavourable for their sport disciplines. This could be the reason why those athletes did not reach international level despite of several years of training.

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FOLIA HISTOCHEMICA
ET CYTOBIOLOGICA
Vol. 42, No. 3, 2004
pp. 181-190
Characteristics of myosin profile in human vastus
lateralis muscle in relation to training background
B. Zawadowska1, J. Majerczak2, D. Semik1, J. Karasinski1, L. Kolodziejski3,
W. M. Kilarski1, K. Duda3 and J. A. Zoladz2
1Department of Cytology and Histology, Institute of Zoology, Jagiellonian University,
2Department of Muscle Physiology, Academy of Physical Education,
3Cancer Institute, Cracow, Poland
Abstract: Twenty-four male volunteers (mean ± SD: age 25.4±5.8 years, height 178.6±5.5 cm, body mass 72.1±7.7 kg) of
different training background were investigated and classified into three groups according to their physical activity and sport
discipline: untrained students (group A), national and sub-national level endurance athletes (group B, 7.8±2.9 years of
specialised training) and sprint-power athletes (group C, 12.8±8.7 years of specialised training). Muscle biopsies of vastus
lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin,
and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC) composition. Significant
differences (P<0.05) regarding composition of muscle fibre types and myosin heavy chains were found only between groups
A (41.7±1.6% of MyHCI, 40.8±4.0% of MyHCIIA and 17.5±4.0% of MyHCIIX) and B (64.3±0.8% of MyHCI, 34.0±1.4%
of MyHCIIA and 1.7±1.4% of MyHCIIX) and groups A and C (59.6±1.6% of MyHCI, 37.2±1.3% of MyHCIIA and 3.2±1.3%
of MyHCIIX). Unexpectedly, endurance athletes (group B) such as long-distance runners, cyclists and cross country skiers,
did not differ from the athletes representing short term, high power output sports (group C) such as ice hockey, karate,
ski-jumping, volleyball, soccer and modern dance. Furthermore, the relative amount of the fastest MyHCIIX isoform in vastus
lateralis muscle was significantly lower in the athletes from group C than in students (group A). We conclude that the myosin
profile in the athletes belonging to group C was unfavourable for their sport disciplines. This could be the reason why those
athletes did not reach international level despite of several years of training.
Key words: Myosin - Muscle - Training - Exercise - Sport
Introduction
Skeletal muscles of mammals are heterogeneous in com-
position of muscle fibres and adaptable - these properties
ensure functional diversity in performing complex
movement patterns. During the last two decades it has
been established that the phenotype of muscle fibres and
their contractile and metabolic proteins as well as fibre
cross-sectional area may change extensively in response
to different work regimes [42, 43]. This ability of muscle
tissue may have practical implication for both sportsmen
and patients.
Skeletal muscle fibres are classified into two major
groups, the slow-twitch and fast-twitch fibres. Their
contractile properties are determined by myosin heavy
chain (MyHC) isoforms: the slow MyHCI and the fast
types MyHCIIA and MyHCIIX [6, 31, 40]. According
to Staron and Pette [43], there is a direct link between
MyHC composition in muscle fibres and their mATPase
activity. Slow muscle fibres express predominantly the
slow type MyHCI isoform with some proportion of fast
type MyHCIIA [5, 6, 15, 16, 36, 40, 45]. Slow fibres rely
more on aerobic metabolism, whereas the fast fibres
depend more on anaerobic metabolism. Thus, slow fi-
bres are important for endurance activities and sports
such as long-distance running, cycling and swimming,
whereas fast fibres are key to power pursuits such as
weight lifting and sprinting [4]. Moreover, IIX fibres are
superior to IIA fibres in respect to maximal power output
and are crucial for sprint strength disciplines [37].
Human slow muscle soleus contains an approximately
equal amount of type I and type IIA myosin isoforms,
while type IIX isomyosin is not expressed in this muscle
[21]. In contrast, human fast muscle such as vastus
Correspondence: B. Zawadowska, Dept. Cytology and Histology,
Institute of Zoology, Jagiellonian University, Ingardena 6,
30-060 Kraków, Poland; e-mail: zaw@zuk.iz.uj.edu.pl

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lateralis expresses all three types of myosin isoforms at
variable proportions [21, 27].
It is well known that vastus lateralis of untrained,
physically active subjects contains approximately 50%
of type I and 50% of type II muscle fibres. For example
in the studies by Andersen and Aagaard [1], Andersen
et al. [4] and Klitgaard et al. [26] the vastus lateralis
possessed approximately 50% slow type I, 40% type IIA
and 10% IIX myosin isoforms. On the other hand, world
class marathon runners and ultra-endurance athletes
have 95% type I fibres [4, 22, 35, 37], whereas sprinters,
power weight lifters and throwers undertaking an an-
aerobic type of strength training contain predominantly
IIA and IIX fibres [2, 3]. The latter athletes possess the
proportion of type I fibres as low as 20% in the muscles
concerned [38]. Hence, skeletal muscles can express
different myosin isoforms depending on the type of
training regime. Another example of plasticity of the
mature skeletal muscles is that long-term endurance
training induces significant conversion from fast to slow
muscle fibres [24, 41, 46]. For example, 8-week training
decreased by 21%-70% type II fibre population in 3 of
4 subjects [7, 25]. Furthermore, MyHC isoform shift
relies on production of a new polypeptide of slow MyHC
in fibres, which are still classified as type IIA fibres.
These changes at the molecular level in type IIA fibres
mark the beginning of their transition towards the slow
type I fibres [7]. Exercise-induced transformation in the
reverse direction (type I to IIA) appears much more
difficult to be achieved and requires rigorous exercise
regimen [4]. According to Esbjörnsson et al. [14] and
Jansson et al. [23], sprint training may cause such trans-
formation in human vastus lateralis muscle. On the other
hand it has recently been shown that each kind of training
(endurance as well as power activities) decreases the
expression of type IIX MyHC isoform [1].
Although top class endurance athletes contain pre-
dominantly type I fibres and the muscles of power
athletes contain predominantly type II fibres [13, 18,
34], it is still not known to what extent the distribution
of muscle fibre types in sportsmen is the effect of in-
herited, genetic predisposition and environmental in-
fluences such as different training regime. Furthermore,
literature on this subject usually describes myosin
profile in muscles of world class sportsmen but little is
known about myosin characteristic in muscles of na-
tional and sub-national level athletes. Those athletes,
despite of many years of training, can not reach interna-
tional level. We postulate that one of the reasons may be
improper myosin profile in their locomotor muscles
crucial for the excellence in their sport.
Hence, the aim of the present study was to analyse the
composition of muscle fibre types and myosin heavy chains
in a thigh muscle vastus lateralis of physically active but
untrained students and of national and subnational level
sportsmen, representing different sport disciplines.
Materials and methods
Subjects. Twenty-four male volunteers (mean ± SD: age 25.4±5.8
years, height 178.6±5.5 cm, body mass 72.1±7.7 kg) of different
training background were investigated and classified into three
groups according to their physical activity and sport discipline.
Group A contained 7 physically active but untrained students of the
Academy of Physical Education in Cracow. Group B included 9
endurance athletes (7.8±2.9 years of specialised training): long dis-
tance runners, cross country skiers and cyclists. Group C consisted
of 8 athletes (12.8±8.7 years of specialised training) performing short
term, high power output sports such as ski jumping, karate, ice-hoc-
key, soccer, modern dance, volleyball and handball. Sportsmen of
group B and C represented national level in their sport disciplines.
All volunteers were subjected to detailed medical interview and
check-up. The Local Ethical Committee approved the investigations.
Muscle biopsy. After local anaesthesia (2 ml of 1% lignocaine),
percutaneous muscle biopsy samples were taken from the middle part
of vastus lateralis m. quadricipitis femoris 15 cm above the upper
margin of patella using ∅ 5 mm Bergström needles [8]. Each biopsy
was transversally divided into two parts. One part was assigned for
histochemical and immunohistochemical analyses and the other part
was used for SDS-PAGE and Western immunoblotting.
Histochemistry and immunohistochemistry. The biopsies were
covered with a mixture of Tissue Tek (TAAB laboratories, UK) and
a talcum powder and rolled into pig colon submucosa. Such speci-
mens were frozen in isopentane cooled with liquid nitrogen and
stored until use. Serial, 10 µm thick cryosections were treated for
mATPase after alkaline (pH 10.4) and acid (pH 4.35 and 4.6)
preincubations [10, 19], and for SDH activity [33]. For immunohis-
tochemistry sections were incubated overnight with mouse monoclo-
nal antibody (Novocastra Laboratories Ltd., Newcastle upon Tyne,
UK) against fast myosin heavy chain (NCL-MHCf, 1:100), slow
myosin heavy chain (NCL-MHCs, 1:150), neonatal myosin heavy
chain (NCL-MHCn, 1:50) or developmental myosin heavy chain
(NCL-MHCd, 1:60) diluted in Tris buffered saline pH 7.2 (TBS). For
visualization of immunoreactions Biotin-Streptavidin kit (DAKO,
Denmark) was applied according to kit instruction (DAKO LSAB 2
System, Peroxidase). After final washes, sections were mounted in
glycerine jelly, viewed and photographed in Axioskop light micro-
scope (Zeiss, Oberkochen, Germany). The point counting method
with A121 square lattice test system was used [48] to establish the
numerical density of the muscle fibre types in relation to the inves-
tigated biopsies. Muscle fibres of type I, IIA+IIX and IIA/I were
counted in sections stained for mATPase activity after alkaline
preincubation. Fibres of type I were also counted in sections immu-
nostained for slow myosin heavy chain while hybrid fibres express-
ing isomyosins I/IIA and fast fibres IIA + IIX were counted in
sections immunostained for fast myosin heavy chain.
SDS-PAGE and Western immunoblotting. Muscle biopsies were
cryosectioned and extracted with 62.5 mM Tris, 10% glycerol, 5%
2-mercaptoethanol, 2.3% SDS, pH 6.8. SDS-PAGE of myosin ex-
tract was performed according to Carraro and Catani [12] with 3%
stacking and 6% separating gels containing 37.5% glycerol at 60 V
for 30 min followed by 180 V for 3 h. For immunoblotting, samples
were transferred onto immobilon-P transfer membrane (Millipore
Corporation, Bedford, USA) at 30 V for 12-15 h [47]. Membrane
was blocked for 1 h in 10% non-fat dry milk in TBS, 0.1%Tween 20
(dilution buffer) and then incubated for 1 h with primary monoclonal
antibody against fast myosin diluted 1:200, or against slow myosin
diluted 1:100 in dilution buffer. Bound primary antibody was de-
tected with goat anti-mouse alkaline phosphatase conjugate (Pierce
Chemical Co., Rockford, IL, USA), diluted 1:2500, followed by
BCIP/NBT (5-bromo-4-chloro-3-indol phosphate/ nitroblue tetrazo-
lium) treatment. The relative amounts of slow and fast MyHC
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B. Zawadowska et al.

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resolved by SDS-PAGE and fast isoforms MyHCIIA and MyHCIIX
analysed by Western immunoblotting were assessed by densitome-
tric analysis of polyacrylamide gels and PVDF membranes, using
CCD camera (Fotodyne Incorporated) and a Gel Pro Analyser soft-
ware.
Statistical analysis. All values are reported as mean±standard devi-
ation (SD). Data was examined by MANOVA and tested for differen-
ces by Tuckey test. A confidence level of P<0.05 was chosen to
indicate statistical significance.
Results
Histochemistry and immunohistochemistry
Histochemical and immunohistochemical examination
of 24 muscle biopsies allowed to classify muscle fibres
into four types, I, I/IIA, IIA/I and IIA+IIX, according to
their MyHC isoform content. Fibres of type I contained
pure slow myosin and were negative in reaction for
mATPase activity after alkaline preincubation (Fig.1 a).
Type II fibres - fast fibres were considered as both
IIA+IIX fibres. They were positive (dark) after alkaline
and negative (white) after acid preincubations (Fig.1 a,
b, c). Both IIA + IIX fibres displayed intense immuno-
staining with antibody against fast myosin (Fig.1d) and
were negative after immunostaining with antibody
against slow MyHC (Fig.1e). Hybrid muscle fibres I/IIA
were detected after immunoreaction against fast myosin
and displayed an intermediate staining intensity between
the fast and slow type fibres (Fig.1d). The I/IIA fibres
were recognized in 22 investigated biopsies (Table 1).
Another hybrid muscle fibres IIA/I were discerned after
reaction for mATPase activity with alkaline preincuba-
tion as the fibres of brown colour in contrast to the black
coloured fibres of type II (Fig.1a). Fibres IIA/I were
present only in four biopsies (Table 1). The metabolic
profile of the particular types of muscle fibres was
evaluated visually on the basis of colour density of
diformazan deposits in reaction for SDH activity. Both
hybrid types of muscle fibres, I/IIA and IIA/I, showed
high oxidative activity similar to type I fibres, while type
II fibres displayed colour density from medium to none
(Fig. 1f). Immunohistochemical analysis of frozen sec-
tions from biopsies of vastus lateralis with monoclonal
antibodies against neonatal and developmental myosin
revealed that none of 24 muscle biopsies contained
neonatal or developmental myosin (not shown).
The mean percentages of slow type I, fast type II and
hybrid (I/IIA + IIA/I together) muscle fibres in vastus
lateralis from students (group A), endurance athletes
(group B) and athletes training sprint strength disciplines
(group C) are summarised in Table 1 and displayed in
Figure 2. Significant differences in muscle fibre type
content existed only between group A and the two other
groups for slow and fast fibres (P<0.02), and between
group A and group C for hybrid fibres (P<0.03).
SDS-PAGE and Western immunoblotting
Electrophoretic analysis of myosin heavy chains in poly-
acrylamide gel revealed the presence of the two most
abundant isoforms of MyHC in each of 24 biopsies of
vastus lateralis. The fastest migrating band represents
slow MyHCI isoform and the slower moving band refers
to fast MyHCIIA isoform. The third isoform, MyHCIIX,
with the slowest electrophoretic mobility was detected
in all biopsies from untrained students (group A), in 3
biopsies from endurance athletes (group B), and in 4
biopsies from athletes performing short term, high
power output sports (group C). An example of MyHC
patterns representative for each group is shown in Figure
3. Densitometric analysis of slow and fast isoforms of
MyHC resolved in polyacrylamide gels showed that
vastus lateralis from the group of students contained
almost equal amount of slow and fast myosin heavy
chains, while in endurance athletes and athletes of group
C slow MyHCI was more abundant than both MyHCIIA
and MyHCIIX (Fig. 4). Differences in MyHC content
were statistically significant at P<0.02 only between the
group of students and the other two groups of sportsmen.
To evaluate more accurately the relative amount of
MyHCIIA and MyHCIIX protein in muscle biopsies,
myosin heavy chains resolved by SDS-PAGE were
transferred onto PVDF membrane and probed with
monoclonal antibody against fast myosin. A typical
example of immunoblot representative for the investi-
gated groups is shown in Figure 5. Densitometric ana-
lysis of MyHC protein bound to PVDF membrane
revealed that MyHCIIA dominated over MyHCIIX in
all investigated biopsies (Fig. 6). However, statistically
significant differences existed only between group A
and the two other groups for MyHCIIA at P<0.01, and
for MyHCIIX at P<0.03. There was no difference be-
tween group B and C for MyHCIIA or MyHCIIX. The
results of SDS-PAGE and Western immunoblotting ana-
lyses for all 24 subjects are summarised in Table 1.
To evaluate the relative proportions of MyHCI,
MyHCIIA and MyHCIIX in biopsies from vastus lat-
eralis, data from Western blotting was expressed as
percentages of total MyHCII estimated by SDS-PAGE.
Therefore, in group A there was 41.7±1 .6% of MyHCI,
40.8±4.0% of MyHCIIA and 17.5±4.0% of MyHCIIX,
in group B: 64.3±0.8% of MyHCI, 34.0±1.4% of
MyHCIIA and 1.7±1 .4% of MyHCIIX, and in group C:
59.6±1.6% of MyHCI, 37.2±1.3% of MyHCIIA and
3.2±1.3% of MyHCIIX.
Analysis of correlation
Three different methods were used for examining
MyHC protein expression in human vastus lateralis
muscle - myofibrillar ATPase assay, immunohisto-
chemical labelling with monoclonal antibodies and
Myosin profile in human skeletal muscle
183

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Table 1. Histochemical, immunohistochemical and electrophoretical characteristics of muscle biopses from vastus lateralis of 24 volunteers
184
B. Zawadowska et al.

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SDS-PAGE followed by Western immunoblotting.
There was a significant positive correlation at P<0.001
between: the relative amount of slow and fast MyHC
resolved by SDS-PAGE, and the number of slow and
fast muscle fibres assayed by mATPase (r=0.74), the
number of slow and fast muscle fibres demonstrated by
monoclonal antibodies and assayed for mATPase
(r=0.95), and the relative amount of slow and fast MyHC
resolved by SDS-PAGE, and the number of slow and
fast muscle fibres demonstrated by monoclonal anti-
bodies (r=0.84) (Fig. 7). High values of correlation
coefficient point out a strong correlation between the
specific parameters estimated with these methods.
Discussion
The main purpose of this study was to characterise
myosin profile of thigh muscle vastus lateralis in na-
tional and subnational level athletes representing differ-
ent sport disciplines in relation to a control group of
physically active but untrained students. This main lo-
comotor skeletal muscle was chosen because it is well
Fig. 1. Histochemical and immunohistochemical analysis of slow I, fast IIA+IIX, and hybrid I/IIA, IIA/I muscle fibres in serial frozen sections
from human vastus lateralis muscle. Myosin ATPase activity after preincubations in buffers at pH: (a) 10.4, (b) 4.35, (c) 4.6. Immunostaining
with monoclonal antibodies against: (d) fast and (e) slow myosin isoforms. Histochemical staining for SDH activity to depict aerobic capacity
of muscle fibres (f). Bar = 50 µm.
Myosin profile in human skeletal muscle
185