Download Skeletal muscle morphology in power-lifters with and without

Histochem Cell Biol (2005) 124: 167–175
DOI 10.1007/s00418-005-0029-5
O R I GI N A L P A P E R
Anders Eriksson Æ Fawzi Kadi Æ Christer Malm
Lars-Eric Thornell
Skeletal muscle morphology in power-lifters with and without
anabolic steroids
Accepted: 25 May 2005 / Published online: 30 July 2005
Springer-Verlag 2005
Abstract The morphological appearance of the vastus
lateralis (VL) muscle from high-level power-lifters on
long-term anabolic steroid supplementation (PAS) and
power-lifters never taking anabolic steroids (P) was
compared. The effects of long- and short-term supplementation were compared. Enzyme-immunohistochemical investigations were performed to assess muscle fiber
type composition, fiber area, number of myonuclei per
fiber, internal myonuclei, myonuclear domains and
proportion of satellite cells. The PAS group had larger
type I, IIA, IIAB and IIC fiber areas (p<0.05). The
number of myonuclei/fiber and the proportion of central
nuclei were significantly higher in the PAS group
(p<0.05). Similar results were seen in the trapezius
muscle (T) but additionally, in T the proportion of fibers
expressing developmental myosin isoforms was higher in
the PAS group compared to the P group. Further, in
VL, the PAS group had significantly larger nuclear domains in fibers containing ‡5 myonuclei. The results of
AS on VL morphology in this study were similar to
A. Eriksson Æ C. Malm Æ L.-E. Thornell (&)
Department of Integrative Medical Biology,
Section for Anatomy, Umeå University,
901 87 Umeå, Sweden
E-mail: [email protected]
Tel.: +46-90-7865142
Fax: +46-90-7865480
A. Eriksson
Department of Health Care,
Section for Medical Science,
Luleå University,
961 36 Boden, Sweden
A. Eriksson Æ L.-E. Thornell
Department of musculoskeletal Research,
Gävle University,
907 13 Gävle, Sweden
F. Kadi
Department of Physical Education and Health,
Örebro University, 701 82 Örebro, Sweden
C. Malm
Winternet, Luleå university, 961 36 Boden, Sweden
previously reported short-term effects of AS on VL. The
initial effects from AS appear to be maintained for
several years.
Introduction
Investigating the mechanisms of long-term supplementation of AS on human trapezius muscle, Kadi (2000)
showed that the enlargement of muscle fibers is accompanied by an increase in the myonuclear number and
satellite cell content. In addition the PAS group have a
significant higher number of cells expressing fetal myosin heavy chain (MyHC) than power lifters not using
anabolic steroids, indicating ongoing new fiber
generation.
The alterations in the number of myonuclei and
satellite cells in response to AS were later confirmed in a
study on the short term effects (20 weeks) of combined treatment with gonadotropin-releasing hormone
(GnRH) agonist and testosterone on the vastus lateralis
muscle (Sinha-Hikim et al. 2002, 2003). However, the
magnitude of muscle fiber hypertrophy was higher in
trapezius from athletes with long-term AS supplementation than in muscles from athletes after 20 week of AS
supplementation. Another difference was the increased
number of centrally located myonuclei in the trapezius
muscle (Kadi 2000). These findings might reflect discrepancies in either the response to AS or differences in
contraction pattern during exercise between the trapezius and the vastus lateralis muscles.
The vastus lateralis is of importance preferentially in
the squat event, when the lifter from an upright position
and with the barbell resting across the back of the
shoulders, sits or ‘‘squats’’ down by doing a flexion in
the knee and hip-joints to a required depth. The lifter
then attempts to stand up again, returning to the original position. Such a lift takes approximately 2–5 s and
requires a maximal explosive force.
168
In a dead lift the lifter takes the barbell from the floor
to an upright standing position until validated by the
referee. In this lift the trapezius muscle performs an
isometric contraction for a total time of approximately
8–10 s. These differences in utilization between vastus
lateralis and trapezius might be reflected at the muscle
fiber level.
The aim of the present investigation was to study the
long-term effects of AS on fiber type composition, fiber
area, number of myonuclei, internal nuclei, nuclear domains and the number of satellite cells in human vastus
lateralis. We also compared the long-term effects of AS
on vastus lateralis to (1) the long-term effects of AS on
the trapezius muscle, previously reported by Kadi
(2000), Kadi et al. (1999b) and (2) to the short-term
effects (20 weeks) of AS on the vastus lateralis muscle
(Sinha-Hikim et al. 2003).
All subjects gave their informed consent to participate in the present study. The Ethical Committee of
Umeå University approved this work. Written consent
in accordance with the policy statement regarding the
use of human subjects was obtained from all the
subjects.
Muscle samples
Biopsies were taken from the upper ventral part of vastus lateralis muscle using forceps. The samples were
mounted in embedding medium (Tissue tek, Miles laboratories, Naperville Ill) and quickly frozen in propane
cooled in liquid nitrogen and stored at 80C until
analyzed.
Enzyme histochemistry
Material and methods
Subjects
Nineteen power-lifters participated in the present study.
Nine of the subjects (31.4±3.3 years) have reported the
use of a wide variety of high doses of testosterone and
anabolic steroids for a period of 9±3.3 years, whereas
ten other power-lifters (27.7± 7.5 years), have never
used these substances. The drug free group had signed a
contract with the local club and Swedish power lifting
federation that committed them never to use any drugs.
Four of them participated voluntarily as controls in
another project aiming to find more effective methods to
detect drugs. We were able to perform this study because
one of the authors (Anders Eriksson, European and
Nordic champion in power lifting 1988) has personal
contacts with the Swedish elite power-lifters.
All athletes were highly competitive and participated
regularly in Swedish and/or international competitions
in power events. They trained regularly four to six times
a week, two to three hours per session. The sessions
consisted in four to seven sets of exercises and three to
twelve repetitions per set.
The nine drug using subjects have been individually
interviewed regarding their steroid usage.
Testosterone was used in combination to a variety of
anabolic steroids (nandrolone, stanozolol, primobolan,
oxymetholone, mastoron, proviron and durobolan). A
mean dosage of 938±527 mg testosterone and anabolic
steroids were self-distributed each week. In addition,
three subjects had used IGF1 (mean dosage 40 mg/day)
and one subject growth hormone. Two of them have
been caught in regular drug testing. The steroid regimen
included both ‘‘staking‘’, or simultaneous use of several
types at high doses, and ‘‘cycling’’, a drug-free period
followed by times when the doses and the types of drugs
taken were increased to a maximum to anticipate peak
performance.
Serial 10-lm thick cross-sections were cut at 20C
using a cryostat microtome, mounted on glass cover
slips, and air-dried at room temperature. The sections
were stained for the demonstration of myofibrillar ATPase (EC 3. 6. 1. 3) after alkaline (pH 10.4 and 9.4) and
acid (pH 4.6 and 4.3) preincubations (Dubowitz 1985).
Visualization of muscle fiber myonuclei were performed
with a Mayer’s hematoxylin (HTX) staining.
Fiber classification
The muscle fibers were classified into types I, IIA, IIAB,
IIB and IIC according to their staining intensity for
myofibrillar ATPase at alkaline and acid pH. For details
see (Kadi 2000).
Immunohistochemistry
Five micrometers thick cross-sections, serial to those
used for enzyme histochemistry, were used for immunohistochemical analysis. Myosin heavy chain (MyHC)
expression was assessed using well-characterized
monoclonal antibodies (mAbs) against human MyHC I
(mAb A4.840) and MyHC I& IIA (mAb N2.261)
(Hughes et al. 1993). The mAb A 4.840 strongly stained
type I fibers, whereas type IIA, IIAB and IIB were
unstained. The mAb N2.261 stained strongly type IIA
fibers, whereas type I and IIAB were equally weakly
stained and type IIB fibers were unstained. Type IIC
fibers were strongly stained with mAb N2.261 and
moderately stained with mAb A4.840. Type IM fibers
exhibited the reversed pattern. For detection of developmental MyHC isoforms sections were stained with
mAb against fetal (NCL-MHCn) MyHCs (Ecob-Prince
et al. 1989). The antibodies were purchased from
Novocastra Laboratories.
169
Identification of the cell border of the muscle fibers
and capillaries was performed with mAb 5H2 against
laminin a2 chain (Sewry et al. 1995). Monoclonal Ab
5H2 labels the basement membrane of muscle fibers
strongly.
For the identification of myonuclei and satellite cells,
sections were double stained with mAb CD 56 against
the Leu 19 antigen and Mayer’s hematoxylin. The
antibodies were obtained from Becton Dickinson. Leu
19 antigen is a cell–cell recognition molecule expressed
during the early stages of fiber formation and in satellite
cells. Myonuclei were stained blue whereas a brown rim
inside the muscle fiber identified satellite cells (Kadi
et al. 1999a). The MHC antibodies were purchased from
the American Type Culture Collection.
The proportion of fibers containing fetal MyHCs was
calculated as follows: [(number of fibers containing fetal
myosin)/(total number of myofibers) · 100].
Immunohistochemical visualization of bound antibody in mAbs against Leu-19, and the MyHCs was
performed using indirect peroxidase-antiperoxidase
(PAP) staining (Sternberger 1979).
Morphometric analysis
Fiber area, fiber types, myonuclei and satellite cells were
analyzed with a light microscope (Zeiss Axiophot, Carl
Zeiss, Oberkochen, Germany) connected to an image
analysis system (IBAS, Kontron elektronic GMBH,
Eching, Germany). Two to four randomly chosen areas
from each biopsy were scanned. For analysis of fiber
area each biopsy was scanned in sections stained for
mAbs against laminin (5H2) and the circumference of
each fiber was traced along the periphery of the basement membrane. Counting of number of myonuclei per
fiber cross-section and frequency of fibers containing
internal myonuclei were performed on sections stained
for HTX. Satellite cell frequency was calculated on
sections stained for Leu 19 as follows: [(satellite cell
number)/(myonuclear number)+satellite cell number) ·
100]. This method has previously been used by Kadi
et al. (1999a) and Kadi and Thornell (2000). The proportion of developmental MyHCs was measured on
whole muscle cross-sections. Classification and measurement of mATPase fiber types and myonuclei were
performed with a 20· objective. Counting of fetal
MyHC containing fibers was performed with a 10·
objective.
The statistical significance of correlations between two
parameters was determined by using Fichers r to z test.
p-values <0.05 were considered statistically significant.
Results
Fiber types
A mosaic pattern of fiber types in cross-sectioned
biopsies was observed in most subjects. The mean values
for all fiber types in both groups were very similar but
the individual variation was large (Table 1, Fig. 1). Type
I and type IIA fibers were the most frequent fiber types
in most subjects but their frequency varied from 28 to
62% for type I fibers and from 4 to 69% for type IIA
fibers. The proportions of type IIAB and type IIB fibers
varied even more. Type IIAB was found in proportions
from 0 to 33% and type IIB from 0 to 50%. Interestingly, type IIB fibers occurred preferentially in two
subjects in the P group. Fiber type grouping occurred in
several of the biopsies from subjects in the PAS group
(Fig. 2) but also to some extent in biopsies from subjects
in the P group.
The number of fibers expressing fetal MyHC varied
considerably between the subjects. In the P group and
in five subjects of the PAS group the proportion of
fibers expressing fetal myosin was between 0 and 2.0%
whereas four subjects in the PAS group had 3.8, 4.1,
12 and 36% of their fibers expressing fetal myosin. The
mean values for percent fibers expressing fetal MyHC
were 6.7±11% in the PAS group and 0.6±0.6% in
the P group (p = 0.12) (Fig. 2, Table 1). No statistically significantly differences were observed in the
proportions of fiber types between the PAS and the P
groups.
Fiber area
Type IIA and type IIAB fibers had the largest mean fiber
area in both groups, but the PAS group had 44% larger
type II fibers than the P group (p<0.001) (Table 2). The
Table 1 Mean fiber type and fiber area distribution. No significantly differences were found between the groups
Fiber Type
type
I
Statistical analysis
Data are presented as means and standard deviations.
The statistical significance of the differences between the
two groups was determined using a t-test for unpaired
data. The correlation coefficient (r) was used to determine the degree of relationship between two variables.
Type
IIA
Type
IIAB
Type
IIB
Type
IIC
Fibers
expressing
fetal myosin
Proportional fiber type distribution
PAS 40±12 40±16 14±11 0.7±1.6 5.0±2.0 6.7±11
P
46±9
35±20 9±11 7±16
3±3
0.6±0.6
Proportional fiber area distribution
PAS 32±16 45±21 15±13 0.7±1.5 5.1±2.5
P
36±9.1 40±21 11±15 9.1±20 2.7±2.5
170
Fig. 1 Immunostained crossections of one PAS subject (A and C)
and two P subjects (B, D, E and F). A and B. Most fibers in the
PAS (A) and the P (B) subject were of type IIA as evidenced by
staining with mAbs N2261 which stains type IIA fibers strongly
(IIA) and type I fibers weakly (I). C and D. The outlines of the
fibers are visualized with an antibody against laminin. Note the
large variation in size of the fibers. Staining within fibers (arrows)
indicate some split fibers. E and F. Serial sections stained with
mAbs A4840 (E) and N2261 (F) of the subject showing the highest
proportion of type IIB fiber. Three fibers are of type I (stained in
both E and F (I)) and one fiber is of type II A (strongly stained in F
(IIA)) the rest are type IIB and type IIAB fibers (IIAB, IIB). Bar:
A, B, E and F 25 lm; C and D 50 lm
171
mean fiber area of both type IIA and IIAB fibers was
significantly different between the groups (p<0.05). Similarly, the mean type I fiber area was 61% larger in the
PAS group than in the P group (p<0.01) (Table 2).
Myonuclei
The mean number of myonuclei/fiber was 32% higher (p
= 0.0001) in the PAS group (6.0±2.2) compared to the
P group (4.5±1.6) (Table 3).
Compared to the P group, the PAS group had significantly higher proportion of fibers containing internal
myonuclei (29±18 vs 9.3 ±10%) (p = 0.007) (Table 3).
When the number of myonuclei from both the PAS
and P groups was plotted against fiber area, a highly
significant correlation was found between the fiber area
and the number of myonuclei/fiber (r = 0.73, p =
0.0002) (Fig. 3). The mean nuclear domain was
1,531±209 lm2 in the PAS group and 1,348±267 lm2
in the P group. The difference was significant in fibers
containing ‡5 myonuclei per fiber (Fig. 4) (p = 0.011).
When both groups were combined, a statistically significant correlation was found between the proportion of
fibers expressing internal myonuclei and fiber area (r =
0.68, p = 0.0009) (Fig. 5).
Satellite cells
The proportions of satellite cells in the PAS and P
groups were practically identical (9.3±4.0 vs 9.4±3.0%,
respectively) (Table 3).
Discussion
To our knowledge, this is the first comparative morphological study of the human vastus lateralis muscle in
two groups of high-level power-lifter athletes. One group
admitted supplementation with testosterone, anabolic
steroids and other banned substances for nearly ten
years and the other group affirmed never taken banned
substances. As we previously have studied the trapezius
muscle from the same individuals (Kadi et al. 1999b) it
allows us now to compare the AS influence on two
muscles used in different ways in power lifting.
The vastus lateralis muscle demonstrated larger
muscle fiber areas than normal in both groups (Staron
et al. 2000). However the PAS subjects differed from the
P subjects in having significantly larger fiber areas, more
myonuclei per fiber and more internal myonuclei. No
differences were seen with regard to fiber type proportions and frequency of satellite cells. All subjects in the
PAS group and seven in the P group had small fibers
expressing fetal myosin but there was no significant
difference between the groups.
The PAS group had 61% larger type I fiber area and
44% larger type II fiber area than the P group. In the
trapezius, the areas were 58 and 33% larger in type I and
type II fibers, respectively (Kadi et al. 1999b). In fact,
AS supplementation, even without strength training has
been reported to induce hypertrophy in human skeletal
muscles (Bhasin et al. 1996; Sinha-Hikim et al. 2002). In
the study by Sinha-Hikim et al. (2002) the muscle fiber
hypertrophy (fiber area) after 20 weeks of AS supplementation (600 mg/week) was 49% in type I fibers and
36% in type II fibers compared to baseline. The dramatic hypertrophic effect on muscle fibers in subjects
supplementing with AS is in accordance with the current
conception on the effects of testosterone and anabolic
steroids, for review see (Herbst and Bhasin 2004).
Maximal force of a muscle is related to the muscle fiber
area, the total muscle area and the fiber types (Bruce
et al. 1997; Bamman et al. 2000).
In the present study we observed that there was a
highly significant positive correlation between fiber area
and total number of myonuclei in the vastus lateralis
muscle (Fig. 3). The PAS subjects, who had the largest
fibers, also had the largest numbers of myonuclei, both
subsarclemmal and centrally located. The same correlation was observed in our study on the trapezius muscle
(Kadi et al. 1999b). In the study from Sinha-Hikim et al.
(2002) the number of myonuclei was significantly increased and was correlated to fiber area. Altogether,
these results support the idea that the number of
myonuclei plays a mechanistic role in muscle fiber
hypertrophy (Edgerton and Roy 1991; Allen et al. 1999;
Kadi 2000).
It is well known that each nucleus supports a certain volume of the cytoplasm with mRNA for turnover
of proteins. This volume is often referred to as a nuclear domain (Cheek 1985). If a myonucleus can expand its nuclear domain by increased synthesis of
mRNA or by more efficient transport of mRNA has
been discussed (Sinha-Hikim et al. 2003). Recently
Kadi et al. (2004) reported that satellite cells are plastic
in response to resistance training and that moderate
changes in skeletal muscle fiber area can be achieved
without addition of new myonuclei. However, our data
suggest that addition of myonuclei is a prerequisite for
more substantial muscle fibers hypertrophy (Kadi et al.
1999b).
In myopathology it is considered pathological when
more than 3% of the fibers contain centrally located
nuclei (Greenfield 1957). Centrally located nuclei in
muscle fibers of strength-trained subjects might be a
phenomenon of adaptation. The centrally located nuclei
might be needed to support extremely large fibers,
preferentially present in the PAS group. Centrally located nuclei will reduce the diffusion distances from a
nucleus to central parts of the myofiber. The observation
of large number of fibers with internal nuclei, 29% in the
PAS group and 9% in the P group, is of interest in this
context. Our previous results on internal nuclei in the
trapezius (25% in the PAS group and 5% in the P group
172
Fig. 2 Serial cross sections
from PAS subject 1(A–H) and
PAS subject 3 (I–P). The
sections were stained with
mAbs N2.261 (A, E, I and M),
A4.840 (B, F, J and N) and
NCL-MHC (C, G, K and O)
and for NADH dehydrogenase
activity (D, H, I and P).
Marked areas in (A–D) and (I–
L) are shown in higher
magnification in (E–H) and
(M–P). In both subjects, there
was a preponderance of type
IIA fibers (strongly stained in
A; E; I and M and unstained in
B; F; J and N). Note type
grouping (*) (group of fibers
show the same staining) in I; J
and L. In PAS subject 1 a
medium sized (enlarged in G)
and a number of small fibers
(arrows in C) show staining for
fetal myosin. In PAS subject
3(K) a high number of small
sized fibers are stained (arrows)
and sometimes form groups of
fibers (area enlarged in O).
Sections A–D and I–L: Bar
10 lm; Sections E–H and M–P:
Bar 25 lm
(Kadi et al. 1999b)) support this concept. We thus propose that the presence of internal nuclei reflects the
limited volume of each nuclear domain, although the
mechanisms of internalization of myonuclei remain unknown.
The PAS group had larger nuclear domains in fibers
containing more than five myonuclei per fiber compared
to the P group (1,531±209 vs 1,348±267 lm2, p<0.01)
(Fig. 4). Further, if combining the results from both
vastus and trapezius, the difference is 13%
(1,656±254 lm2 in the PAS group and 1,463±325 lm2
in the P group) (p<0.05). The suggestion that AS also
affects the size of the myonuclear domains is supported
by the significantly larger nuclear domains in subjects
treated with 300 or 600 mg of testosterone per week
compared to controls (Sinha-Hikim et al. 2003). Also,
strength training will increase myonuclear domains, at
least in older men (Hikida et al. 2000).
Myonuclei in mature muscle fibers are not able to
divide, which means that an increase in myonuclei
number must come from an external source, reviewed by
(Allen 1999). It is generally accepted that these additional nuclei comes from satellite cells (SC) and/or stem
cells (for review see Morgan 2003). In both the PAS and
P groups we observed a larger proportion of SCs than
has been reported for control subjects (for review see
Hawke and Garry 2001). However, no significantly differences were observed between the PAS and the P
173
Table 2 Mean fiber areas for each subject and group values. The PAS group had significantly larger fiber areas for type I, IIA, IIAB and
type IM fibers compared to those of the P group (p<0.05)
PAS
Subject
Type I
SD
Type IIA
SD
Type IIAB
SD
Type IIB
SD
Type IIC
SD
Type IM
SD
1
2
3
4
5
6
7
8
9
Mean
P
1
2
3
4
5
6
7
8
9
10
Mean
4,453
11,588
6,033
6,401
6,592
7,913
5,088
11,251
9,889
7,690*
1,122
3,308
1,773
3,572
1,479
1,777
2,480
5,276
3,906
2,637
14,382
12,159
10,062
10,966
6,581
8,449
10,679
14,709
11,231
11,025*
4,334
3,396
2,604
5,460
1,500
1,971
4,756
3,902
2,439
2,589
10,159
9,300
0.0
11,851
6,382
8,689
10,378
13,044
10,417
10,028*
2,193
2,956
0.0
4,285
1,791
2,336
2,805
3,640
1,394
2,012
0.0
0.0
0.0
13,714
5,934
0,0.
0.0
0.0
0.0
9,826
0.0
0.0
0.0
0.0
395
0.0
0.0
0.0
0.0
5,498
0.0
0.0
0.0
0.0
1,993
12,057
10,875
0.0
0.0
8,309
0.0
0.0
0.0
0.0
4,616
274
1,511
0.0
0.0
5,501
11,575
12,412
13,920
11,300
6,791
6,182
11,769
11,212
8,690
10,428*
8,945
1,616
4,402
4,364
0.0
2,465
6,122
1,769
2,413
2,620
6,188
4,394
4,216
3,948
3,030
5,361
5,461
3,506
6,544
5,044
4,770
1,590
1,337
901
1,124
1,222
1,259
1,626
931
1,280
1,541
1,145
6,109
8,787
7,869
7,146
6,001
7,571
7,428
5,739
9,827
6,621
7,310
1,850
1,995
1,245
2,079
1,236
2,057
1,901
1,246
3,699
1,478
1,293
9,347
8,482
7,319
7,954
6,673
7,829
0.0
5,212
0.0
0.0
7,545
0.0
1,205
2,479
1,662
1,709
1,368
0.0
1,236
0.0
0.0
1,331
0.0
7,877
8,134
7,167
6,852
0.0
0.0
0.0
0.0
0.0
7,508
0.0
1,053
2,566
702
2.0
0.0
0.0
0.0
0.0
0.0
598
0.0
0.0
0.0
0.0
3,401
5,232
0.0
7,063
0.0
0.0
5,232
0.0
0.0
0.0
0.0
2,347
0.0
0.0
0.0
0.0
0.0
1,831
0.0
0.0
0.0
5,785
0.0
7,934
6,220
5,763
8,854
6,910
6,911
0.0
0.0
0.0
0.,0
0.0
4,576
1,738
1,610
3,187
3,549
1,256
Table 3 Mean number of myonuclei per fiber, fibers expressing
internal myonuclei and proportion of satellite cells for each subject.
The abbreviation (*) means significantly different (p<0.05) between the groups
PAS
Subject
Nuclei/fiber
% fibers with
internal nuclei
Satellite cells
% of total nuclei
1
2
3
4
5
6
7
8
9
Mean
P
1
2
3
4
5
6
7
8
9
10
Mean
7.7
7.6
4.8
5.3
5.1
7.0
5.8
6.1
6.8
6.2±1.1*
57.7
19.2
15.2
8.3
6.3
37.0
41.5
42.9
36.1
9.1±5.5*
6.3
15.5
11.1
15.0
3.8
9.2
8.5
7.7
6.3
9.3±4.0
4.9
3.7
4.6
4.4
5.2
6.3
3.7
3.8
6.4
3.6
4.7±1.0
19.2
20.8
3.8
2.3
5.0
29.5
0.0
2.0
2.6
8.1
5.4±5.8
7.3
8.4
8.0
13.8
13.1
11.0
10.9
6.0
4.8
11.1
9.4±3.0
groups, neither in the vastus nor the trapezius muscle.
One possible explanation for the higher number of nuclei per fiber in the PAS group can be an increased
turnover rate of the satellite cells. A significant increase
in satellite cell number has also been observed in young
men after supplementation with 300 and 600 mg of
Fig. 3 Relationship between the mean myonuclei number per fiber
and the cross-sectional area in type I and type II fibers from the
PAS and the P groups. Correlation coefficient r = 0.73, p<0.01
testosterone per week for 20 weeks, even without exercise (Sinha-Hikim et al. 2003). Thus, supplementation of
testosterone and anabolic steroids as well as high-level
resistance training increases the number of satellite cells.
In this study, the fiber typing was based on the
myofibrillar ATPase activity after alkaline and acid
preincubation (Hughes et al. 1993). The accuracy of the
fiber typing was secured on basis of immunohistochemical staining for the different MyHC’s, which is the
basis for the myofibrillar ATPase activity (Schiaffino
and Reggiani 1994). Human muscle type I fibers contain
slow MyHC, the type IIA fibers MyHC IIA and IIB
174
Fig. 4 Muscle fibers separated into two classes on basis of their
mean number of myonuclei. The size of the nuclear domains were
calculated as a range between mean fiber area and number of nuclei
per fiber. One PAS subject (no standard deviation) and seven P
subjects formed the class with less than five myonuclei/fiber)
whereas eight PAS subjects and three P subjects formed the class
with five myonuclei or more. In this latter class, the PAS group had
significantly larger myonuclear domains compared to the P group
(p = 0.011)
Fig. 5 Relationship between the proportion of fibers expressing
internal myonuclei and the mean fiber area in the vastus lateralis
muscles from the PAS and the P groups. Correlation coefficient r =
0.68, (p<0.01)
fibers MyHC IIX. The MyHC IIB, which is the fastest
MyHC in rodents, is not present in human limb muscles
(Smerdu et al. 1994; Ennion et al. 1995). In humans, the
order of contraction force and speed of the MyHC’s is IIIA-IIX where I is the slowest and weakest and IIX is
the fastest and strongest (Hilber et al. 1997; Larsson
et al. 1997).
The strength-trained athletes, both the PAS and the P
subjects, had a high frequency of type II fibers and
mainly type IIA fibers. A variation in fiber type ATPase
activity was commonly observed, which has been related
to the fibers content of different MyHCs (Staron 1997;
Pette and Staron 2001). This is typical for highly trained
subjects, indicating transformation of fibers to optimize
performance (For review see Pette and Staron 2001). In
a previous study, we did not observe a significant difference in proportion of fiber type distribution between
the PAS and the P groups in the trapezius muscle (Kadi
2000). No change in the relative proportion of fiber
types was observed in subjects treated with testosterone
for 20 weeks (Sinha-Hikim et al. 2002). Thus, testosterone and anabolic steroids do not seem to affect the
relative fiber type distribution in human skeletal muscles. From the view of being successful in sports,
examining the individual data for the subjects revealed
that each individual athlete actually showed a personal
muscle profile. A person with higher proportion of type
IIB fibers would, from a theoretical point of view, have a
greater chance of becoming a successful power lifter.
Interestingly, the subject who had the highest percentage
of type IIB fibers (Fig. 1E, F), with approximately half
of the muscle fibers being type IIB, had at the time for
the biopsy the world record in his weight class in squat
and he was not taking AS. Conversely, the subjects with
the largest fiber areas were found in the PAS group,
which means that these subjects in that respect had an
advantage over those in the P group with smaller areas.
Our study also shows that several subjects in the PAS
group had a large amount of small fibers expressing fetal
myosin. Adult muscle fibers do not normally express
fetal myosin. The presence of developing myosin isoforms has been interpreted as signs of hyperplasia
(McCormick and Schultz 1992; McCormick and Thomas 1992; Antonio and Gonyea 1993). A significant
increase in fibers stained for developing isoforms of
myosin, has been demonstrated with strength training
(Antonio and Gonyea 1993; Kadi and Thornell 1999;
McCall et al. 1996; Kadi 2000). These fibers might reflect newly formed fibers or abortively regenerated fibers. In the latter case, failed innervation can cause
degeneration and the new fibers would be of no use for
the athletes. It can be speculated that the increased
number of fibers with fetal myosin is a result from using
AS for almost ten years.
In conclusion, these results suggest that the action
from AS is similar in the vastus lateralis, both long-term
and 20 weeks supplemented, and in the trapezius muscles despite differences in contraction pattern. The results are in agreement according to larger fiber areas,
correlation between myonuclei number per fiber and fiber area and to an increased number of myonuclei and
satellite cells.
Acknowledgements We thank Margareta Enerstedt, Mona Lindström and Lena Carlsson for excellent technical assistance. This
study was supported by grants from the Swedish National Centre
for research in sports (90/98, 79/99), the Swedish Research Council
(12X-03934) and the Medical faculty of Umeå University.
175
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