acta histochem.

104(4) 399405 (2002)

Urban & Fischer Verlag
http://www.urbanfischer.de/journals/actahist

Fibre type composition

of soleus and extensor digitorum longus muscles
in normal female inbred Lewis rats
Toms Soukup1*, Gisela Zacharov1, and Vika Smerdu2
1
Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic, and
2
Institute of Anatomy, Medical Faculty, Ljubljana, Slovenia

Received 19 February 2002; accepted 29 April 2002

Summary
We have analysed the fibre type composition of soleus and extensor digitorum longus (EDL) muscles
of normal female 46-month-old inbred Lewis rats. This rat strain is used in our ongoing study of the
effects of thyroid hormone on myosin heavy chain (MyHC) isoform expression. On the basis of the
mATPase reaction, soleus muscles contained 96.1 2.9% of type 1 fibres supplemented by 2A fibres.
EDL muscles contained type 1 (5.5 1.0%), type 2A (18.8 1.7%) and type 2B (75.7 2.2%) fibres.
Immunohistochemical analysis and SDS gel electrophoresis confirmed that most fibres in the soleus
muscle expressed the type 1 (slow) MyHC isoform and that only a small proportion of fibres
expressed the fast 2a MyHC isoform. Immunohistochemical analysis and SDS gel electrophoresis
demonstrated that almost half of the 2B fibres of EDL muscles expressed the 2x/d MyHC isoform. In
both muscle types, many fibres expressed more than one MyHC isoform. The content of slow fibres
in the soleus muscle of female inbred Lewis rats was slightly higher than that reported for Wistar rats,
but was considerably higher than that of Sprague-Dawley rats, whereas substantial differences were
not found in the proportion of slow and fast fibre types in EDL muscles in these strains.

Key words: inbred Lewis rats skeletal muscles soleus and EDL muscles fibre types mATPase
MyHC isoforms

Introduction
At present, we are studying the effects of innervation
and thyroid hormone levels on fibre type composition
and myosin heavy chain (MyHC) isoform expression in
regenerating and adult slow soleus and fast extensor
digitorum longus (EDL) muscles in rats with an experi-
mentally-altered thyroid status (Zacharov et al., 1999;
Ladecky et al., 2000; Soukup et al., 2001; for review see
Soukup and Jirmanov, 2000). For this purpose, hete-
rochronous isotransplantation was introduced (Jir-
manov and Soukup, 1995), whereby the soleus or EDL
muscle of 24-week-old rats of the Lewis strain are iso-
grafted either into the soleus or EDL muscle of the adult
host inbred recipient of the same strain.
Inbred rats have to be used in order to prevent graft
rejection. Therefore, fibre type composition of the
soleus and EDL muscles of normal nonoperated inbred
Lewis rats are used as a reference for the evaluation of
changes due to transplantation, exogenous nerve inner-
vation and/or altered thyroid hormone status in muscles
of the experimental animals. Although the fibre type

*Correspondence to: Dr. Toms Soukup, Institute of Physiology, Czech Academy of Sciences, Vdensk 1083, CZ-14220 Prague, Czech Republic;
fax: +420-2-4106 2488; e-mail: tmsoukup@biomed.cas.cz

0065-1281/02/104/04-399 $ 15.00/0
400 Soukup et al.

composition of these 2 types of rat muscles is well
known, data for the inbred Lewis strain do not exist as
far as we know. Hence, the aim of the present study was
to determine the fibre type composition of soleus and
EDL muscles of normal nonoperated female rats of the
inbred Lewis strain.
Material and methods
Six female inbred rats of the Lewis strain, 46-month-
old, obtained from an authorised laboratory rat-breed-
ing unit of the Institute of Physiology (Accreditation
number 1020/491/A/00) were used. The Expert Com-
mittee of the Institute of Physiology approved the inves-
tigation and the animals were treated in accordance with
principles of the Care and Use of Animals approved for
the Institute of Physiology by NIH (Bethesda, USA)
under number A5228-01.
The excised soleus and EDL muscles were immedi-
ately frozen in liquid nitrogen and treated as described
previously (Soukup et al., 2001). To asses fibre type
composition of the muscles (1, 2C, 2A and 2B), cryostat
sections were processed for the histochemical assay to
demonstrate myofibrillar adenosine triphosphatase
(mATPase) activity after alkaline (pH 10.3) and acid
(pH 4.5 and 4.3) preincubations (Dubowitz and Brooke,
1973). To demonstrate MyHC isoform expression,
monoclonal antibodies (mAbs) recognising MyHC-1
(BA-D5), MyHC-2a (SC-71), MyHC-2x/d (BF-35 as a
negative marker) and MyHC-2b (BF-F3) were used
(Schiaffino et al., 1986; antibodies were kindly supplied
by Prof. S. Schiaffino, CNR Center of Muscle Biology
and Physiopathology, University of Padova, Italy).
Antibody binding was revealed with the use of a stan-
dard PAP method (Sternberger, 1986; Dako, Glostrup,
Denmark). MyHC isoforms were separated by SDS-
glycerol gel electrophoresis according to the method

Fig. 1. Serial sections of soleus muscle of a 4-month-old female rat of the inbred Lewis strain. Sections were stained with monoclonal anti-
bodies specific for type 1 (BA-D5), type 2a (SC-71), type 1 + 2a + 2b (BF-35; negative marker for 2x/d MyHC) and type 2b (BF-F3) MyHC iso-
forms (Schiaffino et al., 1986). Note that despite different staining intensities, all fibre types reacted with the BF-35 antibody (negative mark-
er for 2X/D fibres) and all fibres did not stain with the BF-F3 antibody. Bar, 100 m.

acta histochemica 104, 4 (2002)

 Fibre types in inbred Lewis rats 401

described by Talmadge and Roy (1993). Running condi-

tions for the minigels were 72 V for 32 h. After separa-
tion, gels were silver-stained (Blum et al., 1987). Per-
centages (%) of muscle fibre types were assessed by the
2-D stereological method using the principles of an
unbiased counting frame (Zacharov and Kubnov,
1995; Zacharov et al., 1997). Stereological measure-
ments were performed on the CAST Grid System
(Olympus, Albertslund, Denmark). Data were expres-
sed as means SD and the significance of differences
was evaluated with the Students t test.
Results
On the basis of mATPase activity, normal soleus muscles
from both legs of 46-month-old female inbred Lewis
strain rats (mean age 21 5 weeks) contained 96.1
2.9% of type 1 fibres. The fibres exhibited high mATPase
activity after acid preincubation and low activity after
alkaline preincubation. The remainder of the fibres were
type 2A fibres with opposite characteristics and only a
Fig. 2. Electrophoretic separation of MyHC isoforms from EDL (lane
b) and soleus (lane c) muscles of a 4-month-old female rat of the
inbred Lewis strain. In lane a, a control muscle preparation (rat
soleus+EDL muscles) was loaded. The 1, 2a, 2x/d and 2b MyHC
bands are labelled.
few were type 2C fibres exhibiting variable alkali-stable
and acid-stable mATPase activity. EDL muscles of nor-
mal 46-month-old female inbred Lewis strain rats con-
tained 5.5 1.0% of type 1 fibres (including a few type
2C fibres), 18.8 1.7% of type 2A fibres and 75.7 2.2%
of type 2B fibres. The latter type was characterized by
medium mATPase activity after preincubation at pH 4.5.

Fig. 3. Serial sections of EDL muscle of a 4-month-old female rat of the inbred Lewis strain. Sections were stained with monoclonal anti-
bodies specific for type 1 (BA-D5), type 2a (SC-71), type 1 + 2a + 2b (BF-35; negative marker for 2x/d MyHC) and type 2b (BF-F3) MyHC iso-
forms (Schiaffino et al., 1986). Fibres expressing 1, 2a, 2x/d or 2b MyHC isoforms are labelled as type 1, 2A, 2X and 2B fibres, respectively.
Bar, 100 m.

acta histochemica 104, 4 (2002)

402 Soukup et al.

Table 1. Percentages of type 1 and type 2 fibres determined on the basis of mATPase activity in the soleus muscle of different rat strains.

Age or/and Sex Type 1 (1C+2C) Type 2 Type 2A Type 2B

body weight

Inbred Lewis
present paper 46 m F 96.1 2.9 3.9 2.9

Wistar (young adult)

Jaweed et al., 1975 200 25 g F ~76 ~24
Pullen, 1977 (CFHB-Wistar) 2.5 m
250300 g F, M 76.3 23.7 0
Soukup et al., 1979 3m F, M 65.6 (8.8) 25.6 0
Melichna et al., 1987 35 d 82.2 1.8 17.8 1.8

Wistar (adult)
Soukup et al., 1979 6.5 m F, M 84.1 (2.1) 13.8 0
Lewis et al., 19941 34 m M 93.1 6.9
Ansved, 1995 45 m M 92 6 (3 3) 55
67 m 97 4 (1 1) 23
Snoj-Cvetko et al., 19962,3 ~5 m M 93.6 1.8 7.7 1.4
Larsson and Yu, 1997 47 m F 95.5 5 (2 1) 34 0
47 m M 92 6 (4 2) 41 0
Zacharov et al., 19974 ~300 g M 91.6 2 8.4 2 0
90.4 3 9.6 3 0
Mean S.D. 93.5 3.7 6.7 3.9

Sprague-Dawley
Ariano et al., 1973 84 16 0
Ho et al., 1983 4.5 m M 83 17
Armstrong and Phelps, 1984 87 4 13 4 0
Gillespie et al., 1987 616 m,
300500 g 80 20
Pousson et al., 1991 4.5 m M 82.8 3 17.2 3
Almeida-Silveira et al., 1994 2 m,
300 g M 85.6 6 (0.6 0.3) 13.8 6
Chopard et al., 20012 1.5 m,
258 g F 83 .5 17 5
Mean S.D. 83.7 2.3 16.0 2.8

WBN/Kob nondiabetic M
Ozaki et al., 2001 10 m F 96.9 3.1 0

Fisher 344
Wigston and English, 19922 6m M 90 3 (2 1) 81 0
1
Wistar-Kyoto strain
2
Based on the immunocytochemical determination of fibre types
3
Area density
4
Right and left limb, respectively
m, age in months; d, age in days; g, weight in grams; F, females; M, males

acta histochemica 104, 4 (2002)

 Fibre types in inbred Lewis rats 403

Table 2. Percentages of type 1 and type 2 fibres determined on the basis of mATPase activity in the EDL muscle of different rat strains.

Age or/and Sex Type 1 Type 2A Type 2B

body weight (1C+2C)

Inbred Lewis
present paper 46 m F 5.5 1.0 18.8 1.7 75.7 2.2

Wistar
Pullen, 1977 (CFHB-Wistar) 9.6 51.2 39.3
Niederle and Mayr, 1978 300350 g M 24 5059 3548
Soukup et al., 1979 3m F, M 4.5 (2.3) 27.8 (30.3 ~ 2X)1 35.1
Green et al., 19842 M 7.7 22.1 70.2
3.1 16.2 80.7
Melichna et al., 1987 35 d 7.6 0.7 58.2 1.5 34.3 1.3
Larsson and Yu, 1997 47 m F 41 14 4 79 6
47 m M 41 21 6 75 6
Mean S.D. 5.7 2.5 36.9 20.1 56.9 21.0

Sprague-Dawley
Ariano et al., 1973 3 59 38
Armstrong and Phelps, 1984 ~450 g 21 42 7 56 8
Eggington, 1990 3 36.2 60.8
Vesely et al., 1999 450 g M 72 45 2 48 2
Chopard et al., 20013 1.5 m,
258 g F 6 1.3
Mean S.D. 4.2 2.2 45.5 9.7 50.7 10

WBN/Kob nondiabetic
Ozaki et al., 2001 10 m F 8.3 48.9 42.8
1
These fibres corresponded to 2A fibres according to the SDH enzyme activity, but their mATPase at pH 4.5 was intermediate between 2A
and 2B fibres
2
Two different groups of control adult rats
3
Based on the immunocytochemical determination of fibre types
m, age in months; d, age in days; g, weight in grams; F, females; M, males

Immunohistochemical analysis and SDS gel elec- Discussion

trophoresis confirmed that in the soleus muscle of nor-
mal female 46-month-old inbred Lewis rats, slow
fibres expressing the type 1 MyHC isoform prevailed
over a small proportion of fast fibres expressing the 2a
MyHC isoform, whereas 2X/D and 2B fibres or corre-
sponding MyHC isoforms were not found in soleus
muscle (Figs. 1, 2). Immunohistochemical analysis and
SDS gel electrophoresis revealed that EDL muscle con-
tained type 1, type 2A, type 2X/D and type 2B fibres
and expressed type 1, 2a, 2x/d and 2b MyHC isoforms
(Figs. 2, 3). Almost half of the histochemically-deter-
mined type 2B fibres corresponded to type 2X/D fibres,
as they expressed the 2x/d MyHC isoform. SDS gel
electrophoresis indicated that the 2x/d isoform was the
second most abundant fast MyHC isoform expressed in
EDL muscles of normal female Lewis rats. In both
soleus and EDL muscles, many fibres expressed more
than one MyHC isoform (Figs. 1, 3).
In the present study, we demonstrated that the fibre type
composition of the typical slow soleus and fast EDL
muscles of female inbred Lewis rats is similar to those
of different outbred rat strains. The proportion of slow
fibres in the soleus muscle of 46-month-old female
Lewis rats was slightly higher than that of Wistar rats
and significantly higher than that of Sprague-Dawley
rats in a comparable age range (Table 1). On the other
hand, the proportion of slow fibres in EDL muscles was
similar in all strains (Table 2).
The fibre type composition of the soleus muscle must
be carefully evaluated as a considerable natural postnatal
increase in the proportion of type 1 fibres resulting from
the transformation of fast type 2A fibres has been report-
ed (Kugelberg, 1976; Soukup et al., 1979; Ho et al.,
1983; Wigston and English, 1992). This is in agreement
with our preliminary results as we found that soleus mus-

acta histochemica 104, 4 (2002)

404 Soukup et al.
cles from 2.53.5-month-old inbred female Lewis rats
contained only approx. 91% of slow type 1 fibres, where-
as those from 78-month-old rats (both males and
females) contained nearly 100% of type 1 fibres. This
implies that the fully differentiated soleus muscles in
inbred Lewis rats become almost uniformly composed of
slow type 1 fibres, which is characteristic for soleus mus-
cle of guinea pig but not for soleus muscle of outbred rat
strains (Ariano et al., 1973). The variability of genetic
factors and probably also the physical status and activity
of the animals can contribute to the natural biological
variability in fibre type composition of individual mus-
cles either among animals of the same strain or among
different strains. Such variability is supposed to be high-
er in outbred than in inbred strains. However, we have
found that a similar variation in fibre type composition of
soleus and EDL muscles also exists in inbred Lewis rats.
Variation in proportion of fibre types was more evident
in developing muscles of young animals than in fully dif-
ferentiated muscles of adult female Lewis rats.
We found no significant differences between the
fibre type composition of the soleus and EDL muscles
from right and left legs, although some variation was
noted in individual rats. Similarly, lack of significant
differences between muscles of both legs has also been
reported for Wistar rats (Zacharov et al., 1997).
Immunohistochemical analysis using monoclonal
antibodies specific for different MyHC isoforms and
SDS gel electrophoresis confirmed the fibre type com-
position of muscles as determined on the basis of mAT-
Pase activity. However, type 2X/D fibres and the
respective 2x/d MyHC isoform as was found in EDL
muscle could only be demonstrated immunohistochem-
ically or electrophoretically. On the basis of the 3 types
of analysis, it is concluded that the soleus muscle of
female inbred Lewis rats largely consists of slow type 1
fibres and only a small proportion of fast 2A fibres is
present, whereas the fast EDL muscle contains all 3 fast
fibre types (2A, 2X/D and 2B) besides a few type 1
fibres. Our present results fully support the finding that
the third fast 2X/D fibre type with characteristics that
are intermediate between that of type 2A and 2B fibres
represents a substantial population of fast fibres in EDL
muscle of normal rats (DeNardi et al., 1993).
Acknowledgments
We are grateful to Ms Vera Semelov, Klra Mrckov, Mrs grafts in rats with experimentally changed thyroid status.
Andreja Omahen, Majda Crnak-Maasarani, Ana Tomazincic
and Mr Marko Slak for their excellent technical assistance
and computer analysis and Dr P Hnk for critical reading of
the manuscript and his valuable comments. The study was
supported by grant 304/00/1653 from the Grant Agency of the
Czech Republic and by grant of the Czech-Slovenian Inter-
governmental S&T Co-operation Program.
acta histochemica 104, 4 (2002)
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