Original
Effects of
Paper
resistance training on fast- and slow-twitch muscles in rats
Biol. Sport 2010;27:221-229
EFFECTS OF RESISTANCE TRAINING ON
FAST- AND SLOW-TWITCH MUSCLES IN RATS
AUTHORS: Seene T. 1, Pehme A. 1, Alev K. 1, Kaasik P. 1, Umnova M.2, Aru M.1
1
2
Accepted
for publication
20.02.2007
Reprint request to:
Teet Seene
University of Tartu
18 Ülikooli, 50090 Tartu, Estonia
Phone: + 372 7 375 364
E-mail: [email protected]
Department of Functional Morphology, University of Tartu, Estonia
Laboratory of Evolutionary Histology, Institute of Ecology & Evolution RAS, Russia
ABSTRACT: The purpose of this study was to investigate the effect of resistance training (RT) on muscle strength,
the dependence of that on the fast-twitch (FT) and slow-twitch (ST) fibers hypertrophy, nuclear domain size,
synthesis and degradation rate of contractile proteins and on the expression of myosin isoforms’. 16 weeks old
Wistar rats were trained on a vertical treadmill for six days a week during six weeks. The power of exercise
increased 4.9% per session. In RT group the mass of studied muscles increased about 10%, hindlimb grip strength
increased from 5.20±0.27 N/100g bw to the 6.05±0.29 N/100g bw (p<0.05). Cross-sectional area and number
of myonuclei of FT and ST fibers in plantaris (Pla) and soleus (Sol) muscles increased, myonuclear domain size
did not change significantly. RT increased the MyHC IId isoforms relative content and decreased that of IIb and
IIa isoforms in Pla muscle, in Sol muscle increased only IIa isoform. In Pla muscle the relative content of myosin
light chain (MyLC) 1slow and 2slow isoforms decreased and that of MyLC 2fast isoforms increased during RT. MyLC
3 and MyLC 2 ratio did not change significantly in Pla but increased in Sol muscle by 14.3±3.4% (p<0.01).
The rat RT programme caused hypertrophy of FT and ST muscle fibers, increase of myonuclear number via fusion
of satellite cells with damaged fibers or formation of new muscle fibers as a result of myoblast fusion and
myotubes formation, maintaining myonuclear domain size.
KEY WORDS: resistance training, muscle strength, MyHC
INTRODUCTION
Resistance training (RT) expands the amount of the myofibrillar
This suggests that additional myonuclei are needed to support
apparatus in order to enlarge fiber cross-sectional area [2].
the enlargement of muscle cells during RT. As each myonucleus may
Also, there is a concomitant alteration in contractile protein
be responsible for the maintenance of a limited volume of sarcoplasm,
phenotype and metabolic enzyme levels, which seem to occur in
a concept of DNA unit or myonuclear domain has been defined as
accordance with activity-induced changes in the muscle’s fiber-
the quantity of cytoplasm regulated by a single myonucleus [11,18].
Increased contractile activity can induce differential expression of
It has been shown that the repetition regime in the resistance
myosin protein isoforms in skeletal muscle. Many different types of
training protocol plays a very important role in the hypertrophy of
studies of the striated muscle focus on the assessment of the
muscle fibers. High numbers of repetitions in resistance training
composition of the myosin heavy chain (MyHC), because of its
did not cause any significant hypertrophy of muscle fibers [9].
important regulatory role in myosin ATPase activity and, therefore,
Animal models have clearly shown that satellite cell activation is
velocity of muscle fiber shortening [5,17]. MyHC is encoded by the
involved and may be a prerequisite for muscle fiber hypertrophy [37].
multigene family, which is mapped to a single chromosome [10,
Using a marker for satellite cells, it has been documented that
15]. The modulation of MyHC protein in adult skeletal muscle is
expression of early markers of myogenesis is activated in satellite
multifactorial, since many factors participate in this process [3,5,15].
cells and muscle fibers in response to RT in humans [23]. Satellite
It has been demonstrated that mechanical loading is more responsible
cells are believed to proliferate and fuse with the existing fibers,
for the modulation of MyHC isoform expression than stimulation
thereby contributing to an increase in myonuclei per muscle fiber
frequency [7]. Pre-translational mechanisms of MyHC protein
[45]. The number of myonuclei increased with fiber hypertrophy and
regulation are highly sensitive to even small amounts of RT [8].
positively correlated with the increased number of satellite cells [24].
As the protein remodelling process consists of protein breakdown
-
-
-
-
-
type profile [32,41].
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221
Seene T. et al.
and synthesis, it is important to know the dynamics of remodelling
12/12 hrs light/dark period. They received diet (SDS-RM1 (C) 3/8,
of myosin isoforms during RT.
Witham, Essex, England) and water ad libitum.
In response to heavy-load RT, the content of fastest MyHC isoforms
Resistance training. The animals were trained on a vertical
decreased and the relative content of slowest isoforms reciprocally
treadmill [33] at a speed of 18 m/min at an 80° angle for
increased in skeletal muscle of small laboratory animals [32] and
a distance of 1.5 m during 5 sec (one run) for six days a week
humans [27].
during six weeks. RT consisted of 2−5 runs per session (Monday – 2,
It is still not fully known how skeletal muscle responds to
Tuesday – 3, Wednesday – 4, Thursday – 5, Friday – 4, Saturday
an increase in mechanical load. It is known that compensatory
– 3), recovery time 1 min 30 sec between runs, and -the peak
hypertrophy is characterized by an increase in muscle mass, muscle
frequency was in the middle of the week. Exercise power increased
protein content, and contractile force, and by a shift from the fast-
0.026 W/per session on average (4.9% per session). The animals
to-slow myosin isoform type in fast-twitch (FT) muscles, but the
carried a progressively heavier extra weight, secured to their tails
exact mechanism of changed MyHC isoforms during RT is poorly
with a belt and elastic tape.
understood [32]. In comparison with MyHC isoforms, much less is
During the first week, rats carried extra weight equal to 30±0.3%
known about changes of myosin light chain (MyLC) isoforms during
of their bw. During the training protocol the extra weight was
adaptation to RT. It has been shown that mechanical activity with
increasing so that in the 6th week it constituted 110% of their bw.
low force causes changes in skeletal muscle MyLC isoforms, including
Total work during the 6-week training period was 1000±20 J and
a depressive effect on the contractile velocity of type IIB fibers [47].
the total energy expenditure 240±4 cal.
It is not known how close a relation exists between the synthesis
Measurement of muscle strength. The hindlimb grip strength was
and degradation rates of MyHC and mixed muscle protein during
measured weekly with Grip Strength Meter 0167-004L (Columbus
adaptation to RT, and for how long after a regular training session
Instruments, Columbus, USA) and expressed as N/100 g bw.
MyHC synthesis rate is depressed. Hypertrophy of different fiber types
Measurement of recovery state. Screening of the amounts of
in FT and slow-twitch (ST) muscles is still unclear
ambulatory and total movements were measured with Opto-
The purpose of this study was to investigate the effect of RT on
Varimex-Mini (Columbus Instruments, Columbus, USA) and
muscle strength and the dependence of that on the FT and ST fibers
expressed as movements/h. Ambulatory activity characterizes
hypertrophy, nuclear domain size, synthesis and degradation rate of
movemental activity of animals; total activity also includes
contractile proteins and on the relative myosin isoforms’ content.
stereotypic (scratching, grooming, digging…) non-ambulatory
We also wanted to clarify the role of satellite cells in RT induced
movements.
skeletal muscle hypertrophy.
Muscle sample preparation. L-[4.5−3H] leucine (170 Ci/mol) was
infused intraperitonally of 1.0 ml for 2 hrs, 250 µCi per 100 g bw
MATERIALS AND METHODS
in control group (n=8) and RT group immediately after the last
Animals were used in accordance with the European Convention
exercise (n=8); 10 h (n=8) and 22 h (n=8) after the last exercise,
for the Protection of Vertebrate Animals Used for Experimental and
for determination of dynamics of MyHC and mixed muscle protein
Other Scientific Purposes and this was controlled by the Committee
synthesis rate.
of Laboratory Animal Science, University of Tartu.
In order to investigate the turnover rate of MyHC protein (control
Animals. The animals used were 16–weeks–old (228± 8 g body
group n=8, and RT group n=8) the double isotope method as
weight (bw), at the beginning on the experiment) male rats of
described by us previously [41] was used. L-[U14C] Lysine (336 mCi/
the Wistar strain (National Laboratory Animals Centre, Kuopio,
mmol) 10 µCi per day was discontinued after five days and L-[4.5–
Finland). Rats were randomly divided into two groups: control
3
group (n=28); and RT group (n=44). At the end of the experiment
The turnover rate of the protein fraction was estimated from
the bw in the control group was 303±6 g, and in RT group 305±7
the 3H/14C ratios. For the same protein turnover rates, the 3H/14C
g (Table 1). All the animals were housed in identical environmental
ratios were expected to be the same. Protein with a higher turnover
conditions in polycarbonate type III cages, at 21ºC, two per cage at
rate would have a greater 3H/14C ratio.
-
H] Lysine (40 Ci/mmol) 100 µCi per day was continued for 12 days.
N
Contr
28
RT
44
Body weight
m. Plantaris
(g)
m. Extensor digitorum
longus
(mg)
m. Soleus
SP activity
303 ± 6
117.4 ±3.1
(mg)
(mg)
(u/mg protein)
229.9 ± 4.6
102.8 ± 2.8
0.32 ± 0.02
305 ± 7
128.6 ± 4.9*
252.1 ± 5.1**
111.6 ± 2.9*
0.25 ± 0.19*
-
Group
Legend: Contr - control group, RT - resistance training group, SP activity - serine proteinase activity, u/mg protein - unit per mg protein,
* - p<0.05 in comparison with subsequent control group, ** - p<0.01 in comparison with subsequent control group
-
-
-
TABLE 1. EFFECT OF RESISTANCE TRAINING ON THE BODY WEIGHT AND MUSCLE WEIGHTS
222
Effects of resistance training on fast- and slow-twitch muscles in rats
The incorporated radioactivity was measured in a liquid
scintillation counter.
myofibrillar protein sample was loaded on 1 mm thick gel per
well. Electrophoresis was performed at a constant current (30
12.5% SDS-PAGE gel electrophoresis was carried out (as
mA) and stopped when the dye front reached the bottom of the
described below), and the identified MyHC bands were sliced and
gels in the vertical slab gel system (Protean II Xi Bio-Rad). The
dissolved in hydrogen peroxide at 50ºC overnight and radioactivity
gels were Coomassie Brilliant Blue R-250 stained.
was determined.
3
H thymidine was infused intramuscularly, 30 µCi per animal
(four animals from each group) 48 h before rats were sacrificed.
Prior to being sacrificed, the animals were anesthetized by
The positions of MyLC isoforms on the gel were identified by their
apparent molecular weight compared to the protein mobility of the
prestained standard (Kallidoscope Prestained Standards, Bio-Rad)
and by reports in literature.
intraperitoneal injection of ketamin (Calysol, Gedeon Richter A.O.
Budapest, Hungary) and diazepam (Lab Renaudin, France).
Then the extensor digitorum longus (EDL), Pla, Sol and gastrocnemius
muscles were quickly removed, trimmed clean of visible fat and
connective tissue, weighed, frozen and stored in liquid nitrogen
pending further processing or fixed for ultrastructural or for histological
studies. Gastrocnemius muscle was used only for measurement of
serine proteinase activity. Three muscle samples were taken from
each muscle.
Separation of total muscle protein. The minced muscle samples
were homogenized in a buffer containing: 50 mM KCl, 10 mM
K2 HPO4, 1 mM EGTA, 1 mM MgCl2, and 1 mM ditiothreitol, at
pH 7.0, and analyzed as total protein fraction. The total muscle
homogenate was dissolved in 0.3 M NaOH and was analyzed for
radioactivity and protein.
Separation of myofibrillar protein. Frozen muscles pulverized
under liquid nitrogen and homogenized in five volumes 20 mM
NaCl, 5 mM Na2HPO4, 1 mM EGTA (pH 6.5). Myofibrillar protein
was extracted with three volumes 100 mM Na4P2O7, 5 mM
EGTA, 1 mM dithiothreitol (pH 8.5) centrifuged, and after 30 min
FIG. 1. ELECTROPHORETICAL SEPARATION OF MYHC AND MYLC
ISOFORMS
Legend: A – MyHC isoforms, B – MyLC isoforms; only region corresponding
to positions of MyLC-s is shown, EDL – extensor digitorum longus muscle,
Pla – plantaris muscle, Sol – soleus muscle
of gentle shaking, was diluted with one volume glycerol and stored
at –80°C. Protein was assayed by using the technique described
MyHC and MyLC isoforms (Fig. 1) were quantified densitometrically
Fractional synthesis rate of muscle proteins. The fractional rate
by a computer-based image analysis system and software (Image
of protein synthesis Ks (expressed by the percentage of the protein
Master 1D, Amersham Pharmacia Biotech) and the percentage
synthesized per day) in each fraction was then calculated from
distribution of the various isoforms was evaluated.
the following relationship: Ks = 100 x Sb/Sa x t, where Sa and Sb
Other
are the specific radioactivities of the total muscle cell protein and
spectrophotometrically in homogenates as described by Millward
protein-bound leucine and t is the incorporation time in days [42].
and Waterlow [29] and DNA unit size in skeletal muscle was
MyHC and MyLC electrophoresis. MyHC composition was
expressed as protein DNA ratio (µg/µg). Serine proteinase
determined by using the SDS-PAGE vertical slab gel system
separation and activity measurement was provided by Dahlmann
(Protean II Xi Bio-Rad) procedures described by Hämäläinen
et al. [12] as described by us earlier [40].
analyses.
DNA
content
was
determined
and Pette [20]. MyHC isoforms were separated by 7.2% SDS-
Corticosterone and testosterone concentration in blood serum
PAGE using 0.75 mm – thick gel. Aliqots containing 0.5 µg of
was measured by competative immunoassay for in vitro diagnostic
myofibrillar proteins were loaded on the gel after being incubated
use with the IMMULITE analyzer (DPC, Los Angeles USA) 24 h
for 10 min at 65°C in sample buffer containing 62.5 mM Tris-HCl
after the last exercise.
of pH 6.8, 20% (vol/vol) glycerol, 5% (vol/vol) 2-mercaptoethanol,
Myonuclear number and fiber size analysis. Muscle samples
2.0% SDS, 0.05% bromphenol blue. Electrophoresis lasted for
were fixed by Tissue Tek O. C. T. Compound 4583 (Miles Inc,
24 h at 120 V. Gels were silver-stained by the method described
USA), immersed in isopentane and stored at −80°C (precooled
by Oakley et al. [30]. The MyLC isoforms were separated by
in liquid nitrogen). Serial 10-µm-thick tissue cross-sections were
a 12.5% SDS-PAGE gel according to Laemmli [26], except that
obtained from each muscle midbelly by cryostat microtome
the glycerol content in the separating gel was 10%. A 10 µg
(Cryo Cut, American Optical Company). The sections were
-
-
-
-
-
by Bradford [6].
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Seene T. et al.
Contr
total activity
1000
900
Contr
RT
450
800
400
700
**
600
movements/h
movements/h
ambulatory activity
500
RT
500
400
300
*
350
300
250
200
150
200
100
100
50
0
0
2h
6h
12 h
24 h
2h
6h
12 h
24 h
FIG. 2. DYNAMICS OF MOTOR ACTIVITY AFTER RESISTANCE TRAINING. MOTOR ACTIVITY OF RATS WAS USED FOR CHARACTERIZATION OF RECOVERY
STATE OF CONTRACTILE MACHINERY AFTER EXERCISE. AMBULATORY ACTIVITY CHARACTERIZES MOVEMENTAL ACTIVITY OF ANIMALS; TOTAL
ACTIVITY INCLUDES ALSO STEREOTYPIC NON-AMBULATORY MOVEMENTS (SCRATCHING, GROOMING, DIGGING…)
Legend: Contr - control group (n = 20), RT - resistance trained group (n = 36), 2 h - 2 hours after last training (n = 36),
12 h - 12 hours after last training (n = 36), 24 h - 24 hours after last training (n = 36), * - p<0.05 in comparison with subsequent level 2 h after exercise,
* - p<0.01 in comparison with subsequent level 2 h after exercise
stained with haematoxylin and eosin. Myonuclear domain size
the cryostat cut cross-section and divided to number of myonuclei
per fiber segment. Muscle fiber types were determined by
the immunohistochemical procedures using myosin antibodies
NCL − MyHCs, NCL − MyHCf, Novocastra Laboratories Ldt,
2,5
% per day
was calculated: crossectional area multiplied to thickness of
3
x
***
2
xxx
***
###
xx
***
xxx
***
###
2h
12 h
24 h
xxx
xxx
x
Contr
xxx
***
xx
*** #
1,5
Newcastle upon Tyne, UK. Myonuclear domain size − cytoplasmic
1
0,5
volume/myonucleus (µm3) of the fiber was determined.
This procedure allowed the delineation of the two major fiber
0
EDL
types, I and II.
Samples were examined with an Olympus BX-40 light microscope
(Tokyo, Japan) with digital camera Olympus DP-10, and
measurements were taken using Olympus DP-soft software.
Ultrastructural studies. Muscle samples for ultrastructural studies
were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmimum
tetroxide, dehydrated in graded alcohol, and embedded in
Epon-812. Ultrathin sections were cut from longitudinally and
transversely oriented blocks and the section from 3−5 blocks from
each animal were stained with uranyl acetate and lead hydroxide
Pla
Sol
FIG. 3. DYNAMICS OF FRACTIONAL SYNTHESIS RATE OF MYHC PROTEIN
IN FT AND ST SKELETAL MUSCLES AFTER RESISTANCE TRAINING
Legend: Contr – control group (n = 8), RT - resistance trained group,
2 h - 2 hours after last training (n = 8), 12 h - 12 hours after last training
(n = 8), 24 h - 24 hours after last training (n = 8), EDL - extensor digitorum
longus muscle, Pla - plantaris muscle, Sol - soleus muscle,
x - p<0.05 in comparison with control group, xx - p<0.01 in comparison
with control group, xxx - p<0.001 in comparison with control group,
** - p<0.01 in comparison with subsequent level 2 h after exercise,
*** - p<0.001 in comparison with subsequent level 2 h after exercise,
# - p<0.05 in comparison with subsequent level 12 h after exercise,
## - p<0.01 in comparison with subsequent level 12 h after exercise,
### - p<0.001 in comparison with subsequent level 12 h after exercise
as described previously [41]. The number of satellite cells
containing a nucleus, per 1000 myonuclei, in experimental and
1800
control groups was estimated by electron microscopy as described
1400
as a ratio of the nucleus-containing satellite cells divided by
1200
the total number of myonuclei, including the nuclei of satellite
1000
-
and were exposed for 8 weeks for detection of incorporation of
-
thymidine into nuclei.
Statistics. Means and standard errors of means were calculated
-
from individual values by standard Excel procedures. Data were
analyzed by SAS procedures, using the analysis of variance
-
(ANOVA) and the Pearson correlation coefficients and partial
correlation
coefficients
were
calculated.
considered significant at p<0.05.
224
Differences
were
µm 2
previously [45]. The satellite cell frequency was determined
cells. Ultrathin sections were covered with photoemulsion
-
1600
Contr
*
*
*
RT
*
800
600
400
200
0
FT
Pla
ST
FT
Sol
ST
FIG. 4. THE EFFECT OF RESISTANCE TRAINING ON THE FT AND ST FIBER
CROSS-SECTION AREA IN DIFFERENT MUSCLES
Legend: Contr - control group (n = 8), RT - resistance trained group (n = 8).
Pla - plantaris muscle, Sol - soleus muscle, FT - fast-twitch, ST - slow-twitch
* - p<0.05 in comparison with control group
Effects of resistance training on fast- and slow-twitch muscles in rats
Contr
5
*
*
*
A
RT
4000
*
4
Contr
4500
RT
µm3 per myonucleus
myonucleus/per cross-section
area
6
3
2
1
3500
3000
2500
2000
1500
1000
500
0
Pla ST
FT
Sol
0
ST
FIG. 5. THE EFFECT OF RESISTANCE TRAINING ON THE NUMBER OF
MYONUCLEI IN FT AND ST FIBERS IN DIFFERENT MUSCLES
CONTR - CONTROL GROUP (N = 8)
RT - RESISTANCE TRAINED GROUP (N = 8).
PLA - PLANTARIS MUSCLE
SOL - SOLEUS MUSCLE
FT - FAST-TWITCH
ST - SLOW-TWITCH
* - P<0.05 IN COMPARISON WITH CONTROL GROUP
FT
B
ST
FT
Pla
Sol
ST
Contr
600
RT
500
Prot/DNA (µg/µg)
FT
400
300
200
100
RESULTS
In RT group, the mass of the studied muscles increased about
10% (Table 1) and hindlimb grip strength increased from
5.20±0.27 N/100g bw to the 6.05±0.29 N/100g bw (p<0.05).
Motor activity, as an indicator of recovery state of muscle
contractile apparatus after resistance training, was lower in RT
group only during a couple of hours after training (Fig. 2).
Mixed muscle protein fractional synthesis rate at the end of the
0
EDL
Pla
Sol
FIG. 6A..THE EFFECT OF RESISTANCE TRAINING ON THE MYONUCLEAR
DOMAIN SIZE IN FT AND ST FIBERS IN DIFFERENT MUSCLES
MYONUCLEAR DOMAIN - VOLUME OF CYTOPLASM PER MYONUCLEUS
FIG. 6B. THE EFFECT OF RESISTANCE TRAINING ON THE DNA UNIT SIZE
IN FT AND ST MUSCLES
Legend: DNA unit size - protein DNA ratio, Contr - control group (n = 8)
RT - resistance trained group (n = 8), EDL - extensor digitorum longus muscle
PLA - PLANTARIS MUSCLE, SOL - SOLEUS MUSCLE, FT - FAST-TWITCH
ST - SLOW-TWITCH
6-week resistance training period increased significantly 24 h after
last training session in EDL muscle from 4.54±0.11 to 4.96±0.12
(p<0.05) in Pla and Sol muscles had only tendency to increase.
Muscle protein degradation rate decreased significantly in Pla muscle
from 3.03±0.08 to 2.71±0.07 (p<0.01). In comparison with the
control group (4.7±0.7 nmol L-1), testosterone concentration
24 hours after last training session was 7.3±1.0 nmol L-1 (p<0.05)
in RT group and corticosterone concentration in control group was
1.20±0.08 µmol L-1 and RT group 1.45±0.09 µmol L-1 (p<0.05).
Serine proteinase activity in the control group was
0.32±0.020 u/mg, and 0.25±0.19 u/mg (p<0.05) in RT group
24 hours after last training session (Table 1).
Fractional synthesis rate of MyHC protein decreased in all studied
muscles in RT group two hours after last session in comparison with
the level control group and started to increase from the 12 h after
-
training (Fig. 3). Comparison of changes in FT and ST muscle fibers’
cross-sectional area and number of myonuclei in Pla and Sol muscle
FT FIBERS IN PLA MUSCLE 24 HOURS AFTER RESISTANCE TRAINING
PENETRATION OF BASAL LAMINA (BL) BETWEEN EXTRASYNAPTIC
SATELLITE CELL AND MUSCLE FIBER (MF). BAR 1 µM; n = 4 per group
in all studied fibers (Figs. 4 and 5). The myonuclear domain size
(Fig. 6A) and DNA unit size (Fig. 6B) did not change significantly in
formation. Incorporation of radioactively labelled thymidine into
ST and FT muscle fibers during RT. Following RT, satellite cells
myonuclei is proof of the above mentioned process (Fig. 8). RT is
separate from muscle fibers basal lamina (Fig. 7), divide and fuse
increased the relative content of MyHC I and IId isoforms and
with damaged fibers, and increase the number of myonuclei in fibers,
decreased that of IIb and IIa isoforms in Pla muscle (Fig. 9).
or form new muscle fibers via myoblast fusion and myotubes
In Sol muscle, RT had tendency to decrease the relative content of
-
-
-
-
during resistance training indicates that a significant increase occured
FIG. 7. ACTIVATION OF SATELLITE CELL UNDER THE BASAL LAMINA OF
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Seene T. et al.
Pla
50
Contr
**
45
RT
40
35
30
***
%
25
**
20
15
***
10
5
0
I
IIa
IId
IIb
Contr
Sol
120
RT
100
FIG. 8. INCORPORATION OF 3H THYMIDINE INTO NUCLEUS OF FT FIBER
80
%
IN PLA MUSCLE 24 HOURS AFTER RESISTANCE TRAINING
TYPICAL POSTMITOTIC ACTIVE NUCLEUS WHICH CONTAINS FEW
CHROMATIN (1) IN REGIONS CLOSE TO CARIOLEMMA. 2 - SILVER
GRANULES UNDER THE NUCLEOLUS; 3 - MYOFIBRILS. BAR 1 µM;
n = 4 per group
60
40
20
***
0
MyHC I isoform and increased that of MyHC IIa isoform (Fig. 9). In
Pla muscle, the relative content of MyLC 1slow and 2slow isoforms
decreased and that of MyLC 2fast isoform increased during RT (Fig.
10). In Sol muscle, the relative content of MyLC 2fast and 3 isoforms
increased during RT (Fig. 10). MyLC 3/MyLC 2 ratio did not change
significantly in Pla muscle but increased in Sol muscle by 14.3±3.4
I
IIa
FIG. 9. THE EFFECT OF RESISTANCE TRAINING ON THE MYHC
ISOFORMS’ COMPOSITION IN FT AND ST MUSCLES
Legend: Contr - control group (n = 16); RT - resistance trained group (n = 16); Pla plantaris muscle; Sol - soleus muscle; I, IIa, IId, IIb - MyHC isoforms
** - p<0.01 in comparison with subsequent control group
*** - p<0.001 in comparison with subsequent control group
% (p<0.01) in comparison with the control group. There was positive
correlation between muscle strength and fractional synthesis rate of
60
MyHC (r=0.74; p<0.01) and negative correlation between muscle
50
(r=−0.71; p<0.01).
40
In FT muscles MyHC turned over faster in RT group in comparison
with control group (Fig. 11). The smaller in muscle the DNA unit
%
strength and relative content of MyHC IIa isoform in FT muscle
size (Fig. 6B), the faster MyHC turnover rate in muscle (Fig. 11).
Contr
Pla
RT
*
30
20
10
DISCUSSION
*
***
0
During RT, skeletal muscles showed marked gains in strength in
MyLC 1slow MyLC 1f ast MyLC 2slow MyLC 2f ast MyLC 3f ast
the group with less repetitions and a slower increase in power per
training session [31]. This is due both to neuronal adaptations
60
and to an increase in the cross-sectional area of muscle.
50
There is consensus in literature that the gain in the cross-sectional
-
to increase after RT, with a tendency for larger increases in type
II than in type I fibers [21]. Our previous study shows that both
-
ST and FT muscles hypertrophy when the power of exercise does
not increase too fast and the number of repetitions per training
-
session is not too high [31]. In the case of high repetions and
a rapid increase of training power, there is no hypertrophy of
-
muscles or gain in strength. Testosterone concentration increased
during RT, but the high level of corticosterone in the group with the
-
rapid increase in training power and volume is responsible for the
226
RT
40
%
area of muscle during RT is mainly due to an increase in myofibrillar
proteins. The cross-sectional area of all fiber types has been shown
Contr
Sol
30
20
10
***
*
0
MyLC 1slow MyLC 1f ast MyLC 2slow MyLC 2f ast MyLC 3f ast
FIG. 10. THE EFFECT OF RESISTANCE TRAINING ON THE MYLC
ISOFORMS’ COMPOSITION IN FT AND ST MUSCLES
Legend: Contr – control group (n = 16); RT - resistance trained group (n = 16); Pla plantaris muscle; Sol - soleus muscle, MyLC 1-3 - MyLC isoforms;
* - p<0.05 in comparison with subsequent control group;
*** - p<0.001 in comparison with subsequent control group
Effects of resistance training on fast- and slow-twitch muscles in rats
1,2
1
found in top-level powerlifters by [43]. The increase in the number
RT
of satellite cells could be due to asymmetric division of satellite cells
*
and will make the muscle more responsive to further adaptation
*
0,8
3H/14C
Contr
[24,46]. The expression of mRNAs for transforming growth factors
(TGF-β2; myostatin, actinin-β, and follistatin), IGF I and II, and
0,6
fibroblast growth factors was investigated in satellite cells in the
0,4
stages from initiation of proliferation to fusion [25].
0,2
It is still not fully known whether character of RT can induce
0
specific types of adaptations or whether the muscle simply responds
EDL
Pla
Sol
FIG. 11. THE EFFECT OF RESISTANCE TRAINING ON THE MYHC
TURNOVER RATE IN FT AND ST MUSCLES
Legend: Contr – control group (n = 8); RT - resistance trained group (n = 8)
EDL - extensor digitorum longus muscle; Pla - plantaris muscle; Sol - soleus muscle
* - p<0.05 in comparison with subsequent control group
in a stereotypic fashion to any increase in the mechanical load.
Our previous studies show that, depending on the character of
mechanical loading, contractile activity can induce differential
expression of myosin protein isoforms in skeletal muscle [32,39].
It was shown by us that different types of mechanical activity affect
the synthesis rate of MyHC and the MyLC in skeletal muscle [38,39].
The reports about myosin adaptation to mechanical load concentrate
mostly on the relative content of isomyosins or MyHC isoforms [9,16].
increased catabolism of muscle protein in this group [31]. Present
However, only a few researchers pay attention to the synthesis of
study show that in the case where RT power is increased up to five
MyHC isoforms in skeletal muscle [1,19]. Although some results
percent per training session, there is less degradation of muscle
suggest that mechanical factors have an important role in controlling
protein. It means that in this case RT has an anticatabolic effect on
the expression of contractile proteins [7], the influence of the quantity
skeletal muscle as show the decrease of serine proteinase activity
and type of mechanical loading on muscle is still unknown. It has
in muscle. A significant increase of FT fibers’ cross sectional area
been suggested that changes in muscle structure, mass, and MyHC
and the number of myonuclei, without changes in nuclear domain
concentration and its isoform expression, induced by a high-resistance
size show the adaptation of skeletal muscle to the RT. About
weight-training programme, are a result of the frequency of contraction
functional significance of nuclear domain size in FT and ST muscle
[14,22,44]. There is no explicit understanding of how the intensity
fibers during adaptation to RT tells us the maintained myonuclear
of RT change the composition of MyHC isoforms. Nor do we know
domain size.
the limits of protein synthesis and adaptative capacity in the case of
It has been expected that increased myonuclear number supplemets
the existing genetic machinery in the synthesis of new contractile
According to the results of the present study, there is reason to
proteins to meet the demands of the mechanical overload [28,36]
believe that changes in MyHC isoforms depend on the proportions
and myonuclear domain size is correlated with muscle fiber type and
of power and volume used during RT. It has been shown that
MyHC expression [35].
the MyHC IIb isoform is highly sensitive to the action of proteinases
The cellular changes occurring during hypertrophic adaptations
[40] and to an increase in exercise training volume [13,39].
to RT essentially confirm the nuclear domain theory, which suggested
All proteins in mammalian muscle fibers are in a continuous
that the cytoplasm-to-myonucleus ratio is related with the myosin
process of being synthesized and subsequently degraded. The balance
type and the amount of protein turnover [4]. It seems to be clear
between protein synthesis and degradation determines whether there
that satellite cells are recruited during hypertrophy of muscle fibers
is either hypertrophy or atrophy in muscle mass. This fundamental
in order to maintain the cytoplasm-to myonucleus ratio.
process also allows qualitative remodeling of the muscle, so that one
It was shown that in the trained subjects who showed muscle
fiber hypertrophy, there was a significant increase in the number of
isoform is replaced by another that is better suited for specific
conditions [2].
Experiments point to the importance of changes in protein turnover
expressed in adult muscle fibers [24]. These findings show an
for the hypertrophic process [50]. RT is increasing the fractional
alternative way for forming new fibers. If the number of newly formed
synthesis rate and the fractional breakdown rate of muscle proteins,
fibers exceeds the number lost by damage, this will lead to an increase
depending on the character of the training [31]. RT with low repetitions
and adequate increase in training power causes an increase in
in the number of fibers [24].
Our present results in laboratory animals during RT indicated that
the synthesis rate and a decreased in the degradation rate of muscle
the satellite cell number under the muscle fiber basal lamina of both
protein [31]. Changes in muscle protein do not clearly reflect
FT and ST fibers increased in the case where the training power was
the process of turnover of certain contractile proteins. 24 h after
increased up to 5% per training session and the number of repetitions
training session MyHC fractional synthesis rate was higher than that
was not too high. This increase in satellite cell number was also
in the control group. In humans the fractional protein synthesis rate
-
-
-
fibers that stained for embryonic and fetal MyHC, normally not
-
-
different character of mechanical loading factors.
Biology
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Seene T. et al.
was found to be higher 48 h after training [34]. Due to the lack of
[49]. Our study shows that there are different mechanisms regulating
pertinent expression data, it is currently not known whether
the expression of MyLC and MyHC fast and slow isoforms in FT and
the absence of mitochondrial adaptations with strength training is
ST muscles during resistance training.
due to transcriptional regulatory events or enhanced mitochondrial
turnover. When power of RT increase up to five percent per training
CONCLUSIONS
session, significant strength gain, muscle hypertrophy, particularly
The rat RT programme during which the power of exercise increased
an increase in FT fibers’ cross-sectional area, without changes in
4.9% per session, caused hypertrophy of both FT and ST muscle
myonuclear domain size were observed in the present study.
fibers, increase of myonuclear number via fusion of satellite cells
The number of satellite cells under the basal lamina of ST and FT
with damaged fibers or formation of new muscle fibers as a result
muscle fibers increased. Satellite cells liberate and fuse with the
of myoblast fusion and myotubes formation, maintaining myonuclear
damaged fibers (incorporation of radioactive thymidine into muscle
domain size. At the same time adaptational changes of contractile
fiber nucleus, proves that). RT protocol used in the present study
apparatus in FT and ST skeletal muscles proceeded via
increases fractional protein synthesis rate in FT muscles, and
the remodelling process of MyHC and MyLC isoforms in different
the MyHC synthesis rate in all studied muscles. Our results show
fibers, and through this process was regulated the turnover rate of
that in FT muscles there is no coordination between expression of
myosin isoforms, the strength generation capability of fibers and
MyHC and MyLC slow and fast isoforms during adaptation to the RT.
the individual characteristics of muscles.
In ST muscle there is an increase in the relative content of fast
isoforms in MyHC and MyLC. It has been shown that variation in Vo
of single fibers expressing similar MyHC isoforms correlated with the
Acknowledgements:
amount of MyLC 3 isoform [48]. In the present study we found the
This study was supported by funds from the Ministry of Education
MyLC 3 to MyLC 2 isoform ratio in ST muscle increased with RT. In
and Research of Estonia, research project number 1787, and by
some studies this ratio did not change during resistance training
the Estonian Scientific Foundation, Grant number 6501.
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-
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