Post on 07-May-2023
ORIGINAL ARTICLE
Effect of different resistance-training regimenson the WNT-signaling pathway
Marcelo Larciprete Leal • Leonardo Lamas • Marcelo Saldanha Aoki •
Carlos Ugrinowitsch • Marcela Sorelli Carneiro Ramos • Valmor Tricoli •
Anselmo Sigari Moriscot
Received: 7 October 2010 / Accepted: 14 February 2011 / Published online: 2 March 2011
� Springer-Verlag 2011
Abstract The purpose of the present study was to eval-
uate the effects of 8 weeks of strength and power training
on the expression of genes related to the canonical WNT
pathway and b-catenin protein levels in physically active
men. Twenty-five subjects (27.4 ± 4.6 years) were bal-
anced based on their relative maximum strength in the
squat exercise (squat 1RM/body mass) and randomly
assigned to strength training (ST) (n = 10), power training
(PT) (n = 10), and control (C) (n = 5) groups. The ST and
the PT groups performed high and low intensity squats,
respectively, thrice a week, for 8 weeks. Muscle biopsies
from the vastus lateralis muscle were collected before and
after the training period. Relative strength and power
increased similarly in both ST and PT groups (P \ 0.001).
Fiber cross-sectional area also increased similarly in both
ST and PT groups. Gene expression and b-catenin protein
expression levels were assessed by real-time PCR and
Western blot. Certain genes were up-regulated in the ST
group (WNT1: 6.4-fold, P \ 0.0001; SFRP1: 3.3-fold,
P \ 0.0001 and LEF1: 7.3-fold, P \ 0.0001) and also in
the PT group (WNT1: 24.9-fold, P \ 0.0001; SFRP1: 2.7-
fold, P \ 0.0001; LEF1: 34.1-fold, P \ 0.0001 and Cyclin
D1: 7.7-fold, P \ 0.001). In addition, the expression of key
WNT pathway genes was substantially more responsive to
PT than to ST (WNT1: P \ 0.0001; LEF1: P \ 0.0001 and
Cyclin D1: P \ 0.001). Finally, the total b-catenin protein
content increased only in the PT group (P \ 0.05). Our
data indicate that a PT regimen triggers greater responses
in key elements of the WNT pathway.
Keywords Skeletal muscle � Gene expression � Strength �Power
Introduction
Skeletal muscle hypertrophy is a widely recognized adap-
tation to resistance training. This adaptation has been
associated with changes in the gene expression profile and
the rate of protein synthesis (Coffey and Hawley 2007;
Toigo and Boutellier 2006). Accordingly, in the past few
years, much attention has been given to the molecular
mechanisms underlying muscle hypertrophy, such as the
expression and activity of MGF, the Akt/mTOR pathway,
myostatin, and atrogin (Glass 2003, 2005; Welle et al.
2007; Williams 2004). However, the molecular basis of the
adaptations produced by different resistance-training regi-
mens such as power and strength training is still elusive.
Previous studies have reported muscle fiber hypertrophy
after a strength-training protocol (Campos et al. 2002;
Lamas et al. 2010; Staron et al. 1990). However, data
regarding power training regimens are scarce. For instance,
Moss et al. (1997) demonstrated that loads correspondent
to the maximum power training intensity induced muscle
Communicated by Hakan Westerblad.
M. L. Leal and L. Lamas contributed equally to the study.
M. L. Leal � M. S. C. Ramos � A. S. Moriscot (&)
Department of Cell and Developmental Biology,
Institute of Biomedical Sciences, University of Sao Paulo,
Av. Prof. Lineu Prestes, 1524. Butanta,
Sao Paulo, SP 05508-900, Brazil
e-mail: moriscot@usp.br
L. Lamas � C. Ugrinowitsch � V. Tricoli
School of Physical Education and Sport,
University of Sao Paulo, Sao Paulo, SP, Brazil
M. S. Aoki
School of Arts, Sciences and Humanities,
University of Sao Paulo, Sao Paulo, SP, Brazil
123
Eur J Appl Physiol (2011) 111:2535–2545
DOI 10.1007/s00421-011-1874-7
hypertrophy. Recently, our research group (Lamas et al.
2010) demonstrated that both strength-training (ST) and
power-training (PT) regimens successfully induced tran-
scription of genes related to the mTOR pathway. None-
theless, the array of pathways modulated by mechanical
stimuli is largely unknown in humans, and the identifica-
tion and characterization of such pathways are important to
provide a better understanding of the molecular mecha-
nisms related to the specific outcomes of different resis-
tance-training regimens. In this context, the wingless-type
MMTV integration site family (WNT) has recently been
shown to be involved in adaptations to mechanical over-
load. For instance, this pathway was activated during
skeletal muscle hypertrophy induced by an ablation pro-
tocol, which overloaded the plantaris muscle in mice
(Armstrong and Esser 2005).
Canonical WNT signaling is transduced through Friz-
zled receptors and the LRP5/LRP6 co-receptor to the
b-catenin cascade (Bhanot et al. 1996; Pinson et al. 2000).
b-catenin is a multifunctional protein that can act as a
co-factor modulating transcription in the nucleus (Logan and
Nusse 2004; Novak and Dedhar 1999; Shapiro 1997). In
the absence of WNT signaling, b-catenin is phosphorylated
and rapidly targeted for proteosomal degradation (Li et al.
1999; Amit et al. 2002; Gao et al. 2002); however, in the
presence of canonical WNT signaling, Disheveled (DVL)
is phosphorylated, leading to the inhibition of glycogen
synthase kinase 3b (GSK3 b) and the disintegration of the
multiprotein complex that includes GSK3 b, axin, ade-
nomatous polyposis coli (APC) and casein kinase 1 (CK1).
The latter event inhibits the degradation of b-catenin,
allowing its cytoplasmic accumulation and subsequent
nuclear translocation. Once in the nucleus b-catenin forms
a complex with transcription factors of the T-cell specific
transcription factor/lymphoid enhancer-binding factor
(TCF/LEF) family and thereby induces transcription of
WNT target genes, such as c-Myc and Cyclin D1 (Logan
and Nusse 2004; Shtutman et al. 1999). Although c-Myc
and Cyclin D1 are thought to function as cell cycle regu-
lators, recent evidence suggests that these proteins can also
regulate cell size (Hesketh and Whitelaw 1992; Montagne
2000; Piedra et al. 2002).
In a study by Armstrong and Esser (2005), it was
observed that key components of the WNT pathway were
up-regulated in overloaded plantaris muscles, i.e., the
ligand receptor frizzled 1, DVL1, b-catenin, LEF1 and the
target gene Cyclin D1. In addition, Steelman et al. (2006)
showed modulation of specific WNT-related genes in
myostatin knockout mice. Since it is well known that such
animals exhibit marked muscle growth, this study also
supports the hypothesis that WNT pathway activation may
be associated with skeletal muscle hypertrophy (Steelman
et al. 2006).
Meanwhile, other studies have shown that endogenous
inhibitors such as b-catenin-interacting protein (ICAT)
(Tago et al. 2000) and secreted frizzled-related proteins
(SFRP) (Kawano and Kypta 2003) may reduce activity in
the WNT pathway. Therefore, a better understanding of
WNT signaling requires a proper investigation of its
endogenous inhibitors, since these may blunt the muscle
hypertrophy response.
Although some data are available on WNT modulation in
animal models, it is still unclear whether WNT pathway
components are regulated following different resistance-
training regimens in humans. In addition, it has not been
established whether ST and PT regimens can differentially
modulate WNT genes in humans. Since different resistance-
training regimens may promote specific adaptations, we
hypothesized that gene and protein expression related to
WNT signaling might be differentially modulated by the ST
and PT regimens. Therefore, we decided to investigate the
effects of different resistance-training regimens (ST or PT for
8 weeks) on the gene and protein expression of the canonical
WNT pathway components in physically active men.
Methods
Experimental design
This study featured a 3 9 2 repeated measures design, with
the groups strength training (ST), power training (PT) and
control (C), and time (pre- and post-test) as factors. Sub-
jects performed, both in the pre- and post-tests, a 1 repe-
tition maximum (RM) squat exercise, after which a muscle
biopsy was taken. Figure 1 gives a pictorial view of the
study design. This study was conducted according to the
Declaration of Helsinki and was approved by the research
ethics committee from the Institute of Biomedical Sci-
ences, University of Sao Paulo, Brazil. Participants were
informed about the procedures and risks before giving
written consent.
Subjects
For structural/functional analysis and gene expression
determination, twenty physically active male subjects with at
least 2 years of experience in strength training were assigned
to either an ST group (n = 10) (height 177.6 ± 4.1 cm,
body mass 74.4 ± 7.6 kg, and age 22.3 ± 3.6 years), or a
PT group (n = 10) (178.1 ± 6.5 cm, 76.4 ± 9.1 kg, and
24.7 ± 4.3 years). It was a requirement that they had not
participated in any strength-training program in the last
6 months before joining the present study. Subjects in
training groups were classified in quartiles according to their
relative strength in the squat exercise (1RM/body mass).
2536 Eur J Appl Physiol (2011) 111:2535–2545
123
Then, subjects from each quartile were randomly assigned to
groups to ensure similar initial conditions between groups.
An independent t test revealed no differences in initial rel-
ative strength values between trained groups (P [ 0.05, data
not shown). The C group (n = 5) (176.3 ± 3.4 cm,
77.4 ± 7.2 kg, and 27.1 ± 4.3 years) was composed of
healthy sedentary males. For Western blot analysis, we
evaluated 10 subjects either in ST group (n = 5) or PT group
(n = 5).
One repetition maximum test and power production
during the high velocity squat
The 1 repetition maximum (1RM) and the high velocity
squat (HVS) were performed on a Smith machine (Naka-
gym�, Sao Paulo, Brazil). Body position and foot place-
ment were determined with measuring tapes fixed on both
the bar and on the ground. In addition, an adjustable-height
wooden seat was placed behind the subjects to keep the bar
displacement and knee flexion angle constant on each squat
repetition. Subjects’ settings on the Smith machine were
recorded to guarantee the same positioning across testing
sessions. The 1RM squat test was performed according to
standard procedures described by Brown and Weir (2001).
Subjects ran for 5 min on a treadmill at 9 km.h-1, followed
by lower limb light stretching exercises and two light squat
warm-up sets. Individuals performed five repetitions with
50% of the estimated 1RM in the first set, followed by a set
of three repetitions with 70% of the estimated 1RM. A
3 min interval was allowed between sets. After the second
warm-up set, subjects rested for 3 min. Then, they were
given up to five trials to achieve the 1RM load (e.g.
maximum weight that could be lifted once with the proper
technique), with a 3 min interval between attempts. Max-
imum strength values were normalized for body weight for
further analysis.
To assess lower limb power, the mechanical power
produced during the HVS was determined from changes in
the Smith machine bar position through six repetitions.
Before the actual assessment, subjects underwent two
familiarization sessions in which they were considered
acquitted with the test if intra- and inter-day variation was
lower than 3%. Vertical displacement of the bar was col-
lected at a frequency of 50 Hz and a finite differentiation
technique was used to estimate bar velocity and accelera-
tion. Next, bar force and power were calculated using
standard procedures (Bosco et al. 1995). Subjects per-
formed two sets of six repetitions with 26.5 kg as warm-up
with a 1 min rest interval. Then they executed two addi-
tional sets of six repetitions at 50% of the squat 1RM, as
fast as possible, with a 3 min rest interval. The highest
relative mean concentric power obtained in a single repe-
tition of the HVS was considered for data analysis.
Training regimens
The ST group trained with intensities between 10RM and
4RM, while the PT group trained with intensities varying
from 30 to 60% of 1RM. Both groups were instructed to
perform each repetition as fast as possible, and trained with
a similar number of sets per week. However, training
intensities and external movement velocity were different
between them. Training intensities progressed through the
8-week period following a periodized model and were
about 40% higher in the ST group compared with the PT
group (Table 1). Load adjustments were performed every
4 weeks for both the ST and PT groups. The ST exercise
load was based on the maximum load that could be sup-
ported to complete the desired number of repetitions, while
the PT exercise load was based on a percentage of the squat
1RM. The differences in the method for determining the
training load between groups were due to two factors:
(a) the maximum number of repetitions performed at a
specific percentage of the 1RM load (e.g., 70% of the
1RM) has a very high variability between individuals.
Therefore, determining the maximum weight to perform a
Fig. 1 Time line of the study design
Eur J Appl Physiol (2011) 111:2535–2545 2537
123
specific number of repetitions increases the precision of the
ST training load; and (b) maximum power is usually gen-
erated with exercise loads between 30 and 60% of the squat
1RM for physically active individuals.
Muscle biopsy sampling
Unilateral pre- and post-test muscle samples were taken
from the midportion of the vastus lateralis muscle of the
right leg using the percutaneous biopsy technique with
suction. Immediately after the procedure, the muscle
sample was removed from the needle and frozen in liquid
nitrogen. The pre-test biopsy was done 48 h after the squat
1RM pre-test and 4 days before the start of the training
period. The post-test biopsy was taken through an incision
adjacent to the pre-test site 48 h after the last training
session. It should be mentioned that the muscle samples
from PT and ST groups in the pre- and post-test conditions
were also used as a source of tissue in a previous study
(Lamas et al. 2010).
Measurement of fiber cross-sectional area
Images of fiber sections were captured on a computer with
the imaging software MetaMorph version 6.1 (Universal
Imaging Corp.�, USA). The cross-sectional area of all
muscle fibers was determined using computerized pla-
nimetry, in the 4.3 pH image (Image PRO Plus�, Media
Cybernetics�, USA). A range of 100–261 and 109–306
fibers were assessed per subject in the pre- and post-test
muscle samples, respectively.
Reverse transcription
Total cellular RNA was isolated from pre- and post-test
muscle samples using the TRIzol reagent (Invitrogen�,
Carlsbad, CA, USA). Total RNA (1 lg) was typically used
in a reaction containing oligo DT (0.5 lg/ll), 10 mM of
each dNTP, 5 9 first-strand buffer, 0.1 M dithiothreitol
and 200 U reverse transcriptase (SuperScript II, Invitro-
gen�, Carlsbad, CA, USA). Reverse transcription was
performed at 70�C for 10 min followed by 42�C for 60 min
and 95�C for 10 min.
Primer design
Primer sets were designed using Primer Express Software
(Applied Biosystems�, Foster City, CA, USA). The
sequences of primers for each gene are shown in Table 2.
Real-time polymerase chain reaction (PCR)
All samples were analyzed in duplicate, and the emitted
fluorescence was quantified with an ABI Prism 7300
sequence detector (Applied Biosystems�, Foster City, CA,
USA) based on current methodology (Bustin 2002).
Table 1 Training regimens for strength-training and power-training groups
Monday Wednesday Friday
Training protocol—strength training group
Week 1 4 9 10 4 9 10 4 9 10
Week 2 2 9 10; 3 9 8 3 9 8; 2 9 6 2 9 10; 3 9 8
Week 3 3 9 8; 3 9 6 3 9 8; 3 9 6 3 9 8; 3 9 6
Week 4 2 9 10; 2 9 8; 2 9 6 2 9 10; 2 9 8; 2 9 6 2 9 10; 2 9 8; 2 9 6
Week 5 1 9 8; 3 9 6; 3 9 4 3 9 6; 4 9 4 4 9 6; 3 9 4
Week 6 3 9 6; 3 9 4 4 9 6; 4 9 4 4 9 6; 4 9 4
Week 7 3 9 6; 3 9 4 3 9 6; 3 9 4 3 9 6; 3 9 4
Week 8 2 9 6; 2 9 4 2 9 6; 2 9 4 2 9 6; 2 9 4
Training protocol—power training group
Week 1 4 9 8 (30%) 4 9 8 (30%) 4 9 8 (30%)
Week 2 2 9 8 (30%); 3 9 8 (40%) 3 9 8 (40%); 2 9 6 (50%) 2 9 8 (30%); 3 9 8 (40%)
Week 3 3 9 8 (40%); 3 9 6 (50%) 3 9 8 (40%); 3 9 6 (50%) 3 9 8 (40%); 3 9 6 (50%)
Week 4 2 9 8 (30%); 2 9 8 (40%); 2 9 6 (50%) 2 9 8 (30%); 2 9 8 (40%); 2 9 6 (50%) 2 9 8 (30%); 2 9 8 (40%); 2 9 6 (50%)
Week 5 2 9 8 (30%); 3 9 8 (40%); 2 9 6 (50%) 3 9 8 (40%); 4 9 6 (60%) 3 9 8 (30%); 2 9 6 (50%); 2 9 6 (60%)
Week 6 3 9 8 (30%); 3 9 6 (60%) 4 9 8 (40%); 4 9 6 (50%) 4 9 8 (30%); 4 9 6 (60%)
Week 7 3 9 8 (40%); 3 9 6 (60%) 2 9 8 (40%); 4 9 6 (60%) 2 9 8 (40%); 4 9 6 (60%)
Week 8 2 9 8 (40%); 2 9 6 (50%) 2 9 8 (40%); 2 9 6 (50%) 2 9 8 (40%); 2 9 6 (50%)
2 9 10 number 2 represents the number of sets, while number 10 describes the number of repetitions. Percentage within parentheses indicates the
training load (1RM %)
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Analyses of amplification plots were performed using
Applied Biosystems sequence detection software, and the
results were expressed using the comparative cycle
threshold (CT) method described in the manufacturer’s user
bulletin no. 2 (Applied Biosystems�, Foster City, CA,
USA).
The CT represents the PCR cycle at which an increase in
reporter gene fluorescence above a baseline signal can first
be detected. The DCT values were calculated for every
sample for each gene of interest as follows: (CT Target-CT
Endogenous control). Ribosomal protein large P0 (RPLP0) was
used as the endogenous control gene. This gene has been
previously used as an endogenous control in exercising
humans (Sorensen et al. 2009; Leger et al. 2006) and we
have consistently found that its expression does not vary
after either ST or PT regimens (CT means ± SD for pre-
and post-test conditions were 20.15 ± 1.02 and
20.19 ± 0.74 respectively, data not shown).
The relative changes in the expression levels of each
specific gene were calculated by subtracting the DCT of the
pre-test condition (used as the calibrator) from the corre-
sponding DCTs of the two experimental groups. The values
and ranges given were determined as follows: 2�DDCT with
DDCT ± SD = (CT Target-CT Endogenous control)Post-test-
(CT Target-CT Endogenous control)Pre-test, with the SD being the
standard deviation of the mean DDCT value (user bulletin
no. 2, Applied Biosystems�, Foster City, CA, USA).
Expression values are shown as mean plus standard devi-
ation of fold differences relative to the pre-test condition,
which was arbitrarily set to 1.
Protein expression
Total protein from vastus lateralis muscle was obtained
using 30 mg of tissue to 1 ml of digestion buffer (KCl
90 mM, Hepes 10 mM, MgCl2? 3 mM, EDTA 5 mM,
glycerol 1%, DTT 1 mM, SDS 0.04%). Protein concentra-
tion was determined by the Bradford method. Twenty lg of
total protein was subjected to electrophoresis in 5% stacking
and 15% resolving polyacrylamide-SDS gels, and the sep-
arated proteins were transferred to a nitrocellulose mem-
brane (Bio-Rad Laboratories�, Hercules, CA, USA). The
membrane was stained with Ponceau solution to demon-
strate that the protein concentration was similar among
lanes. The membrane was then washed with TBST (Tris
50 mM, NaCl 150 mM, pH 7.5 and Tween-20, 2%) for
10 min at room temperature, after which it was incubated
overnight at 4�C with rabbit polyclonal antibodies against
b-catenin (SC-7,199, 1:700; Santa Cruz Biotechnology
Inc.�, CA, USA) in TBST. After washing, the membrane
was incubated with a secondary anti-rabbit IgG conjugated
with peroxidase (Jackson Immuno Research Laboratories�,
PA, USA; 1:10,000 dilution in TBST) for 1 h at room
temperature. Following further washing with TBST the
membrane was incubated with ECL detection reagent
(Amersham Biosciences�, Little Chalfont, Buckinghamshire,
UK), which produced a chemiluminescent signal that was
detected by exposure to X-ray film. The protein bands were
quantified by densitometry and expressed as percentages.
Statistical analysis
A standard descriptive statistical analysis (average and
standard deviation) was followed by the Shapiro–Wilk and
Levene tests to confirm data normality and variance
homogeneity, respectively. A mixed model was produced
for each dependent variable, with groups and time as fixed
factors, and subjects as a random factor (Ugrinowitsch
et al. 2004) to test for differences in mRNA and b-catenin
protein expression. Whenever a significant F value was
Table 2 Primer sequences used for real-time PCR
Genes Forward (50-30) Reverse (50-30)
WNT1 CAACCGAGGCTGTCGAGAAA AGGATTCGATGGAACCTTCTGA
Frizzled 1 CCTCCACGGTCACCTTCAAC TTTGGCATTGGTTATCTCACTCA
SFRP1 ACGGTGTGTCCTCCCTGTGA GCAAACTCGCTGGCACAGA
DVL1 CACACGGTCAACAAGATCACCTT GGCGAGATTGCTGCAGAGAT
b-catenin AGCTGATTTGATGGAGTTGGACAT GTAAGACTGTTGCTGCCAGTGACTA
ICAT AGCACCTGTTTGCCTGAAGT CTGGCTGTCAGGTTTGATCC
LEF1 TCCAGCTCCTGATATCCCTACTTT CTGACCTTGCCAGCCAAGAG
c-Myc ACTTACAACACCCGAGCAAG CGAGTCGTAGTCGAGGTCAT
Cyclin D1 TCGTGGCCTCTAAGATGAAGGA AGGTTCCACTTGAGCTTGTTCAC
RPLP0 CGACCTGGAAGTCCAACTAC ATCTGCTGCATCTGCTTG
Genbank Accession codes for WNT1, frizzled 1, SFRP1, DVL1, b-catenin, ICAT, LEF1, c-Myc, Cyclin D1 and RPLP0 genes are NM_005430,
NM_003505, NM_003012, NM_182779, NM_001904, NM_020248, NM_016269, NM_002467, NM_053056 (RPLP0 NM_001002 transcript
variant 1, NM_053275 transcript variant 2), respectively
Eur J Appl Physiol (2011) 111:2535–2545 2539
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obtained, a post-hoc test with a Tukey adjustment was
performed for multiple comparison purposes. The signifi-
cance level was set at P \ 0.05.
Results
Relative strength, relative concentric power, and fiber
cross-sectional area after ST and PT regimens
Progressive resistance training resulted in significant
increases in relative strength and relative mean concentric
power from the pre- to the post-test condition (P \ 0.001,
main time effect) in both trained groups (ST, 24 and 7%;
PT, 14 and 11%, respectively). However, there were no
significant differences between trained groups in the
post-test condition, for both relative strength and mean
concentric power (P [ 0.05; Fig. 2a, b). Muscle fiber
cross-sectional area also increased significantly in both
trained groups in the post-test (P \ 0.05) as compared to
the pre-test condition (Fig. 2c), although with no signif-
icant differences between groups in the last assessment
(P \ 0.05).
Gene expression after ST and PT regimens
Several genes encoding proteins involved in the canonical
WNT-signaling pathway were analyzed. Figures 3, 4 and 5
show the fold changes in mRNA levels of the selected
WNT pathway-related genes, normalized for RPLP0 and
relative to pre-test levels for the ST, PT, and C groups.
Firstly, we highlight gene expression profiles related to
ligand-receptor coupling and endogenous inhibition of
WNT receptor binding. The results showed that, in the
trained groups, mRNA levels for the ligand WNT1
increased 6.4-fold (P \ 0.0001) in the ST group and 24.9-
fold (P \ 0.0001) in the PT group (Fig. 3a). Although
mRNA levels for the frizzled 1 receptor were not signifi-
cantly different from pre- to post-test in both ST and PT
groups (i.e., interaction effect; P [ 0.05), a main time
effect was observed (P \ 0.01, Fig. 3b). Meanwhile,
mRNA levels for the WNT ligand pathway inhibitor
SFRP1 increased 3.3-fold (P \ 0.0001) in the ST group
and 2.7-fold (P \ 0.0001) in the PT group (Fig. 3c). In the
control group, expression levels of these genes were not
significantly altered (Fig. 3a, b and c).
Subsequently, we analyzed downstream genes of the
WNT pathway (b-catenin, LEF1 and Cyclin D1). Although
b-catenin mRNA levels were not significantly different
from the pre-test condition in both ST and PT groups, a
main time effect was observed (P \ 0.001, Fig. 4a). The
mRNA levels of the transcription factor LEF1 increased
7.3-fold (P \ 0.0001) in the ST group and 34.1-fold
(P \ 0.0001) in the PT group (Fig. 4b). Interestingly, only
PT induced an increase (7.7-fold; P \ 0.001) in Cyclin D1
gene expression (Fig. 4c). In the control group, expression
levels of these genes were not significantly altered (Fig. 4a,
b and c).
Lastly, we grouped together genes whose expression
was not significantly altered by either training regimen.
These included DVL1, ICAT and c-Myc genes (Fig. 5a, b
and c, respectively).
Fig. 2 Relative maximum strength (a), relative concentric power
(b) and fiber cross-sectional area (c) for strength-training (ST,
n = 10, black bars) and power-training (PT, n = 10, gray bars)
groups in the pre- and post-test conditions (mean ± SD). Main time
effect, post-test greater than pre-test *P \ 0.01
2540 Eur J Appl Physiol (2011) 111:2535–2545
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b-catenin protein expression
After 8 weeks of training, the total protein expression of
b-catenin was quantified in the ST and PT groups. No
significant changes in b-catenin protein expression levels
were observed in the ST group; however, these levels were
significantly increased after training in the PT group
(P \ 0.05, Fig. 6).
Fig. 3 Gene expression in strength training (ST, n = 10, black bars),
power training (PT, n = 10, gray bars) and control (C, n = 5, whitebars) groups pre- and post-8-week training period (mean ± SD). The
genes analyzed are represented on the y axis. These were WNT1 (a),
Frizzled 1 (b) and secreted frizzled-related protein 1 (SFRP1) (c),
respectively. aPost-test greater than pre-test P \ 0.0001. bPost-test
greater than control group at post-test P \ 0.0001. cPower post-test
greater than strength post-test P \ 0.0001. dMain time effect, post-
test greater than pre-test P \ 0.01
Fig. 4 Gene expression in strength training (ST, n = 10, black bars),power training (PT, n = 10, gray bars) and control (C, n = 5, white bars)
groups pre- and post-8-week training period (mean ± SD). The genes
analyzed are represented on the y axis. These were b-catenin (a), LEF1
(b) and Cyclin D1 (c), respectively. aPost-test greater than pre-test
P \ 0.0001. bPost-test greater than control group at post-test P \ 0.0001.cPower post-test greater than strength post-test P \ 0.0001. dMain time
effect, post-test greater than pre-test P \ 0.001. ePost-test greater than
pre-test P \ 0.001. fPost-test greater than control group at post-test
P \ 0.01. gPower post-test greater than strength post-test P \ 0.001
Eur J Appl Physiol (2011) 111:2535–2545 2541
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Discussion
The main finding of the present study was that key genes
related to the canonical WNT pathway were up-regulated
after 8 weeks of ST and PT regimens. In addition, it should
be highlighted that the PT regimen was a much more potent
stimulus to elevate the expression of WNT pathway genes
and b-catenin protein levels, compared to the ST regimen.
These results corroborate our initial hypothesis that distinct
resistance-training regimens preferentially modulate gene
and protein expression of WNT pathway components.
We employed these different training regimens with the
aim of determining their effect on the gene expression
profile of the WNT pathway, which has been recently
associated with a hypertrophic response in skeletal muscle
in animal models. The WNT-signaling pathway was ini-
tially associated with colon cancer and embryonic devel-
opment (Moon et al. 1997; Peifer and Polakis 2000) and it
has been extensively demonstrated that abnormal activa-
tion of this pathway can lead to the development of
numerous types of tumors, including colorectal cancer
(Morin et al. 1997). In addition, canonical WNT target
genes such as c-Myc and Cyclin D1 have been associated
with activation of the cell cycle (Hesketh and Whitelaw
1992; Montagne 2000; Piedra et al. 2002). These genes
were also identified as potential modulators of cardiac
hypertrophy; for example, in the heart, c-Myc expression
has been found to increase quickly (*3 h) after a hyper-
trophic stimulus imposed by pressure overload (Izumo
et al. 1988). Similarly, another study, which also used the
cardiac pressure overload model, showed that knock down
of Cyclin D2 expression attenuates the hypertrophic
response (Angelis et al. 2008). Thus, it is now clear that
these WNT target genes are strongly modulated in the
cardiac hypertrophic model. Nevertheless, the role of WNT
signaling in skeletal muscle is still unclear, especially in
humans. In fact, to the best of our knowledge, this is the
first study to describe the effects of resistance-training
programs on the WNT pathway in humans.
Fig. 5 Gene expression in strength training (ST, n = 10, black bars),
power training (PT, n = 10, gray bars) and control (C, n = 5, whitebars) groups pre- and post-8-week training period (mean ± SD). The
genes analyzed are represented on the y axis. These were DVL1 (a),
ICAT (b) and c-Myc (c), respectively. There were no differences
between training conditions and regimens
Fig. 6 Effects of strength training (ST, n = 5, black bars) and power
training (PT, n = 5, gray bars) on total b-catenin protein expression
after an 8-week training period. A representative Western blot is
shown and bars represent the means ± SD of densitometric analysis
of b-catenin/alpha-actinin ratio in pre- and post-test conditions
expressed as arbitrary units. Power post-test greater than power pre-
test; *P \ 0.05. Power post-test greater than strength post-test;�P \ 0.05
2542 Eur J Appl Physiol (2011) 111:2535–2545
123
In the present study, we detected a more marked acti-
vation of WNT gene expression by the PT regimen when
compared to the ST regimen, which is likely to have
resulted from their distinct mechanical characteristics. For
instance, PT is performed using light loads and high
movement velocities, while the ST regimen involves using
high loads, which consequently lead to a greater time under
tension and lower movement velocity. Although it is well
accepted that these differences would eventually lead to
specific outcomes, we did not observe differences between
groups in performance indices (i.e., strength and power)
and muscle fiber cross-sectional area in the post-test con-
dition. Furthermore, resistance-training guidelines suggest
that heavy resistance training, as in our ST regimen, would
produce greater muscle fiber hypertrophy and molecular/
cellular modulation than the PT regimen (Fry 2004; Rata-
mess et al. 2009). On the other hand, emerging data indi-
cate that divergent resistance-training approaches can elicit
similar anabolic responses (Burd et al. 2010). These
investigators showed that similar changes occur in acute
protein synthesis response, despite large discrepancies in
training regimens.
Several studies reported muscle fiber hypertrophy after a
strength-training protocol (Campos et al. 2002; Lamas
et al. 2010; Staron et al. 1990). However, data regarding
power training are scarce. For example, Malisoux et al.
(2006) reported an average of 25% hypertrophy across fiber
types using an unloaded power training (i.e., plyometrics
only). Moss et al. (1997) also demonstrated that loads
corresponding to the maximum power training intensity
produced hypertrophy gains of 2.8% (P \ 0.001). There-
fore, it appears that muscle hypertrophy occurs even when
exercising with low loads if movement velocity is high.
Indeed, there are a couple of studies showing that high
movement velocities produce greater muscle hypertrophy
than low movement velocity, when the workload is
equalized between them (Farthing and Chilibeck 2003;
Shepstone et al. 2005). In the present study, muscle fiber
hypertrophy response was also similar between groups.
The cross-sectional area of type I, IIa and IIb fibers
increased by 15, 18 and 41%, respectively, for the ST
group, and by 15 and 19% for type IIa and IIb fibers,
respectively, for the PT group. Type I fibers decreased the
cross-sectional area by 5% after the PT, but this effect
seems to be within the normal measurement variation,
taking into consideration the variability of our control
group data. Although a similar muscle fiber hypertrophy
response was observed, it is possible that specific outcomes
of PT and ST regimens could be achieved after the period
of 8 weeks.
Out of nine genes analyzed, the PT regimen produced
greater increments in three key genes of the WNT pathway
as compared to the ST regimen: those encoding the ligand
WNT (*24- vs. *6-fold), the transcriptional factor LEF-1
(*34- vs. *7-fold) and the WNT pathway target gene
Cyclin D1 (*7.7-fold vs. no significant change under the
ST regimen). Accordingly, b-catenin protein levels were
significantly elevated only in the PT group, although
b-catenin mRNA levels were not significantly different
between groups. Such differences between mRNA and
protein expression levels are extensively observed in the
literature in many biological systems and might indicate
the involvement of post-transcriptional mechanisms, such
as increases in translational efficiency obtained by maxi-
mizing recruitment of elongation initiation factors. How-
ever, such differences also may reflect a gap in time
between mRNA levels and protein content. The elevated
level of b-catenin protein along with increased levels of
LEF-1 (its heterodimerization partner) mRNA reinforces
the effective nuclear targeting induced by PT. Furthermore,
activation of the Cyclin D1 (a major target of b-catenin/
LEF heterodimers) gene in the PT group strongly suggests
a specific biological role for the WNT pathway in skeletal
muscle adaptations to the high mechanical overload.
In our study, we also addressed the genes involved in
repression of the WNT pathway, namely, SFRP and ICAT.
SFRP competes with WNT for binding to the frizzled
receptor (Kawano and Kypta 2003), while ICAT interferes
with the b-catenin/TCF interaction and antagonizes WNT
signaling (Tago et al. 2000). Interestingly, while ICAT
gene expression was not affected by training, we observed
a marked increase in SFRP1 gene expression. Although
one might suggest that the increase in SFRP1 gene
expression would not be expected, it should be emphasized
that activation of the WNT pathway might trigger a cellular
negative feedback loop, ensuring that the system will not
be over-stimulated. Indeed, we detected several LEF-
responsive elements along the SFRP1 gene regulatory
region (data not shown), suggesting that the SFRP1 gene
could be a direct target for the LEF transcriptional factor.
The reason, as well as the physiological relevance, for
the greater responsiveness of the WNT pathway to PT over
ST is currently unknown. The total number of repetitions
and range of motion cannot account for such differences
since these parameters were balanced between groups. On
the other hand, it is possible that muscle action velocity
plays a role in the differential effects of ST and PT regi-
mens. For example, it has been shown that high velocity
eccentric muscle actions result in greater hypertrophy
(Farthing and Chilibeck 2003) than low velocity eccentric
muscle actions, probably due to greater protein remodeling
(Shepstone et al. 2005). It should be emphasized that
eccentric actions constitute a potent mechanical stimulus
since they induce muscle damage (Chapman et al. 2008)
and activate mechanical transduction mechanisms (O’Neil
et al. 2009), which can be associated with protein synthesis
Eur J Appl Physiol (2011) 111:2535–2545 2543
123
and muscle hypertrophy (Morgan and Partridge 2003). In
addition, satellite cells are well known to participate in
skeletal muscle repair; therefore, one can speculate that PT,
due to its high velocity/eccentric component, leads to a
more intense recruitment of satellite cell activity. Accord-
ingly, cyclin D1, the expression of which was markedly
increased only in PT, is involved in progression through the
cell cycle from G1 to S (Alao 2007) and could be involved
in higher satellite cell proliferation in PT. Alternatively, it
is possible that cyclin D1 is actually involved in non-
proliferative events leading to muscle fiber hypertrophy. It
is worth recalling that heart hypertrophy can be elicited by
activation of WNT target genes (Angelis et al. 2008; Izumo
et al. 1988), which is associated with an increase in cyclin
D expression (Angelis et al. 2008). Therefore, it is possible
to imagine cyclin D1 not only as a driver of the cell cycle
but also as a cell mass regulator.
Although this study sheds light into new aspects on
WNT pathway regulation by resistance training, we should
acknowledge important intrinsic limitations in the experi-
mental design, which in a certain extent restrict our precise
understanding on WNT pathway regulation by resistance
training. For example, increased number of biopsies would
greatly strengthen our knowledge upon the kinetics of
WNT-related genes after a single bout (i.e., 6, 12, and
24 h) and also the same procedure after the last bout.
Similarly, it would be very interesting to have an additional
biopsy just before the last bout, so it would be possible to
compare the effect of the resistance training, not consid-
ering the effect of the last bout.
Different training regimens would be primarily expected
to promote specific outcomes including maximized
strength gains in ST and increased power output in PT. In a
certain way, our data oppose this assumption since PT and
ST resulted in similar strength and power gains in the
period of 8 weeks. Interestingly, despite the fact that nei-
ther of the training regimens employed is designed to
promote a significant hypertrophic response, both indeed
induced a similar increase in muscle fiber cross-sectional
area. It is not clear at this point why these training regimens
are driving different outcomes in terms of WNT pathway-
related gene/protein expression modulation. Accordingly,
we also do not know the biological relevance of such
cellular alterations. One aspect that might be important is
the fact the subjects used in the present study, although not
engaged in specific strength training in the last 6 months,
are not sedentary, they are in fact physically active. This
might result in an additional period (more than 8 weeks) to
observe functional differences.
In summary, our results clearly demonstrate that resis-
tance-training regimens (ST and PT) activated expression of
components of the WNT-signaling pathway at the gene and
protein levels. Interestingly, the PT regimen induced a much
greater gene expression response and exclusively increased
b-catenin protein expression levels when compared with the
ST regimen, raising the possibility that the former elicits
WNT signaling in a preferential manner. This study provides
insight into the molecular basis of differential training
outcomes of specific regimens. Future studies should investi-
gate the effects of longer periods of different resistance-training
modes on training outcomes and molecular adaptations related
to muscle hypertrophy. In addition, future studies could focus
not only on the gene expression profile but also on measure-
ments of muscle protein expression.
Acknowledgments The authors express their gratitude to Antonio
Garcia Soares for excellent technical assistance. This work was
supported by FAPESP (Fundacao de Amparo a Pesquisa do Estado de
Sao Paulo, Brazil), Grants: 08/58415-3, 07/52288-7, 06/00302-3 and
06/61523-7.
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