Eur J Appl Physiol

DOI 10.1007/s00421-013-2733-5

Original Article

Acute lowload resistance exercise withand withoutblood flow

restriction increased protein signalling andnumber ofsatellite
cells inhuman skeletal muscle
MathiasWernbom WilliamApro GranPaulsen
TormodS.Nilsen EvaBlomstrand TrulsRaastad

Received: 3 January 2013 / Accepted: 16 September 2013

Springer-Verlag Berlin Heidelberg 2013

Abstract post-exercise, but back to baseline at 48h. The number of

PurposeTo investigate hypertrophic signalling after a
single bout of low-load resistance exercise with and with-
out blood flow restriction (BFR).
Methods Seven subjects performed unilateral knee exten-
sions at 30% of their one repetition maximum. The sub-
jects performed five sets to failure with BFR on one leg,
and then repeated the same amount of work with the other
leg without BFR. Biopsies were obtained from m. vastus
lateralis before and 1, 24 and 48h after exercise.
Results At 1-h post-exercise, phosphorylation of p70S6K-
Thr389
and p38MAPKThr180/Tyr182 was elevated in the
BFR leg, but not in the free-flow leg. Phospho-p70S6K-
Thr389
was elevated three- to fourfold in both legs at 24-h
Communicated by Martin Flueck.
M.Wernbom G.Paulsen T.S.Nilsen T.Raastad resistance exercise Anabolic signalling P70S6 kinase
Department ofPhysical Performance, Norwegian School ofSport
Sciences, Oslo, Norway
M.Wernbom(*)
Lundberg Laboratory forHuman Muscle Function andMovement
Analysis, Department ofOrthopaedics, Sahlgrenska University
Hospital, The Sahlgrenska Academy atUniversity ofGothenburg,
Grna Strket 12, 413 45Gteborg, Sweden
e-mail: mathias.wernbom@orthop.gu.se
W.Apro E.Blomstrand
strand Laboratory, Swedish School ofSport andHealth
Sciences, Stockholm, Sweden
W.Apro
Department ofClinical Sciences, Intervention andTechnology,
Karolinska Institutet, Stockholm, Sweden
E.Blomstrand
Department ofPhysiology andPharmacology, Karolinska
Institutet, Stockholm, Sweden
visible satellite cells (SCs) per muscle fibre was increased
for all post-exercise time points and in both legs (33
53%). The proportion of SCs with cytoplasmic extensions
was elevated at 1-h post in the BFR leg and the number of
SCs positive for myogenin and/or MyoD was increased at
1- and 24-h post-exercise for both legs combined.
Conclusion Acute low-load resistance exercise with BFR
resulted in early (1h) and late (24h) enhancement of phos-
pho-p70S6KThr389, an early response of p38MAPK, and an
increased number of SCs per muscle fibre. Enhanced phos-
pho-p70S6KThr389 at 24-h post-exercise and increases in SC
numbers were seen also in the free-flow leg. Implications of
these findings for the hypertrophic effects of fatiguing low-
load resistance exercise with and without BFR are discussed.
Keywords Blood flow restricted exercise Ischaemic
Introduction
Resistance exercise with low loads in combination with
blood flow restriction (BFR), also referred to as strength
training with vascular occlusion, has repeatedly been
shown to induce muscle hypertrophy in untrained individu-
als (e.g., Takarada etal. 2002; Nielsen etal. 2012; Lauren-
tino etal. 2012). In some studies, BFR resistance exercise
(BFRRE) has been applied very frequently (12 sessions
per day) for 13weeks, resulting in significant increases in
muscle cross-sectional area (CSA), muscle fibre CSA and
strength despite the very short training periods (Abe etal.
2005; Fujita etal. 2008; Nielsen etal. 2012).
However, marked hypertrophy has been noted even
with only 2 sessions per week of BFRRE with multiple

13

sets (Takarada etal. 2002, 2004; Laurentino etal. 2012),
and the rates of quadriceps CSA gains observed in these
studies (0.110.22% per calendar day) are at least equal
to the rates typically resulting from multiple-set heavy
resistance exercise at the same frequency of training
(reviewed by Wernbom etal. 2008). This observation
indicates that fatiguing low-load BFRRE, like conven-
tional heavy resistance training, can induce long-lasting
elevations in muscle protein synthesis (MPS) and ana-
bolic signalling.
Also similarly to heavy resistance exercise, low-load
BFRRE appears to increase MPS at least in part through
the mTOR-p70S6K pathway (Fujita etal. 2007; Fry etal.
2010). However, studies on acute anabolic signalling with
BFRRE have to date mainly focused on the early time
period (03h) after exercise, thus little is known about the
time course of hypertrophic signalling in the later part of
the recovery after an acute bout of low-load strength train-
ing with BFR.
In addition to protein synthesis, long-term load-induced
muscle hypertrophy is usually accompanied by myonu-
clear addition, which in turn is dependent on satellite cell
(SC) activation (Kadi etal. 1999). There is evidence that
in humans, large increases in myofibre CSA in response
to strength training are dependent on myonuclear addition
(Kadi etal. 1999; Petrella etal. 2008) and that muscle fibre
area is correlated with the number of myonuclei per muscle
fibre (Kadi etal. 1999; Nielsen etal. 2012).
Currently, relatively little is known about the effects of
BFRRE on SCs, myonuclei, and myogenic regulatory fac-
tors (MRFs). Drummond etal. (2008) reported increases
in the SC activity markers p21 and MyoD, as well as
decreased myostatin mRNA at 3h after BFRRE (4 sets of
1530 repetitions at 20% of 1RM), while the mRNA for
myogenin, cyclin D1 and the insulin-like growth factor 1
(IGF-1) isoform MGF did not change. Interestingly though,
the control training at the same load without occlusion
induced similar changes as the BFRRE. In contrast, Manini
etal. (2011) found no significant changes in the mRNA for
MyoD, myogenin, myostatin, and IGF-1 8h after acute
BFRRE. A recent study demonstrated remarkable increases
in the numbers of Pax-7 positive SC and in myonuclei with
a short-term period of BFRRE (Nielsen etal. 2012). How-
ever, the effects of a single bout of BFRRE on SC numbers
have not yet been investigated.
Therefore, the aim of the current investigation was to
study the acute response to a BFRRE session, with refer-
ence to anabolic signalling and the number of SCs, up to
48-h post-exercise. Our hypothesis was that exercise with
BFR induces a more pronounced and long-lasting effect
than exercise with normal blood flow with regard to ana-
bolic signalling.
13
Eur J Appl Physiol
Materials andmethods
Subjects
Six male subjects and one female subject (meanSE,
26 1year, height 1793cm, body mass 804kg)
were recruited from the student populations at the Nor-
wegian School of Sports Science in Oslo and the School
of Sports Science at the University of Gothenburg. All
subjects were exercising on a regular basis (e.g., running,
cycling, cross-country skiing, alpine skiing, skating, trek-
king) and had previous experience with resistance training,
but some were not performing heavy strength training at
the time of the study (n=2), while others were very well
trained in this regard (n=3), and still others were moder-
ately trained (n=2).
The subjects were informed of the experimental risks
and signed an informed consent document prior to the
investigation. The study was approved by the Regional Eth-
ics Committee of Southern Norway and complied with the
standards set by the Declaration of Helsinki regarding the
use of human subjects.
Experimental design
A within-subjects study design was used in which one leg
was randomised to exercise with partial BFR and the other
leg to exercise without BFR. Four out of the seven subjects
performed the BFR exercise with their dominant leg. To
match the volume of work, and because occlusion reduces
the number of repetitions to failure at low loads (Wernbom
etal. 2006, 2009), the BFR leg was always exercised first.
All subjects participated in a familiarisation session one
week (range 411days) before the main experiment. In the
main trial, the subjects were tested for maximal voluntary
isometric contraction (MVC) strength immediately before
the exercise bout, at 1- and 2-min post-exercise, and at 4,
24, 48, 72, 96 and 168h post-exercise. Detailed strength
and recovery data for the subjects have been reported else-
where (Wernbom etal. 2012).
Biopsies were obtained from vastus lateralis before and
1, 24 and 48h after exercise. The pre-biopsy, as well as the
24 and the 48-h biopsies were collected before strength
testing commenced, while the 1-h post-exercise samples
were taken after the exercise protocols had been completed.
The subjects were studied in a fed state, and they were
instructed to consume breakfast ~3h before reporting to
the laboratory on the days when biopsies were taken. The
rationale for this was that overnight fasting is not represent-
ative of the typical strength training situation, and that we
wanted to see if previous findings of BFR resistance train-
ing in the fasted state on muscle signalling would extend
Eur J Appl Physiol
also to training in the fed state. Nevertheless, it should be
noted that the subjects were studied 3h after feeding with
regard to the pre-exercise biopsy and the 24 and 48h biop-
sies, and 4h after feeding with regard to the 1-h post-
exercise biopsy.
The participants were instructed not to change their die-
tary habits during the time course of the study, and were
asked to refrain from any drinks that contained caffeine or
alcohol, as well as not to take anti-inflammatory drugs and
nutritional supplements (e.g., amino acids, creatine, and
exogenous antioxidants) that could impact on the results of
the investigation. They were also instructed not to perform
any strenuous activities involving their quadriceps muscles
during the last 72h before the main test.
Muscle function testing andfamiliarisation protocols
Muscle function testing and familiarisation protocols have
been reported in detail previously (Wernbom etal. 2012).
Briefly, 411days before the main exercise test, the sub-
jects were tested for their unilateral 1RM strength for each
leg in a variable resistance knee extension machine (Leg
Extension model 66478, Gym2000, Geithus, Norway). The
1RM was determined according to the procedures of Staron
etal. (1990). Following the 1RM testing and the practice of
MVCs, the subjects were familiarised with BFR resistance
exercise and performed two sets of BFRRE with submaxi-
mal effort with each leg.
Main exercise andtesting protocols
At a subsequent session after the 1RM testing, with ~7days
between the sessions (range 411), the subjects reported to
the laboratory for the main test. The subjects warmed up
5min on a stationary bicycle at a light load (~75 Watts) and
then moved to the knee extension apparatus. After warm-
ing up with three submaximal isometric contractions with
gradually increasing effort for each contraction, the sub-
jects performed two MVCs.
After 5min of rest, the subjects performed unilateral
knee extension exercise at 30% of 1RM during partial
BFR (see further below). After inflation of the tourniquet,
the subjects did as many repetitions as possible for a total
of five sets for the occluded leg. The cuff pressure was
maintained during the whole exercise bout, including the
rest periods and was released after the post-exercise test-
ing had been completed (total time of partial BFR: ~9min).
The subjects remained seated in the machine during the
rest periods, and the rest period between each set was
45s for both the occluded leg and the free-flow leg. The
range of motion was between 100 and 20 of knee flexion
(0 =full extension) and the cadence was 20 repetitions
per minute (1.5s each for the concentric and the eccentric
muscle actions, respectively) which was controlled with a
metronome set at 40bpm. No rest was permitted between
the repetitions, and the subjects were instructed to stop the
eccentric phase just before the weight stack touched down
to avoid relaxation of the quadriceps. The number of com-
plete repetitions performed in each set was noted.
When the five sets for the occluded leg had been com-
pleted, and the muscle function tests had been performed,
the pressure cuff was deflated. After 10min rest, the sub-
jects performed exactly the same number of sets and repeti-
tions and muscle function testing for the other leg as with
the occluded leg, but without BFR.
Blood flow restriction
A curved tourniquet cuff of 135mm in width (width of the
pneumatic bag: 100mm) was connected to a surgical tour-
niquet system (Zimmer A.T.S. 2000, Zimmer Patient Care,
Dover, OH) with automatic regulation of the pressure. The
cuff was wrapped around the proximal part of the thigh and
inflated to a pressure of 90100mm Hg just before exercise
with vascular occlusion and kept inflated during the rest
periods in between the sets. With the same cuff, we previ-
ously demonstrated that this level of pressure was sufficient
to markedly affect muscle performance when compared
with free-flow conditions (Wernbom etal. 2009, 2012).
Pressures in this range in combination with wide cuffs have
also successfully been used in training studies on BFRRE
(Laurentino etal. 2012; Nielsen etal. 2012).
Doppler ultrasound measurements of the femoral artery
blood flow performed during the familiarisation session
revealed that for the male subjects, full occlusion occurred
on average at ~180mm Hg, and for the female subjects at
~160mm Hg. Because too high pressures with a wide cuff
result in the subject being unable to complete more than
23 sets to failure (Wernbom, unpublished observations),
we decided to use a pressure of 100mm Hg for the male
subjects and 90mm Hg for the female subjects, corre-
sponding to about 50% of the full occlusion pressure plus
an additional 10mm Hg.
During the familiarisation session, we observed a ~50
60% decrease in femoral artery blood flow in our subjects
with cuff pressures of 90100mm Hg at rest in the seated
position before exercise. In addition to achieving full
occlusion at lower pressures than narrow cuffs, wide cuffs
reduce the variability in full occlusion pressures and make
these less dependent on limb circumference (Crenshaw
etal. 1988; Graham etal. 1993).
Muscle biopsy sampling
Biopsies were obtained from m. vastus lateralis in the free-
flow leg before exercise and from both legs 1, 24 and 48h
13

after exercise and care was taken to ensure that the time
points of the post-exercise biopsies were respected. The
rationale for this design was that investigating muscle dam-
age at the cellular level after BFRRE was also a research
aim of this project (see Wernbom etal. 2012), therefore,
we wanted to exclude the possibility that any damage seen
in the first post-exercise muscle sample from the BFR leg
might have been caused by previous biopsy sampling. Dur-
ing the biopsy, subjects laid supine, and the procedure was
performed with a Bergstrm needle under local anaesthesia
(Xylocain adrenaline, 10mgml1 +5gml1; Astra- fluoride membranes (Bio-Rad Laboratories) at a constant
Zeneca, Sdertlje, Sweden).
The first muscle sample from each leg was taken from
the vastus lateralis approximately midway between the ori-
gin and the insertion, from a location which was distal to
the part that was compressed by the tourniquet during train-
ing (occluded leg). In the free-flow leg, this incision was
also used for the second biopsy (1-h post-exercise) but the
needle was angled in a different direction so that the sam-
ple was taken about 5cm from the first. The second (24h)
and third incisions (48h) were placed approximately 3 and
6cm proximally to the first incision. Care was taken to
avoid affected tissue from earlier biopsies.
Originally, biopsies were taken from a further three par-
ticipants, i.e., ten subjects in total, but unfortunately sam-
ples were lost for some of the subjects due to technical
problems. Complete sets of biopsies (including both limbs
and all time points) were achieved in 7 of the subjects for
all analyses, except protein signalling at 48-h post-exercise
(n=6).
Tissue processing forprotein signalling
Muscle biopsy specimens were freeze-dried, freed of
blood and connective tissue, and then homogenised in
ice-cold buffer (80l/mg dry weight) containing 2mM
HEPES (pH 7.4), 1mM EDTA, 5mM EGTA, 10mM
MgCl2, 50mM -glycerophosphate, 1% TritonX-100,
1mM Na3VO4, 2mM dithiothreitol, 20g/ml leupep-
tin, 50g/ml aprotinin, 1% phosphatase inhibitor cock-
tail (Sigma P-2850), 40g/l PMSF. Homogenates were
then centrifuged at 10,000g for 10min at 4C to remove
cellular debris and the resulting supernatant was stored at
80C. Protein concentrations were determined in ali-
quots of supernatant diluted 1:10 in distilled water using
a bicinchoninic acid assay (Pierce Biotechnology, Rock-
ford, IL, USA). Samples were diluted in Laemmli sample
buffer (Bio-Rad Laboratories, Richmond, CA, USA) and
homogenising buffer to obtain a final protein concentra-
tion of 1.5g/l. Following dilution, the samples were
heated at 95C for 5min to denature proteins present in
the supernatant. Samples were then kept at 20C until
further analysis.
13
Eur J Appl Physiol
Immunoblot analysis
Samples containing 30g total proteins were separated
by SDS-PAGE on Criterion cell gradient gels (420%
acrylamide; Bio-Rad Laboratories) for separation of all
proteins investigated. Electrophoresis was performed on
ice at 200V for 120min, following which the gels were
equilibrated in transfer buffer (25mM Tris base, 192mM
glycine, and 10% methanol) for 30min to optimise trans-
fer. The proteins were then transferred to polyvinylidine
current of 300mA and 100V for 3h at 4C, after which
the membranes were stained with MemCodeTM Revers-
ible Protein Stain Kit (Pierce Biotechnology) (Anthara-
vally etal. 2004) to confirm equal loading of the samples.
All samples were run simultaneously on multiple gels,
however, with all samples from each subject loaded on the
same gel always beginning with the pre-exercise sample
followed by the free-flow leg and BFR leg samples for each
time point.
Membranes were blocked for 1h at room temperature
in Tris-buffered saline (TBS; 20mM Tris base, 137mM
NaCl, pH 7.6) containing 5% non-fat dry milk Subse-
quently, the membranes were incubated overnight with
commercially available primary polyclonal phosphospe-
cific antibodies (mTOR Ser2448; p70S6K Thr389; eEF2
Thr56; AMPK Thr172; ERK1/2 Thr202/Tyr204; p38MAPK
Thr180/Tyr182; Akt Ser473; p90RSK Thr573; Cell Signaling
Technology Inc., Danvers, MA, USA).
Antibodies were diluted 1:1,000 (1:500 for p90RSK
Thr573 and 1:4,000 in the case of eEF2 Thr56) in TBS sup-
plemented with 0.1% Tween-20 containing 2.5% non-fat
dry milk (TBSTM). After incubation with these primary
antibodies, the membranes were washed with TBSTM and
then incubated for 1h at room temperature with second-
ary anti-rabbit IgG antibodies conjugated with horseradish
peroxidase (diluted 1:10,000; Cell Signaling Technology
Inc.). Next, the membranes were washed serially (twice
for 1min followed by three times for 15min) with TBS
TM followed by 4 additional washes with TBS for 5min
each. Finally, membranes with the antibodies bound to the
phosphorylated proteins were visualised by chemilumines-
cent detection on a Molecular Imager ChemiDocTM XRS
system and the bands were analysed using Quantity One
version 4.6.3 software (Bio-Rad Laboratories). All values
are expressed relative to total protein levels measured with
MemCode Reversible Protein Stain Kit.
Immunohistochemistry
Serial cross-sections (8m) were incubated with antibodies
(ab). To visualise SCs/myoblasts, sections were analysed for
immunoreactivity against NCAM/CD56 (monoclonal ab,
Eur J Appl Physiol
ab9018, Abcam; 1:200). To follow the activation and differ-
entiation of the satellite cells, polyclonal antibodies against
MyoD and myogenin were incubated along with NCAM.
Double staining with NCAM and MyoD was performed on
one section, and double staining with NCAM and myogenin
on a neighbouring section (Hanssen etal. 2012). Alexa
Fluor 488 and Alexa Fluor 594 (goat anti-mouse and goat
anti-rabbit; Invitrogen-Molecular Probes, Eugene, Oregon,
USA) were used as secondary antibodies. The sections were
finally counterstained with DAPI (for nuclear staining) and
mounted under coverslips (ProLong Gold Antifade Reagent
with DAPI, P36935, Invitrogen-Molecular Probes). Images
of the stained cross-sections were captured using an Axi-
ocam camera (Zeiss, Oberkochen, Germany) mounted on
a Axioskop-2 light microscope (Zeiss). Multiple images
(10, 20, and 40 objectives) were taken so that the
whole muscle biopsy cross-section was captured.
We decided to use NCAM/CD56 instead of PAX7 to iden-
tify satellite cells/myoblasts. The rationale for this was in part
based on the observations that NCAM and PAX7 together
mark ~9495% of the satellite cells in human skeletal mus-
cle (Verdijk etal. 2007; Lindstrm and Thornell 2009), with
NCAM alone marking an additional ~5% of the satellite
cells and myoblasts (Lindstrm and Thornell 2009). Because
PAX7 is downregulated in satellite cells which undergo dif-
ferentiation (Zammit 2008), the ~5% satellite cells which
are marked by NCAM alone probably represent satellite cells
which are differentiating, possibly to become new myonuclei
or replace myonuclei due to increased turnover (Lindstrm
etal. 2010). Thus, NCAM can follow activated human satel-
lite cells for a longer time course than PAX7.
A nucleus with DAPI staining surrounded by NCAM
staining and which was localised to the border of the mus-
cle fibre was considered as a satellite cell (Petrella etal.
2008; Mackey etal. 2011; Hanssen etal. 2012). An MRF-
positive satellite cell was identified as NCAM staining
around myogenin and/or MyoD staining (Hanssen etal.
2012). For SC counts, all myofibres on the sections were
included (37949 fibres, meanSE, range 441875).
Out of 49 sections used for SC counts, four contained less
than 100 myofibres (44, 78, 83 and 94 fibres, respectively).
Mackey etal. (2009) reported that a minimum of 50 type
I and 75 type II myofibres were required to reliably deter-
mine SC numbers for each type. Because we did not distin-
guish between type I and type II fibres in the counts of SCs,
our samples included sufficient numbers of muscle fibres in
48 out of the 49 sections. Data are presented as number of
positive cells per myofibre.
Statistical analyses
A two-way repeated measure ANOVA and Bonferronis
post hoc test were used to assess the effects of blood flow
restriction and time. If there was a tendency for a time
effect, but no group or interaction effects were detected, the
data was pooled (free-flow and BFR) and analysed with a
one-way repeated measure ANOVA. Effect size (Cohens d)
was calculated as the difference between means divided by
the standard derivation. Data are presented as meanSE.
The level of significance for all statistical analyses was
p<0.05. GraphPad Prism 6 (San Diego, CA, USA,
http://www.graphpad.com) was used for statistical analyses.
Results
Exercise performance
As previously reported (Wernbom etal. 2012), the number
of repetitions performed before concentric torque failure in
the BFR leg decreased from 285 in the 1st set to 61
in the 5th set, and the total number of repetitions was 57.
For further details, see Wernbom etal. (2012).
Protein signalling
The phosphorylations (p) of Akt (Ser473) and mTOR
(Ser2448) were not significantly altered in any leg at any
post-exercise time points. In contrast, after 1h of recovery,
p-p70S6K (Thr389) was 2.7-fold higher than baseline in the
BFR leg (p<0.05), whereas it was unaltered in the free-
flow leg (Fig.1). Although not significantly different with
ANOVA, p-p70S6K tended to be greater in the BFR leg
than in the free-flow leg at 1h (p=0.11, effect size=1.0).
At 24-h post-exercise, p-p70S6K (Thr389) was 3.3-fold
higher in the occluded leg (p<0.05) and 3.5-fold higher
in the free-flow leg (p<0.05) as compared to pre-exer-
cise, with no difference between the conditions. At 48h,
20
Phosphorylation of S6K1 at T389
Freeflow
BFR * *
*
15
(arbitrary units)
10
5
0
Pre 1h Post 24h Post 48h Post
Fig.1Phosphorylation of p70S6K at Thr389 in the BFR and free-
flow resistance exercised leg at baseline and 1, 24 and 48h following
exercise. An immunoblot from one subject is shown above the graph.
*p<0.05 versus baseline
13
 Eur J Appl Physiol

Table1Phosphorylation (in Kinase Baseline 1-h post 24-h post 48-h post
arbitrary units) of selected
kinases at different time points Free flow BFR Free flow BFR Free flow BFR
(meanSE)
p-AMPK (Thr172) 7913 678 6410 6612 4811 6313 435
p-Akt (Ser473) 6013 377 5010 5514 456 5114 4612
p-mTOR (Ser2448) 13423 14312 14810 14313 12615 1418 13215
p-p90S6K (Thr573) 7913 5517 4820 8219 4913 5115 6824
p-eEF2 (Thr56) 10015 816 659 857 7413 9112 979
p-ERK1/2 9323 6417 5517 6921 5415 7119 9330
(Thr202/Tyr204)

70 Freeflow *

% change in satellite cells per fibre

BFR *
Phosphorylation of p38 at T180/Y182

20 60
Freeflow
BFR * * *
50
*
upperband (arbitrary units)

15
* 40

10 30

20
5
10
0
0
Pre 1h Post 24h Post 48h Post
1h Post 24h Post 48h Post

Fig.3Changes in satellite cells per fibre. *p<0.05 versus baseline

Phosphorylation of p38 at T180/Y182

20 Freeflow
BFR
lowerband (arbitrary units)

15 * eukaryotic elongation factor 2 (eEF2) at Thr56, no significant

10
5
0
Pre 1h Post 24h Post
Fig.2Phosphorylation of p38 at Thr180/Tyr182 in the BFR and free-
flow resistance exercised leg at baseline and 1, 24 and 48h following
exercise. An immunoblot from one subject is shown above the graph.
Upper panel upper band, possibly p38gamma, lower panel lower
band, possibly p38alpha/beta. *p<0.05 versus baseline
p-p70S6K (Thr389) was no longer elevated over baseline for
any of the legs. The phosphorylations of ERK1/2 (Thr202/
Tyr204) and p90RSK (Thr573) did not change significantly
as compared to baseline at any time point following exer-
cise regardless of the conditions (Table1).
Regarding p-p38MAPK (Thr180/Tyr182), two bands were
seen, and both increased at the 1h time point (p<0.05)
for the BFR leg, but did not change significantly at any
other post-exercise time points regardless of the conditions
(Fig. 2). For p-AMPK (Thr172) and phosphorylation of the
changes were observed over time in any condition (Table1).
Satellite cells
The mean number of NCAM-positive SCs per muscle fibre
before exercise was 0.20 (SE=0.02). The numbers of SCs
48h Post per muscle fibre were significantly increased after exercise
for all post-exercise time points and in both legs (3353%,
p<0.05, Fig.3). The relative proportion of SCs with visible
cytoplasmic extensions before exercise was 18.83.0%
(mean SE). The proportion of SCs with extensions
was increased at 1-h post-exercise in the BFR leg to
33.12.2% (p<0.05, effect size=2.05), but the values at
24h (18.35.5%) and 48h (24.24.1%) were not signif-
icantly different from pre-exercise (p>0.05). The percent-
age of SCs with extensions was not significantly changed in
the free-flow leg at any time point (22.82.7, 27.33.6
and 25.53.6% at 1, 24 and 48-h post-exercise, respec-
tively, p>0.05). No significant differences were observed
between the BFR and the free-flow legs for any post-exercise
time point. MRF-positive SCs tended to increase in both legs
(Fig.4) and the elevations at 1- and 24-h post-exercise were
significant when both legs were combined (p<0.05 for 1h
and p<0.01 for 24-h post-exercise).

13
Eur J Appl Physiol
Proportion of MRF + SC (%) 25 Freeflow
BFR
20
* exercise, as well as longer-lasting (at least 2427h) eleva-
15
* on BFRRE versus free-flow exercise at 30% of 1RM (Wer-
10
5
0
Pre 1h Post 24h Post
Fig.4Changes in the proportions of MRF-positive satellite cells
(expressed as % of the total number of satellite cells). *p<0.05 ver-
sus baseline for both legs combined
Discussion
There were several notable findings in the present study:
first, there was a prolonged increase (up to at least 24h in
the fed state) in the phosphorylation of p70S6K at Thr389 in
the BFR leg. Phospho-p70S6K (Thr389) was elevated also
in the free-flow leg at 24h, but not at 1h. Second, the phos-
phorylation of p38MAPK, which can positively impact on
the mTOR-p70S6K pathway in different cell types (Cully
etal. 2010), including skeletal muscle cells (Zbinden-Fon-
cea etal. 2012), increased at 1h in the BFR leg but not
at the later time points. Third, there was a marked increase
in the number of visible satellite cells after both the BFR
and the free-flow exercise protocols, and also an increase
in the number of MRF-positive satellite cells for both legs
combined.
Protein signalling
The increase in the p-p70S6K (Thr389) at 1-h post-exercise
in the occluded leg is in line with Fry etal. (2010), who
also found elevated p-p70S6K (Thr389) at 1-h post-BFRRE
in a group of older men. However, BFRRE did not signifi-
cantly increase the p-p70S6K (Thr389) at 1-h post-exercise
in young men (Gundermann etal. 2012). The subjects in
Gundermann etal. (2012) were studied after an overnight
fast and the load was lower than in the present study (20%
of 1RM vs. 30% of 1RM). Also, all 5 sets were performed
to failure in our study, whereas the BFRRE protocol used
by Gundermann etal. (2012) was standardised to 30-15-
15-15 repetitions. Thus, the protocol used by Gundermann
etal. (2012) may have been submaximal, at least in the first
few sets. Still, Fujita etal. (2007), Fry etal. (2010) and
Gundermann etal. (2012) all reported elevated p-p70S6K
(Thr389) at 3-h post-BFRRE.
The finding of elevated p-p70S6K (Thr389) at 24h not
only in the BFR leg but also in the free-flow leg deserves
comment. Burd etal. (2010b, 2011) recently demonstrated
that 4 sets of knee extensions to failure at 30% of 1RM
caused increased MPS and p-p70S6K (Thr389) at 4-h post-
tions in MPS and mTOR signalling. Based on our studies
nbom etal. 2006, 2009), and feedback from the subjects,
we estimate that during the first 2 sets with the free-flow
leg, our subjects had only ~35 repetitions left before fail-
ure. After the first 2 sets, it became gradually easier for the
48h Post subjects to match the work of the BFR leg.
The fact that the subjects had to work quite hard with
thefree-flow leg at 30% of 1RM is an important point,
because a free-flow protocol at 20% of 1RM which is
work-matched to a BFRRE protocol at the same load does
not induce increases in protein synthesis (Fujita etal. 2007)
or in satellite cells and myonuclei (Nielsen etal. 2012),
while 3 sets of unilateral knee extensions to failure at 30%
of 1RM can induce hypertrophy (Mitchell etal. 2012).
The delay in the increase of p-p70S6K (Thr389) in the
free-flow leg versus the BFR leg also merits a comment.
Because we observed signs suggesting mild muscle damage
especially in the BFR leg (Wernbom etal. 2012), it is pos-
sible that our BFRRE protocol stimulated an early increase
in p-p70S6K through some damage-related mechanism(s).
The late elevations in p-p70S6K (Thr389) in both legs are
consistent with the reports of Burd etal. (2010a, 2011), and
may in part be explained by enhanced amino acid sensi-
tivity (Burd etal. 2011). Although not significant, p-eEF2
(Thr56) tended to be reduced at 1h in the BFR leg. A
dephosphorylation of eEF2 activates the enzyme, which in
turn can lead to an elevated protein synthesis via increased
translation elongation.
The phosphorylations of Akt and mTOR were not sig-
nificantly altered in either leg at any post-exercise time
points. We also did not observe a p-ERK1/2 response to
our BFRRE protocol. In contrast, Fry etal. (2010) reported
elevated p-ERK1/2 (Thr202/Tyr204) at 1h after BFR resist-
ance exercise in older men, and Gundermann etal. (2012)
noted a tendency for p-ERK1/2 to increase at 3-h post-exer-
cise. Moreover, Burd etal. (2010b) reported that p-ERK1/2
(Thr202/Tyr204) was elevated at 4-h post-resistance exer-
cise after 4 sets of 30% at 1RM to failure. However, other
human resistance training studies (e.g. Karlsson etal. 2004;
Tannerstedt etal. 2009) indicate that phosphorylation of
ERK1/2 is quickly induced by exercise and then decreases
rapidly back to baseline within 60min. Therefore, we can-
not rule out that an earlier and/or a later phosphorylation of
ERK1/2 may have occurred in the present investigation, but
was not detected in our 1-h post-exercise biopsies.
The increased p-p38MAPK at an early post-exercise
time point in response to BFR exercise is a novel finding.
Activation of p38 has been demonstrated in skeletal mus-
cle after mechanical stretch (Wretman etal. 2001) and p38
13

is strongly phosphorylated in skeletal muscle after acute
eccentric exercise (Wretman etal. 2001; Tannerstedt etal.
2009; ONeil etal. 2009). Notably, p38 has been demon-
strated to be both necessary and sufficient for activation
of the mTOR pathway, and activation of p38 increases the
size of some types of human cells, whereas inhibition of
p38 reduces cell size (Cully etal. 2010). Interestingly, a
p38-mTOR signalling module in skeletal muscle, which is
responsive to the forcetime integral, was recently identi-
fied with factor analysis (Rahnert and Burkholder 2013).
However, p38 is also involved in muscle wasting
induced by cytokines such as TNF-alpha, and by reactive
oxygen species, via the induction of MuRF1 and atrogin-
1/MAFbx. These divergent cellular responses to p38 may
be related to the existence of four different isoforms (alpha,
beta, delta and gamma), which sometimes have opposite
functions (Lovett etal. 2010). The isoform involved in the
positive effects on mTOR-p70S6K signalling and cell size
is possibly p38alpha (Zheng etal. 2011), while p38beta is
involved in the proteolytic pathways (Zhang and Li 2012)
and can inhibit mTOR signalling (Zheng etal. 2011).
However, p38beta may also activate the mTOR pathway
under some circumstances (Wu etal. 2011). Interestingly,
p38gamma is required for maintenance of the size of slow
skeletal muscle (Foster etal. 2012) and is associated with
an endurance phenotype (Scharf etal. 2013).
We observed two bands in the Western blots for p38.
Boppart etal. (2000) reported one lower and one upper
band in human skeletal muscle and found these to be rep-
resentative of p38alpha and p38gamma, respectively.
Similarly, Scharf etal. (2013) reported two major bands
of phosphorylated p38 in mouse skeletal muscle, where
the lower band was p38alpha and the upper band was
p38gamma. However, although p38beta is less expressed
than p38alpha, these two isoforms are similar in size on
Western blots (Foster etal. 2012; Zhang and Li 2012).
Thus, BFRRE may activate p38alpha and/or beta, as well
as p38gamma, but this possibility was not further explored
in our study. Nevertheless, it seems plausible to speculate
that a p38gamma response may in part underlie the adap-
tations in muscle endurance reported in several studies on
BFRRE (e.g., Takarada etal. 2002; Nielsen etal. 2012).
The phosphorylation of AMPK did not change signifi-
cantly, but if anything, it tended to be decreased with BFR
training at 24- and 48-h post-exercise (3946%). Also
of note, p-AMPK was not elevated at 1h. Norrbom etal.
(2011) reported that p-AMPK (Thr172) was increased three-
fold at 2h after 45min of ischaemic knee extensor endur-
ance exercise and also tended to be increased immediately
after exercise. As with p-ERK1/2, we cannot rule out that
p-AMPK (Thr172) may have increased temporarily. How-
ever, Fry etal. (2010) reported no increase in p-AMPK
(Thr172) immediately after a bout of BFR resistance
13
Eur J Appl Physiol
exercise, as well as 13-h post-exercise, in older men. Col-
lectively, these findings suggest that brief BFRRE may not
induce phosphorylation of AMPK, or that it does so only
transiently, in contrast to longer-lasting BFR endurance
exercise.
Satellite cell numbers
The 3353% increases in the number of visible SCs per
muscle fibre post-exercise after a single bout of BFRRE,
as well as low-load RE without BFR, are novel findings.
SC numbers have been reported to increase by ~30140%
within 2448h in response to an acute bout of maximum
eccentric exercise (Dreyer etal. 2006; Paulsen etal. 2010;
McKay etal. 2010). Nielsen etal. (2012) reported that SCs
per muscle fibre were 34 times higher after 5days (7 ses-
sions) of BFRRE when compared with baseline values.
Thus, both a single bout and multiple BFRRE sessions can
cause elevations in SC numbers, although the magnitude of
the response appears to be greater with multiple frequent
training sessions.
An intriguing finding in our current study is the ~33
36% elevation in the number of visible SCs already 1h
after exercise. We believe that this early increase does not
reflect an actual elevation in cell number, but is caused by
the swelling of the activated cells (Figs.5, 6). In support of
this notion, the proportion of SCs with cytoplasmic exten-
sions was increased at 1-h post-exercise in the BFR leg.
In the free-flow leg, the increases in SCs with extensions
were not significant, although a trend was observed at 24-h
post-exercise (46% increase, p=0.03 with a t test, effect
size =0.97). SCs are normally spindle-shaped cells and
Fig.5Example of an NCAM-positive cell, possibly a satellite cell,
marked with NCAM (fluorescent green) and Dapi (blue). Note the
long cytoplasmic extension, which indicates activation from the qui-
escent state (colour figure online)
Eur J Appl Physiol
Fig.6Two NCAM-positive cells, presumably satellite cells. Note
the long extensions
cytoplasmic extensions/processes are regarded as struc-
tural markers of an activated SC (Anderson 2000; Hawke
and Garry 2001; Holterman and Rudnicki 2005). Notably,
SCs have been shown to be activated and enlarged within
10min after injury (Anderson 2000).
In an electron microscopy study on ischaemiareperfu-
sion in primate skeletal muscle (Gregory and Mars 2004),
it was reported that the quiescent SCs in the pre-ischaemia
samples had a length of about 6.5m, whereas the length
of early activated SCs at 6h after tourniquet-induced
ischaemia (3h) was about 1015m (i.e., the length was
doubled). Increases in the widths of the early activated SCs
were noted, and the relative area occupied by the nucleus
in the SC was only slightly reduced, indicating that the
absolute volumes of the nuclei were also increased with SC
activation, in addition to the expanded cytoplasm. Reorien-
tation of some SCs to the long axis of the muscle fibres was
also observed.
All these effects would increase the likelihood of detect-
ing SCs in immunohistochemistry sections. In addition,
NCAM stains a greater part of the SC than Pax7, which
is confined to the nucleus (Mackey etal. 2009). Regard-
ing the later increases in SC number in the current study,
we assume that these were at least in part caused by
proliferation.
The use of NCAM as a satellite cell marker has been ques-
tioned recently (e.g., Lindstrm and Thornell 2009). How-
ever, staining for NCAM and PAX7 co-localises in ~95%
of all SCs in human muscle samples (Verdijk etal. 2007;
Lindstrm and Thornell 2009). Furthermore, Dreyer etal.
(2006) reported no incidences where an NCAM-positive
cell was located outside the basal lamina. Similarly, Mackey
etal. (2011) found only one interstitial NCAM+cell per
250 muscle fibres, i.e., 0.004 NCAM+cells per myofibre,
as compared to 0.110.15 sublaminar NCAM+cells per
myofibre. These findings suggest that NCAM stained cells
outside the myofibres are rare, on the order of ~3% of the
total population of NCAM+cells.
Lindstrm and Thornell (2009) also emphasised stain-
ing of the basal lamina to exclude NCAM+interstitial cells
and small muscle fibres from being counted as satellite
cells. However, as noted above, interstitial NCAM+cells
are rare. Moreover, a SC rests in an indentation in the
muscle fibre (Hawke and Garry 2001) and does not dis-
tort the external surface of the myofibre (Muir etal. 1965).
Hence, NCAM+SCs may be tentatively identified by
morphological criteria even without basal lamina staining,
although this is admittedly difficult and requires a skilled
observer. Therefore, we are confident that the exercise-
induced increases in NCAM+cells in the present study
were mainly due to increases in the numbers of visible
NCAM+satellite cells. With this in mind, we put priority
on MyoD and myogenin stainings to learn more about the
fates of some of the activated SCs.
The number of MRF-positive satellite cells increased
~twofold for both legs combined at 1- and 24-h post-exer-
cise(Fig. 4). An example of an MRF-positive SC is shown in
Fig. 7. One of the subjects had an unusually high number of
MRF+SCs already at baseline (18%), but he too increased
MRF+SCs after exercise. If his results are excluded,
MRF+SCs increased about three- to fourfold, from ~2.5%
at baseline to 910% at 24h for both legs combined. The
elevated number of MRF+SCs after a single bout in both
legs suggests that myonuclear addition could occur within a
few sessions not only with BFRRE, but also with fatiguing
low-load resistance exercise without BFR.
The pre-exercise value of 0.20 SCs per fibre may seem
high as compared to other studies (e.g., Mackey etal.
2011). However, strenuous exercise, particularly strength
training, is known to increase SC numbers (Petrella etal.
2008; Mackey etal. 2011). The highest SC numbers before
exercise were observed in three individuals who had 0.27,
0.27 and 0.21 SCs per myofibre, and these subjects also
had the greatest 1RM knee extension values. Thus, the
relatively high training status of our subjects may in part
explain the high SC numbers, as well as the occurrence of
SC cytoplasmic extensions in the pre-exercise samples.
Mild muscle damage asa stimulus inBFR resistance
exercise?
A current model of SC activation is that Ca2+influx via
stretch-activated ion channels (SACs) and L-type voltage-
gated calcium channels results in the production of nitric
oxide (NO). NO activates matrix metalloproteinases, which
release hepatocyte growth factor, which in turn activates the
SC (Hara etal. 2012). Interestingly, inhibition of NO syn-
thase inhibited the rapid increases in cytoplasmic volumes
13

Fig.7Example of a satellite
cell/myoblast positive for both
NCAM (fluorescent green) and
myogenin (red) (colour figure
online)
associated with activation of SCs after crush injury (Ander-
son 2000). In addition to being markers of activation, cyto-
plasmic processes allow for chemotaxis of the SC along the
myofibre (Hawke and Garry 2001).
We previously observed signs of mild damage already in
1-h post-exercise muscle samples from the BFR leg (Wer-
nbom etal. 2012). We, therefore, speculate that the early
increase of SCs with cytoplasmic extensions in the BFR leg
may have been caused by NO release, possibly associated
with mild muscle damage. Muscle damage-related events
might also have participated in the early elevations of
p-p70S6K and p-p38 in the BFR leg. Peroxynitrite, a reac-
tion product of NO and superoxide, was recently shown to
13
Eur J Appl Physiol
be crucial for load-induced mTOR-p70S6K signalling and
muscle hypertrophy (Ito etal. 2013).
We documented signs of mild damage (muscle sore-
ness, elevated membrane permeability and decreased mus-
cle strength) not only in the BFR leg but also in the free-
flow leg (Wernbom etal. 2012). Intriguingly, links between
exercise-induced muscle soreness, elevated NO levels and
decreased muscle torque have been proposed (Radak etal.
2012). Therefore, we hypothesise that damage-related
pathways (e.g., activation of SACs and/or increased mem-
brane permeability) and associated NO release may have
been involved also in the later increases in SC numbers and
p70S6K signalling in our study.
Eur J Appl Physiol
Limitations
We did not measure muscle protein synthesis; hence, we
do not know whether the anabolic signalling translated into
elevations of muscle protein synthesis. However, training
periods with low-load BFR resistance exercise protocols
similar to the one in the present study results in myofibre
hypertrophy (Nielsen etal. 2012).
Because we did not have a control group, we cannot
exclude the possibility that the biopsy procedure per se
might to some extent have influenced the results. Nev-
ertheless, it has been shown that when performed at least
13cm apart, repeated needle biopsies alone do not result
in elevated satellite cell numbers (Crameri etal. 2004;
van de Vyver and Myburgh 2012) or in MAPK signalling
(Guerra etal. 2011).
We also cannot rule out that the free-flow leg might
have benefitted from a systemic response elicited by the
BFR exercise, as cross-transfer effects of bilateral BFRRE
have been demonstrated (Madarame etal. 2008). However,
unilateral strength training results in blunted hormonal
responses when compared with bilateral exercise (Migiano
etal. 2010). Because some systemic factors may originate
from the working muscles (Pedersen 2011), the smaller
muscle mass involved in unilateral when compared with
bilateral knee extensions would probably result in blunted
remote cross-transfer effects regardless of whether the
active factors are released from the muscles or via endo-
crine organs. Notably, the unilateral knee extension model
has successfully been used in studies with a within-subjects
design to differentiate between various resistance exercise
protocols in terms of increases in MPS, protein signalling,
and muscle size (Burd etal. 2010a, b, 2011; Mitchell etal.
2012).
Finally, it is possible that the MVCs performed could
have contributed to the signalling and SC responses. How-
ever, the overall number of MVCs was limited, and the
two immediate post-exercise MVCs were performed in
a fatigued state, especially in the BFR leg (~38% of pre-
exercise MVC, see Wernbom etal. 2012). Therefore, we
consider the effects of the MVCs to be marginal. Never-
theless, it cannot be excluded that the MVCs and/or cross-
transfer effects may have attenuated differences between
the two legs.
Conclusions
In conclusion, an acute bout of multiple sets to failure of
unilateral low-load resistance exercise with BFR can result
in an early enhanced phosphorylation of p70S6K (Thr389)
which lasts up to a least 24-h post-exercise in the fed state,
and in an early phosphorylation of p38MAPK as well as
a greater number of visible satellite cells per muscle fibre.
Our findings may help explain why BFR resistance training
at 2050% of 1RM can induce hypertrophy even with only
2 sessions per week (Takarada etal. 2002, 2004; Lauren-
tino etal. 2012).
Notably, the free-flow leg also demonstrated increased
p-p70S6K (Thr389) and satellite cells numbers. This indi-
cates that performing low-load knee extensions close to
concentric failure can trigger multiple hypertrophic path-
ways even without BFR. Therefore, our findings agree with
and extend those of Burd etal. (2010b, 2011) and Mitchell
etal. (2012).
AcknowledgmentsThe authors thank the subjects for their time
and effort. This project was in part supported by a grant from the
Swedish National Centre for Research in Sports (Grant: CIF 125/05).
Conflict of interestThe authors declare no conflicts of interest.
References
Abe T, Yasuda T, Midorikawa T, Sato Y, Kearns CF, Inoue K, Koizumi
K, Ishii N (2005) Skeletal muscle size and circulating IGF-1 are
increased after two weeks of twice daily kaatsu resistance train-
ing. Int J Kaatsu Train Res 1:714
Anderson JE (2000) A role for nitric oxide in muscle repair: nitric
oxide-mediated activation of muscle satellite cells. Mol Biol Cell
11:18591874
Antharavally BS, Carter B, Bell PA, Krishna Mallia A (2004) A high-
affinity reversible protein stain for Western blots. Anal Biochem
329:276280
Boppart MD, Asp S, Wojtaszewski JF, Fielding RA, Mohr T, Good-
year LJ (2000) Marathon running transiently increases c-Jun
NH2-terminal kinase and p38 activities in human skeletal muscle.
J Physiol 526:663669
Burd NA, Holwerda AM, Selby KC, West DW, Staples AW, Cain NE,
Cashaback JG, Potvin JR, Baker SK, Phillips SM (2010a) Resist-
ance exercise volume affects myofibrillar protein synthesis and
anabolic signalling molecule phosphorylation in young men. J
Physiol 588:31193130
Burd NA, West DW, Staples AW, Atherton PJ, Baker JM, Moore DR,
Holwerda AM, Parise G, Rennie MJ, Baker SK, Phillips SM
(2010b) Low-load high volume resistance exercise stimulates
muscle protein synthesis more than high-load low volume resist-
ance exercise in young men. PLoS ONE 5(8):e12033
Burd NA, West DW, Moore DR, Atherton PJ, Staples AW, Prior T,
Tange JE, Rennie MJ, Baker SK, Phillips SM (2011) Enhanced
amino acid sensitivity of myofibrillar protein synthesis persists
for up to 24h after resistance exercise in young men. J Nutr
141:568573
Crameri RM, Langberg H, Magnusson P, Jensen CH, Schrder HD,
Olesen JL, Suetta C, Teisner B, Kjaer M (2004) Changes in sat-
ellite cells in human skeletal muscle after a single bout of high
intensity exercise. J Physiol 558:333340
Crenshaw AG, Hargens AR, Gershuni DH, Rydevik B (1988) Wide
tourniquet cuffs more effective at lower inflation pressures. Acta
Orthop Scand 59:447451
Cully M, Genevet A, Warne P, Treins C, Liu T, Bastien J, Baum B,
Tapon N, Leevers SJ, Downward J (2010) A role for p38 stress-
activated protein kinase in regulation of cell growth via TORC1.
Mol Cell Biol 30:481495
13

Dreyer HC, Blanco CE, Sattler FR, Schroeder ET, Wiswell RA (2006)
Satellite cell numbers in young and older men 24 hours after
eccentric exercise. Muscle Nerve 33:242253
Drummond MJ, Fujita S, Abe T, Dreyer HC, Volpi E, Rasmussen BB
(2008) Human muscle gene expression following resistance exer-
cise and blood flow restriction. Med Sci Sports Exerc 40:691698
Foster WH, Tidball JG, Wang Y (2012) p38 activity is required
for maintenance of slow skeletal muscle size. Muscle Nerve
45:266273
Fry CS, Glynn EL, Drummond MJ, Timmerman KL, Fujita S, Abe T,
Dhanani S, Volpi E, Rasmussen BB (2010) Blood flow restriction
exercise stimulates mTORC1 signaling and muscle protein syn-
thesis in older men. J Appl Physiol 108:11991209
Fujita S, Abe T, Drummond MJ, Cadenas JG, Dreyer HC, Sato Y,
Volpi E, Rasmussen BB (2007) Blood flow restriction during
low-intensity resistance exercise increases S6K1 phosphorylation
and muscle protein synthesis. J Appl Physiol 103:903910
Fujita T, Brechue WF, Kurita K, Sato Y, Abe T (2008) Increased
muscle volume and strength following six days of low-intensity
resistance training with restricted muscle blood flow. Int J Kaatsu
Train Res 4:18
Graham B, Breault MJ, McEwen JA, Mcgraw RW (1993) Occlusion
of arterial flow in the extremities at subsystolic pressures through
the use of wide tourniquet cuffs. Clin Orthop 286:257261
Gregory MA, Mars M (2004) Mobilisation of satellite cells follow-
ing ischaemia and reperfusion in primate skeletal muscle. S Afr J
Sports Med 16(1):1724
Guerra B, Gmez-Cabrera MC, Ponce-Gonzlez JG, Martinez-Bello
VE, Guadalupe-Grau A, Santana A, Sebastia V, Via J, Calbet
JA (2011) Repeated muscle biopsies through a single skin inci-
sion do not elicit muscle signaling, but IL-6 mRNA and STAT3
phosphorylation increase in injured muscle. J Appl Physiol
110:17081715
Gundermann DM, Fry CS, Dickinson JM, Walker DK, Timmerman
KL, Drummond MJ, Volpi E, Rasmussen BB (2012) Reactive
hyperemia is not responsible for stimulating muscle protein syn-
thesis following blood flow restriction exercise. J Appl Physiol
112:15201528
Hanssen KE, Kvamme NH, Nilsen TS, Rnnestad B, Ambjrnsen
IK, Norheim F, Kadi F, Halln J, Drevon CA, Raastad T (2012)
The effect of strength training volume on satellite cells, myogenic
regulatory factors, and growth factors. Scand J Med Sci Sports.
doi:10.1111/j.1600-0838.2012.01452.x
Hara M, Tabata K, Suzuki T, Do MK, Mizunoya W, Nakamura M,
Nishimura S, Tabata S, Ikeuchi Y, Sunagawa K, Anderson JE,
Allen RE, Tatsumi R (2012) Calcium influx through a possible
coupling of cation channels impacts skeletal muscle satellite cell
activation in response to mechanical stretch. Am J Physiol Cell
Physiol 302:C1741C1750
Hawke TJ, Garry DJ (2001) Myogenic satellite cells: physiology to
molecular biology. J Appl Physiol 91:534551
Holterman CE, Rudnicki MA (2005) Molecular regulation of satellite
cell function. Semin Cell Dev Biol 16:575584
Ito N, Ruegg UT, Kudo A, Miyagoe-Suzuki Y, Takeda S (2013) Acti-
vation of calcium signaling through Trpv1 by nNOS and perox-
ynitrite as a key trigger of skeletal muscle hypertrophy. Nat Med
19:101106
Kadi F, Eriksson A, Holmner S, Thornell LE (1999) Effects of ana-
bolic steroids on the muscle cells of strength-trained athletes.
Med Sci Sports Exerc 31:15281534
Karlsson HK, Nilsson PA, Nilsson J, Chibalin AV, Zierath JR, Blom-
strand E (2004) Branched-chain amino acids increase p70S6k
phosphorylation in human skeletal muscle after resistance exer-
cise. Am J Physiol Endocrinol Metab 287:E1E7
Laurentino GC, Ugrinowitsch C, Roschel H, Aoki MS, Soares AG,
Neves M Jr, Aihara AY, Fernandes Ada R, Tricoli V (2012)
13
Eur J Appl Physiol
Strength training with blood flow restriction diminishes myosta-
tin gene expression. Med Sci Sports Exerc 44:406412
Lindstrm M, Thornell LE (2009) New multiple labelling method for
improved satellite cell identification in human muscle: applica-
tion to a cohort of power-lifters and sedentary men. Histochem
Cell Biol 132:141157
Lindstrm M, Pedrosa-Domellf F, Thornell LE (2010) Satellite
cell heterogeneity with respect to expression of MyoD, myo-
genin, Dlk1 and c-Met in human skeletal muscle: application to
a cohort of power lifters and sedentary men. Histochem Cell Biol
134:371385
Lovett FA, Cosgrove RA, Gonzalez I, Pell JM (2010) Essential role
for p38alpha MAPK but not p38gamma MAPK in Igf2 expres-
sion and myoblast differentiation. Endocrinology 151:43684380
Mackey AL, Kjaer M, Charifi N, Henriksson J, Bojsen-Moller J,
Holm L, Kadi F (2009) Assessment of satellite cell number and
activity status in human skeletal muscle biopsies. Muscle Nerve
40:455465
Mackey AL, Holm L, Reitelseder S, Pedersen TG, Doessing S, Kadi
F, Kjaer M (2011) Myogenic response of human skeletal muscle
to 12weeks of resistance training at light loading intensity. Scand
J Med Sci Sports 21:773782
Madarame H, Neya M, Ochi E, Nakazato K, Sato Y, Ishii N (2008)
Cross-transfer effects of resistance training with blood flow
restriction. Med Sci Sports Exerc 40:258263
Manini TM, Vincent KR, Leeuwenburgh CL, Lees HA, Kavazis AN,
Borst SE, Clark BC (2011) Myogenic and proteolytic mRNA
expression following blood flow restricted exercise. Acta Physiol
201:255263
McKay BR, Toth KG, Tarnopolsky MA, Parise G (2010) Satellite cell
number and cell cycle kinetics in response to acute myotrauma in
humans: immunohistochemistry versus flow cytometry. J Physiol
588:33073320
Migiano MJ, Vingren JL, Volek JS, Maresh CM, Fragala MS, Ho JY,
Thomas GA, Hatfield DL, Hkkinen K, Ahtiainen J, Earp JE,
Kraemer WJ (2010) Endocrine response patterns to acute unilat-
eral and bilateral resistance exercise in men. J Strength Cond Res
24:128134
Mitchell CJ, Churchward-Venne TA, West DW, Burd NA, Breen L,
Baker SK, Phillips SM (2012) Resistance exercise load does not
determine training-mediated hypertrophic gains in young men. J
Appl Physiol 113:7177
Muir AR, Kanji AH, Allbrook D (1965) The structure of the satellite
cells in skeletal muscle. J Anat 99:435444
Nielsen JL, Aagaard P, Bech RD, Nygaard T, Hvid LG, Wernbom M,
Suetta C, Frandsen U (2012) Proliferation of myogenic stem cells
in human skeletal muscle in response to low-load resistance train-
ing with blood flow restriction. J Physiol 590:43514361
Norrbom J, Sllstedt EK, Fischer H, Sundberg CJ, Rundqvist H,
Gustafsson T (2011) Alternative splice variant PGC-1{alpha}-b
is strongly induced by exercise in human skeletal muscle. Am J
Physiol Endocrinol Metab 301:E1092E1098
ONeil TK, Duffy LR, Frey JW, Hornberger TA (2009) The role of
phosphoinositide 3-kinase and phosphatidic acid in the regulation
of mammalian target of rapamycin following eccentric contrac-
tions. J Physiol 587:36913701
Paulsen G, Egner IM, Drange M, Langberg H, Benestad HB, Fjeld
JG, Halln J, Raastad T (2010) A COX-2 inhibitor reduces mus-
cle soreness, but does not influence recovery and adaptation after
eccentric exercise. Scand J Med Sci Sports 20:e195e207
Pedersen BK (2011) Muscles and their myokines. J Exp Biol
214:337346
Petrella JK, Kim JS, Mayhew DL, Cross JM, Bamman MM (2008)
Potent myofiber hypertrophy during resistance training in humans
is associated with satellite cell-mediated myonuclear addition: a
cluster analysis. J Appl Physiol 104:17361742
Eur J Appl Physiol
Radak Z, Naito H, Taylor AW, Goto S (2012) Nitric oxide: is it the
cause of muscle soreness? Nitric Oxide 26:8994
Rahnert JA, Burkholder TJ (2013) High frequency electrical stimula-
tion reveals a p38-mTOR signaling module correlated with force-
time integral. J Exp Biol 216:26192631
Scharf M, Neef S, Freund R, Geers-Knrr C, Franz-Wachtel M, Bran-
dis A, Krone D, Schneider H, Groos S, Menon MB, Chang KC,
Kraft T, Meissner JD, Boheler KR, Maier LS, Gaestel M, Scheibe
RJ (2013) MAPKAPK2/3 regulate SERCA2a expression and
fiber type composition to modulate skeletal muscle and cardio-
myocyte function. Mol Cell Biol 33:25862602
Staron RS, Malicky ES, Leonardi MJ, Falkel JE, Hagerman FC,
Dudley GA (1990) Muscle hypertrophy and fast fiber type con-
versions in heavy resistance-trained women. Eur J Appl Physiol
60:7179
Takarada Y, Sato Y, Ishii N (2002) Effects of resistance exercise com-
bined with vascular occlusion on muscle function in athletes. Eur
J Appl Physiol 86:308314
Takarada Y, Tsuruta T, Ishii N (2004) Cooperative effects of exer-
cise and occlusive stimuli on muscular function in low-intensity
resistance exercise with moderate vascular occlusion. Jpn J Phys-
iol 54:585592
Tannerstedt J, Apr W, Blomstrand E (2009) Maximal lengthen-
ing contractions induce different signaling responses in the type
I and type II fibers of human skeletal muscle. J Appl Physiol
106:14121418
Van de Vyver M, Myburgh KH (2012) Cytokine and satellite cell
responses to muscle damage: interpretation and possible con-
founding factors in human studies. J Muscle Res Cell Motil
33:177185
Verdijk LB, Koopman R, Schaart G, Meijer K, Savelberg HH, van
Loon LJ (2007) Satellite cell content is specifically reduced in
type II skeletal muscle fibers in the elderly. Am J Physiol Endo-
crinol Metab 292:E151E157
Wernbom M, Augustsson J, Thome R (2006) Effects of vascular
occlusion on muscular endurance in dynamic knee extension
exercise at different submaximal loads. J Strength Cond Res
20:372377
Wernbom M, Augustsson J, Raastad T (2008) Ischemic strength train-
ing: a low-load alternative to heavy resistance exercise? Scand J
Med Sci Sports 18:401416
Wernbom M, Jrrebring R, Andreasson MA, Augustsson J (2009)
Acute effects of blood flow restriction on muscle activity and
endurance during fatiguing dynamic knee extensions at low load.
J Strength Cond Res 23:23892395
Wernbom M, Paulsen G, Nilsen TS, Hisdal J, Raastad T (2012) Con-
tractile function and sarcolemmal permeability after acute low-
load resistance exercise with blood flow restriction. Eur J Appl
Physiol 112:20512063
Wretman C, Lionikas A, Widegren U, Lnnergren J, Westerblad H,
Henriksson J (2001) Effects of concentric and eccentric contrac-
tions on phosphorylation of MAPK(erk1/2) and MAPK(p38) in
isolated rat skeletal muscle. J Physiol 535:155164
Wu XN, Wang XK, Wu SQ, Lu J, Zheng M, Wang YH, Zhou H,
Zhang H, Han J (2011) Phosphorylation of raptor by p38{beta}
participates in arsenite-induced mTORC1 activation. J Biol Chem
286:15011511
Zammit PS (2008) The muscle satellite cell: the story of a cell on the
edge! In: Schiaffino S, Partridge T (eds) Skeletal muscle repair
and regeneration. Springer, The Netherlands, pp 4564
Zbinden-Foncea H, Deldicque L, Pierre N, Francaux M, Raymack-
ers JM (2012) TLR2 and TLR4 activation induces p38 MAPK-
dependent phosphorylation of S6 kinase 1 in C2C12 myotubes.
Cell Biol Int 36:11071113
Zhang G, Li YP (2012) p38beta MAPK upregulates atrogin1/MAFbx
by specific phosphorylation of C/EBPbeta. Skelet Muscle 2(1):20
Zheng M, Wang YH, Wu XN, Wu SQ, Lu BJ, Dong MQ, Zhang H,
Sun P, Lin SC, Guan KL, Han J (2011) Inactivation of Rheb by
PRAK-mediated phosphorylation is essential for energy-deple-
tion-induced suppression of mTORC1 Nat Cell Biol 13:263272
13