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J Physiol 590.17 (2012) pp 4351–4361
RAPID REPORT
Proliferation of myogenic stem cells in human skeletal
muscle in response to low-load resistance training with
blood flow restriction
Jakob Lindberg Nielsen1 , Per Aagaard1 , Rune Dueholm Bech2 , Tobias Nygaard3 , Lars Grøndahl Hvid1 ,
Mathias Wernbom4 , Charlotte Suetta5 and Ulrik Frandsen1
1
Institute for Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark
Department of Orthopaedic Surgery, Odense University Hospital, Odense, Denmark
3
Department of Orthopaedic Surgery, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
4
Norwegian School of Sport Sciences, Oslo, Norway
5
Department of Clinical Physiology and Nuclear Medicine, Bispebjerg University Hospital, Copenhagen, Denmark
The Journal of Physiology
2
Key points
• In the last decade muscle training performed using a combination of low external loads and
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•
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•
partial restriction of blood flow to the exercising limb has gained increasing interest, since it
leads to significant gains in muscle strength and muscle mass.
The cellular mechanisms responsible for the muscular adaptations induced by this training
paradigm are not fully understood.
This study shows that 3 weeks of high-frequency, low-intensity muscle exercise with partial
blood flow restriction induces increases in maximal muscle strength accompanied by highly
marked gains in muscle fibre size.
Furthermore, the results indicate that these muscular adaptations rely on a considerable
upregulation in myogenic satellite cells number, resulting in nuclear addition to the exercised
myofibres.
The results contribute to a better understanding of the physiological mechanisms underlying
the gain in muscle strength and muscle mass observed with blood flow restricted low-intensity
resistance exercise.
Abstract Low-load resistance training with blood flow restriction has been shown to elicit substantial increases in muscle mass and muscle strength; however, the effect on myogenic stem
cells (MSCs) and myonuclei number remains unexplored. Ten male subjects (22.8 ± 2.3 years)
performed four sets of knee extensor exercise (20% 1RM) to concentric failure during blood
flow restriction (BFR) of the proximal thigh (100 mmHg), while eight work-matched controls
(21.9 ± 3.0 years) trained without BFR (control, CON). Twenty-three training sessions were
performed within 19 days. Maximal isometric knee extensor strength (MVC) was examined
pre- and post-training, while muscle biopsies were obtained at baseline (Pre), after 8 days intervention (Mid8) and 3 (Post3) and 10 days (Post10) post training to examine changes in myofibre
area (MFA), MSC and myonuclei number. MVC increased by 7.1% (Post5) and 10.6% (Post12)
(P < 0.001) with BFR training, while type I and II MFA increased by 38% (Mid8), 35–37%
(Post3) and 31–32% (Post10) (P < 0.001). MSCs per myofibre increased with BFR training
from 0.10 ± 0.01 (Pre) to 0.38 ± 0.02 (Mid8), 0.36 ± 0.04 (Post3) and 0.25 ± 0.02 (Post10)
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DOI: 10.1113/jphysiol.2012.237008
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(P < 0.001). Likewise, myonuclei per myofibre increased from 2.49 ± 0.07 (Pre) to 3.30 ± 0.22
(Mid8), 3.20 ± 0.16 (Post3) and 3.11 ± 0.11 (Post10), (P < 0.01). Although MFA increased in
CON at Mid8, it returned to baseline at Post3. No changes in MSC or myonuclei number were
observed in CON. This study is the first to show that short-term low-load resistance exercise
performed with partial blood flow restriction leads to marked proliferation of myogenic stem
cells and resulting myonuclei addition in human skeletal muscle, which is accompanied by substantial myofibre hypertrophy.
(Received 27 May 2012; accepted after revision 12 July 2012; first published online 16 July 2012)
Corresponding author U. Frandsen: Institute for Sports Science and Clinical Biomechanics, University of Southern
Denmark, Odense, Denmark. Email: ufrandsen@health.sdu.dk
Abbreviations: BFR, blood flow restricted; CSA, cross sectional area; MFA, myofibre area; MSC, myogenic stem cell;
MVC, maximal isometric voluntary contraction; RM, repetition maximum; VL, m. vastus lateralis.
Introduction
Repetitive muscle loading performed using a combination
of low external load (20–50% 1RM) and blood flow
restriction (BFR) has recently gained interest, as it appears
to increase human skeletal muscle mass and maximal
muscle strength to a similar or greater extent (Takarada
et al. 2002) as seen with heavy-load resistance training
(Aagaard et al. 2001). In addition, BFR training appears
to show superior results on these parameters compared
to low-load resistance training without BFR (Abe et al.
2006; Holm et al. 2008), although recent results have
suggested a hypertrophic role of low-intensity resistance
training as well (Mitchell et al. 2012). However, the
underlying mechanisms responsible for the adaptive
changes in muscle morphology in response to BFR
training remain largely unknown. Recent studies show
increased protein synthesis following acute bouts of
BFR training, accompanied by post-translation regulation
in the AKT/mTOR pathway (Fujita et al. 2007; Fry
et al. 2010). Moreover, a reduced expression of the
proteolysis-related genes FOXO3a, Atrogin and Murf-1,
as well as the negative regulator of muscle mass, myostatin, recently were observed 8 and 48 h after acute BFR
exercise (Manini et al. 2011; Laurentino et al. 2012).
In contrast, mRNA expression of other myogenic- and
proteolysis-related targets did not change or differ between
BFR and non-occluded exercise conditions (Drummond
et al. 2008; Manini et al. 2011). The activation and
proliferation of MSCs have been implied to be involved
in accelerated hypertrophy signalling in human skeletal
muscle, where the amount of myonuclei in the myofibre has been proposed to impose a ceiling effect on
myofibre hypertrophy (Kadi et al. 2004; Petrella et al.
2008).
Myogenic stem cells (MSCs) are quiescent cells
positioned between the sarcolemma and the basal lamina
of myofibres (Mauro, 1961) that provide the only source of
myogenic-derived nuclei with the ability of mitogenesis. It
is well-documented that MSCs activate and proliferate in
response to prolonged heavy-resistance training in human
skeletal muscle (Kadi & Thornell, 2000; Kadi et al. 2004;
Olsen et al. 2006; Petrella et al. 2008; Mackey et al. 2010).
The newly formed daughter cells differentiate to become
fusion-capable myoblasts that either return to quiescence
or irreversibly withdraw from the cell cycle to fuse with
pre-existing myofibres with the purpose of assisting in
myofibre regeneration or providing myonuclei addition.
In human intervention studies, addition of myonuclei
only seems to take place concurrently with marked myofibre hypertrophy (Kadi & Thornell, 2000; Kadi et al.
2004; Olsen et al. 2006; Petrella et al. 2008; Mackey et al.
2010), which suggests that these factors are interlinked
in human skeletal muscle. Hence, MSCs most likely play
an essential role in conditions of amplified muscle protein
synthesis by providing additional DNA content for mRNA
transcription. However, the MSC response including the
aspect of myonuclei addition has not yet been examined
during and after BFR training.
The aim of the present study was to investigate whether
the hypertrophy response observed with BFR training
involves MSC proliferation and myonuclei addition.
To our knowledge the present data are the first to
demonstrate that substantial MSC proliferation and myonuclei addition can be evoked by short-term (3 weeks) BFR
training in human skeletal muscle, which is accompanied
by significant gains in myofibre size and contractile muscle
function.
Methods
Subjects
Twenty healthy male subjects were included in the study,
twelve performing blood flow restricted training (BFR)
(body mass 82.3 ± 13.7 kg; height 181.2 ± 6.4 cm; age
22.8 ± 2.1 years) (mean ± SD), while eight served as
controls performing non-occluded work-matched bouts
of exercise (CON) (body mass 80.2 ± 11.4 kg; height
182.9 ± 8.8 cm; age 21.9 ± 3.0 years) (mean ± SD). None
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Elevation of myogenic stem cells with blood flow restricted exercise
of the subjects had participated in systematic strength
training within a year prior to the study and they did not
participate in any structured training regimes in addition
to the present intervention. The study was approved by the
local Ethics Committee (S-200900070) in accordance with
the Declaration of Helsinki, and written informed consent
was obtained from subjects prior to inclusion.
Protocol overview
Muscle mechanical function was tested using isokinetic
dynamometry before (Pre) and 5 and 12 days after
cessation of training (Post5 and Post12). Muscle biopsies
were obtained from m. vastus lateralis (VL) 3 days before
training (Pre), at day 8 during training (Mid8) as well as 3
and 10 days post-training (Post3 and Post10) in BFR subjects, and at Pre, Mid8 and Post3 in control subjects. One
week prior to the intervention period, subjects underwent
a familiarization session for the strength test procedures
and had their 1RM determined. All measurements were
obtained in the experimental leg, which was chosen by
paired within group randomization between the dominant
and non-dominant leg. Subjects were carefully instructed
not to deviate from their normal pattern of food intake,
not to engage in any supplementary training and to refrain
from any alcohol intake during the intervention period.
All strength measurements and biopsy samplings were
conducted at the same time point of the day to control
for diurnal variations.
Training intervention
The subjects participated in a 3 week supervised training
programme consisting of 23 training sessions. Training
was conducted once per day (Mon–Wed) and twice
per day (Thu–Fri) during the first week, and twice
per day (Mon–Fri) during week 2 and 3. Exceptions
were Mon–Tue in week 2 as well as Friday in week
3 when only a single training session was conducted.
Successive training sessions were separated by at least
4 h (Abe et al. 2006). Before each training session,
a pneumatic cuff (15.0 cm width) (9-7350-003, Delfi
Medical, Vancouver BC, Canada) was placed around the
proximal portion of the thigh. The cuff was connected
to a computerized tourniquet system (Zimmer A.T.S.
750, Warsaw, IN, USA) that ensured automatic regulation
of cuff pressure. Following a brief warm up (15–20
repetitions without load) and inflation of the cuff to
100 mmHg, BFR subjects performed four sets of unilateral
dynamic knee extensions (Cybex VR4850, Medway, MA,
USA) at 20% 1RM to concentric failure. Successive sets
were separated by 30 s rest periods. Restriction of muscular
blood flow was maintained for the entire training session
(7.91 ± 1.06 min, including rest periods), and was released
immediately upon completion of the fourth set. Control
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subjects performed a work-matched exercise protocol
without BFR.
Assessment of maximal isometric muscle strength
Knee extensor MVC were performed at 70 deg knee
joint angle (0 deg = full extension) using an isokinetic
dynamometer (Kinetic Communicator 500H, Chattecx
Corp., Hixson, TN, USA) (Aagaard et al. 2001). Subjects
were placed with the lateral epicondyle of the knee aligned
with the rotational axis of the dynamometer. The hip
and thigh were carefully fastened to the dynamometer,
while the lower leg was attached to the dynamometer
lever arm 2 cm above the medial malleolus. Subjects
performed a standardized 5 min warm-up on a stationary
bike, followed by ∼10 submaximal dynamic contractions
in the dynamometer. The test consisted of five 3 s maximal
isometric contractions (45 s pause), during which subjects
were instructed to contract as hard as possible. Strong
verbal encouragement and online visual feedback of the
exerted force was provided. All trials with visible countermovement contractions were disregarded and repeated.
Force signals were sampled at 1000 Hz and corrected for
gravity of the lower leg (Aagaard et al. 2001). The trial with
the highest MVC was selected for further analysis.
Muscle biopsy sampling
Muscle biopsies (∼150 µg) were obtained from VL muscle
using a 5 mm Bergström biopsy needle under sterile
conditions and local anaesthesia (1% lidocaine, Amgros
742122, Copenhagen, Denmark), as described in detail
previously (Aagaard et al. 2001). Biopsies were obtained
from the same region and depth of the VL muscle and
placed approximately 2–3 cm apart. The sequence of
the four biopsies was randomized. Muscle samples were
aligned and mounted in Tissue-Tec (4583, Sakura Finetek,
Alphen aan den Rijn, The Netherlands) and subsequently
frozen in isopentane pre-cooled with liquid nitrogen and
stored at –80◦ C for later analysis.
Immunofluorescence microscopy
Transverse serial sections (8 µm) of the embedded
muscle biopsy specimen were cut at −22◦ C using a
cryostat (HM560; Microm, Walldorf, Germany) and were
mounted on glass slides.
Immunohistochemical stainings were fixed for 10 min
at room temperature in a 4% formaldehyde fixation
buffer containing 0.05% Triton X-100 (Sigma-Aldrich,
St. Louis, MO, USA). After fixation the staining procedure
consisted of three steps that were identical. Each step
was initiated with a 1:10 wash buffer (Dako, S3006,
Glostrup, Denmark) and subsequently blocked (Dako,
X0909) for 10 min. Next primary and secondary antibodies were applied for 60 min incubation separated by
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a wash sequence. MSCs were visualized with an antibody
against Pax7 (Pax7, Hybridoma Bank, Iowa City, IA, USA
1:100), while laminin (Dako, Z0097, 1:100) and MHC-I
(M8421, 1:2000) were added in that order for distinction of
the myofibre border and myofibre type slow, respectively.
Specific secondary antibodies (order listed: Alexa-555
goat anti-mouse (Invitrogen, A21424, Life Technologies
Denmark, Naerum, Denmark, 1:1000), Alexa-488 goat
anti-rabbit (Invitrogen, A11034, 1:1000) and Alexa-350
goat anti-mouse (Invitrogen, A11045, 1:500)) were
applied after each primary antibody. Finally, sections were
mounted with a fluorescent anti-fade medium containing
DAPI (which stains nuclei) (Invitrogen, P36935), and subsequently slides and coverglass were pasted together and
stored protected from light at 5◦ C.
Biopsy stainings were visualized on a computer screen
using a light microscope (Carl Zeiss Axio Imager M1,
Germany) and a high-resolution AxioCam (Carl Zeiss),
and all morphometric analysis were performed using
a digital analysis program (Carl Zeiss, AxioVision 4.6).
Type I (stained) and type II (unstained) myofibres were
differentiated, and MFA was determined. On average
371 ± 103 and 618 ± 158 myofibres (mean ± SD) were
analysed per biopsy for the assessment of MFA and fibre
type distribution, respectively. Furthermore, nuclei were
identified either as MSC or myonuclei using the following
criteria: MSC-derived nuclei had to stain positive for Pax7
and be placed within the basal lamina; nuclei with a sublaminar placement were considered myonuclei (Fig. 1).
The number of Pax7+ nuclei was expressed relative to the
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number of type I and II myofibres, myofibre area and the
proportion of myonuclei, while the number of myonuclei
was expressed relative to the number of type I and II myofibres as well as MFA (Mackey et al. 2010). A total of 75
myofibres for each fibre type were analysed per biopsy in
accordance with previous reliability analysis (Mackey et al.
2009) to ensure a reliable estimate of MSC and myonuclei
number. Myofibres were analysed in three or more separate
areas of the cross section. All analyses were carried out
manually by the same investigator, who was blinded with
respect to subject-ID and time point. Nine data points are
reported at Mid8 in the BFR group as one subjects failed
to turn up to this biopsy sampling, while a dataset was
omitted from CON due to inadequate biopsy quality.
Statistical analysis
Statistical analysis was performed using a linear
mixed-model. Variables were analysed with subject-ID
as a random effect and time and group as fixed effects.
Myofibre types were analysed separately. Variables with
skewed distributions were appropriately transformed.
Furthermore, training data were analysed with a one-way
ANOVA, while associations between relevant parameters
were evaluated with linear regression and Pearson’s
product–moment correlation. All statistical analyses were
performed with STATA 10.1 (StataCorp, College Station,
TX, USA). Values are presented as means ± SEM, unless
otherwise stated. The level of statistical significance was
set at P ≤ 0.05.
Results
Subjects
Two subjects from the BFR group left the project prematurely for reasons not related to the intervention. The
remaining subjects (BFR: n = 10; CON: n = 8) completed
22.8 ± 0.4 (mean ± SD) of 23 possible training sessions.
No changes in body weight were observed during the intervention.
Training progression
Figure 1. Representative immunohistochemical staining (BFR,
Mid8) containing Pax7 (red), laminin (green) and MHC I/Dapi
(blue)
Arrows denote Dapi+ /Pax7+ myogenic stem cells and stars mark
myonuclei (scale bar = 50 µm).
Averaged over the intervention period subjects performed
40.5 ± 5.2, 12.1 ± 3.9, 8.0 ± 3.1 and 6.6 ± 3.5 repetitions
in the first, second, third and fourth set, respectively;
this summed to a total of 66.2 ± 11.3 repetitions per
session (mean ± SD). The mean training load (20% 1RM)
was 19.5 ± 2.6 and 20.9 ± 4.0 kg (mean ± SD) during
BFR intervention and in controls, respectively. Total
work increased proportionally with the total repetitions
performed, as training-load and range of motion remained
unchanged during the intervention. To evaluate changes
within the training period, the 23 training sessions were
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respectively (P < 0.001). No changes were observed in
CON (Fig. 2).
Myofibre cross sectional area and fibre
type distribution
Figure 2. Maximal isometric knee extensor muscle strength at
baseline (Pre), and 5 and 12 days after cessation of training
(Post5 and Post12)
Pre to Post differences: ∗ P < 0.001. Mid5 to Post12 difference:
†P < 0.05. Values are means ± SEM; BFR: n = 10; C: n = 8.
divided into three phases (A: session 1–7, B: session
8–16, C: session 17–23). Total repetitions per training
session increased from A (51.5 ± 6.7) to B (66.3 ± 3.7)
and C (75.9 ± 2.2) (P < 0.01) (mean ± SD), while also
increasing between B and C (P < 0.01). Repetitions per
set increased from A to B and C in all sets (P < 0.05) and
between B and C in set 1 and 3 (P < 0.05). Training data
are presented for the BFR group only, as all training in
CON was work-matched.
Maximal isometric quadriceps strength
Knee extensor MVC increased with BFR training
from 271.6 ± 47.5 N m (Pre) to 290.7 ± 54.1 (Post5)
and 300.4 ± 60.9 N m (Post12) (mean ± SD) (Fig. 2),
corresponding to relative increases of 7.0 and 10.6%,
Type II MFA was larger than type I MFA at Pre
in both intervention groups (P < 0.01). Type I MFA
increased with BFR training from 3781 ± 191 µm2
(Pre) to 5201 ± 321 (Mid8), 5103 ± 256 (Post3) and
4960 ± 226 µm2 (Post10), corresponding to relative
increases of 37.6, 35.0 and 31.2% (P < 0.001) (Fig. 3A).
Likewise type II MFA increased from 4170 ± 192 µm2
(Pre) to 5772 ± 390 (Mid8), 5718 ± 300 (Post3) and
5512 ± 253 µm2 (Post10), corresponding to relative
increases of 38.4, 37.1 and 32.2% (P < 0.001) (Fig. 3B).
In CON, MFA increased from 4041 ± 250 and 4975 ± 350
to 5058 ± 582 and 6396 ± 620 µm2 at Mid8 for type I and
type II myofibres, corresponding to increases of 25.2 and
28.6% (P < 0.05), but returned to baseline level values
at Post3 (Fig. 3A and B). In both groups myofibre type
distribution remained unchanged during the intervention
period.
Myogenic stem cell content
Pax7+ cells per type I myofibre increased with BFR training
from 0.11 ± 0.01 (Pre) to 0.41 ± 0.04 (Mid8), 0.36 ± 0.05
(Post3) and 0.26 ± 0.03 (Post10), corresponding to
increases of 292.4, 242.6 and 143.5% (P < 0.001) (Fig. 4A
and C). Pax7+ cells per type II myofibre increased
from 0.10 ± 0.01 (Pre) to 0.37 ± 0.03 (Mid8), 0.35 ± 0.03
(Post3) and 0.23 ± 0.02 (Post10), corresponding to
relative gains of 276.2, 264.7 and 143.9% (P < 0.001)
(Fig. 4B and D). No changes were observed in CON
(Fig. 4A–D).
Figure 3. Myofibre cross sectional area at baseline (Pre), 8 days into the training intervention (Mid8),
and 3 and 10 days after cessation of training (Post3 and Post10)
A, BFR and CON response in type slow myofibres; B, BFR and CON response in type fast myofibres. Pre to
Mid8/Post differences: ∗ P < 0.001. Pre to Mid8 difference: ∗∗ P < 0.01. Between group difference: †P < 0.05.
Values are means ± SEM; BFR: n = 10 at Mid8 n = 9; C: n = 7.
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In BFR-subjects Pax7+ cells expressed relative to
type I MFA (mm2 ) increased from 26.5 ± 2.4 cells mm−2
(Pre) to 79.0 ± 10.7 (Mid8), 71.2 ± 8.1 (Post3) and
52.0 ± 3.6 cells mm−2 (Post10) corresponding to gains
of 198.5, 168.8 and 96.5% (P < 0.001) (Fig. 4E).
Likewise, Pax7+ cells increased in type II myofibres
from 22.9 ± 2.8 cells mm−2 (Pre) to 62.2 ± 7.5 (Mid8),
60.2 ± 5.1 (Post3) and 43.7 ± 3.4 cells mm−2 (Post10),
corresponding to relative gains of 171.7, 163.1 and 91.0%
(P < 0.001) (Fig. 4F). No changes were observed in CON
for this parameter (Fig. 4E and F).
The ratio of Pax7+ cells to myonuclei number increased
with BFR training in type I myofibres from 4.6 ± 0.4 (Pre)
to 13.7 ± 1.6 (Mid8), 12.4 ± 1.7 (Post3) and 8.5 ± 0.7
(Post10), corresponding to relative gains of 196.9, 170.0
and 84.2% (P < 0.001) (Fig. 4G). In type II myofibres
the ratio of Pax7+ cells to myonuclei number increased
from 3.6 ± 0.4 (Pre) to 11.0 ± 1.1 (Mid8), 10.5 ± 1.0
(Post3) and 7.3 ± 0.5 (Post10), corresponding to increases
of 203.1, 189.8 and 102.3% (P < 0.001) (Fig. 4H). No
changes were observed in CON for this parameter (Fig. 4G
and H).
Myonuclei content
BFR training lead to an increased number of myonuclei per type I myofibre from 2.34 ± 0.08 (Pre) to
3.11 ± 0.21 (Mid8), 2.98 ± 0.18 (Post3) and 2.99 ± 0.10
(Post10), corresponding to relative gains of 33.2, 27.4 and
28.0% (P < 0.001) (Fig. 5A). Likewise, in type II myofibres the number of myonuclei per myofibre increased
from 2.65 ± 0.11 (Pre) to 3.43 ± 0.25 (Mid8), 3.43 ± 0.16
(Post3) and 3.23 ± 0.13 (Post10), corresponding to
relative increases by 29.5, 29.6 and 22.0% (P < 0.001)
(Fig. 5B). No changes were observed in CON for this
parameter (Fig. 5A and B).
MFA per myonuclei decreased for type I myofibres
during BFR training from 2075 ± 165 to 1816 ± 76 µm2
between Post3 and Post10, corresponding to a 12.5%
decrease (P < 0.05) (Fig. 5C). In CON an increase in myonuclei domain from 2052 ± 77 to 2536 ± 202 µm2 and
from 2123 ± 61 to 2728 ± 217 µm2 was observed between
Pre and Mid8 in type I and II myofibres, respectively,
corresponding to increases of 23.6 and 28.5% (P < 0.01)
(Fig. 5C and D). However, myonuclei domain returned to
baseline levels in CON at Post3 for both myofibre types
(Fig. 5C and D).
Correlations
Following BFR training a positive relationship emerged
between the relative change in myonuclei number per fibre
and MFA (r = 0.51; P < 0.01), and a similar relationship
was observed between the relative change in MSC per fibre
and MFA (r = 0.58; P < 0.01).
Discussion
The present study is the first to examine the effect of
low-load resistance exercise with blood flow restriction
on MSC and myonuclei number in human skeletal
muscle. Several notable findings emerged. Firstly, it was
demonstrated that the expression of Pax7+ cells was
markedly increased (∼3- to 4-fold up-regulated) relative
to baseline values in response to 19 days of BFR resistance
training and continued to remain elevated in the following
10 days of detraining. Secondly, BFR training led to an
increased number of myonuclei per myofibre, strongly
indicating the presence of MSC/myoblast fusion with
existing myofibres. Thirdly, type I and II myofibre area
increased markedly already in the very initial phase
of training (5 days of training; Mid8) irrespectively of
intervention modality; however, MFA remained elevated
throughout the study period with BFR training only, thus
returning to baseline levels in controls at the end of the
training period (19 days of training, Post3). Thus, the present data demonstrate that low-load BFR training can elicit
marked MSC proliferation as well as myonuclei addition
in human skeletal muscle, which is accompanied by substantial gains in MFA.
The increase in MFA (∼30–40%) presently observed in
the BFR group is highly unique taking into consideration
the low intensity of loading (≤20% 1RM) and short
duration of training (19 days). Previous studies utilising
BFR-exercise have reported substantial increases in
anatomical quadriceps CSA using MR imaging (Takarada
et al. 2002; Abe et al. 2006), while training using low-load
training without BFR combined with normal or high
training frequency showed only minor (3%) or no change
in quadriceps CSA (Abe et al. 2006; Holm et al. 2008),
although more substantial gains in anatomical quadriceps
volume (∼7%) recently have been reported as well
(Mitchell et al. 2012). It is well-documented that heavy
resistance training can lead to significant gains in MFA.
Thus, 15–20% increases in mean MFA were reported
Figure 4. Pax7 positive cells at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and
10 days after cessation of training (Post3 and Post10)
A and B, Pax7+ myogenic stem cells per myofibre, type I and II myofibres (group response). C and D, Pax7+
myogenic stem cells per myofibre, type I and II myofibres (individual response). E and F, Pax7+ myogenic stem
cells per myofibre cross sectional area (mm2 ) (group response). G and H, Pax7+ myogenic stem cells to myonuclei
ratio (group response). Pre to Mid8/Post differences: ∗ P < 0.001. Mid8/Post3 to Post12 differences: ‡P < 0.01.
Between group difference: †P < 0.05. Values are means ± SEM; BFR: n = 10 at Mid8 n = 9; C: n = 7.
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after 12–16 weeks of heavy resistance training in young
untrained men (Aagaard et al. 2001; Kadi et al. 2004;
Olsen et al. 2006), while Petrella and co-workers reported
an MFA increase of ∼37% in individuals characterized
as hypertrophy responders after 16 weeks of heavy-load
resistance training (Petrella et al. 2008). In contrast,
12 weeks of high-volume low-load resistance training
(15.5% 1RM) without BFR did not lead to any change in
MFA (Mackey et al. 2010). However, increased mean MFA
(∼18%) recently was reported following 10 weeks (30
sessions) of low-load (30% 1RM) knee extensor training
performed until fatigue onset (Mitchell et al. 2012).
Interestingly, a large increase in the MFA of both myofibre types in the BFR group was observed after only 8 days
of the intervention (Mid8), and remained elevated at 3 and
10 days after cessation of the training intervention lasting
19 days. Control subjects matched the increase at Mid8,
but MFA returned to baseline level at Post3. The transitory
change in MFA observed in the control subjects indicates
that the cell volume was influenced by factors related to the
early habituation to exercise other than protein accretion.
Such factors could include cell swelling and/or changes
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in glycogen/mitochondria content. However, the latter
factors seem unlikely to affect MFA in the present range, as
an increase in glycogen content and mitochondria volume
is thought to increase MFA by less than 5% (Nygren
et al. 2000). Lasting cellular swelling (∼3 days) could be
explained by hypoxia-induced modification of homeostasis regulating membrane channels (Korthuis et al.
1985), stretch-induced opening of membrane channels
(Singh & Dhalla, 2010) or microfocal damage to the
plasma membrane (Grembowicz et al. 1999).
It is likely that the initial increase in MFA observed
following 5 days of intervention (Mid8) irrespectively
of training modality was influenced by exercise-induced
cell-swelling, and as the observed MFA increase during
such a short time frame is unlikely to be accounted
for by a positive protein turnover alone. This notion is
further supported by the finding that MFA returned to
reach baseline values during the latter period of training
in the control subjects. Conversely, it is plausible that
the late-phase gain in MFA observed with BFR training
occurred mainly due to accumulation of myofibrillar
proteins, as supported by the finding that MFA remained
Figure 5. Myonuclei number at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and
10 days after cessation of training (Post3 and Post10)
A and B, myonuclei number per myofibre, type I and II myofibres. C and D, cross sectional area (µm2 ) per myonuclei,
type I and II myofibres. Pre to Mid8/Post differences: ∗ P < 0.001, ∗∗ P < 0.01. Mid8 to Post10 difference: ‡P < 0.05.
Between group difference: †P < 0.05. Values are means ± SEM; BFR: n = 10 at Mid8 n = 9; C: n = 7.
C 2012 The Authors. The Journal of Physiology
C 2012 The Physiological Society
J Physiol 590.17
Elevation of myogenic stem cells with blood flow restricted exercise
elevated 3–10 days post-training along with a 7–11%
persistent increase in MVC. It cannot be excluded that
this gain in MVC could arise at least in part from
elevated neuromuscular activity, but this parameter has
been reported to remain unchanged after low-load BFR
training and to only increase with heavy resistance training
(Kubo et al. 2006).
Furthermore, indirect evidence for longitudinal myofibrillar protein accretion following low-load BFR training
is provided in several acute studies where an augmented
protein turnover was observed. Thus, mixed muscle
protein synthesis appears to increase with acute BFR
exercise in parallel with increased signalling in anabolic
mitogen-activated protein kinase pathways related directly
to protein synthesis along with a down-regulated
proteolysis and reduced myostatin signalling (Fujita et al.
2007; Drummond et al. 2008; Fry et al. 2010; Manini et al.
2011; Laurentino et al. 2012). In contrast, mixed protein
synthesis and related signalling pathways were largely
unaffected after acute low-intensity exercise without BFR
(Fujita et al. 2007; Drummond et al. 2008; Fry et al.
2010; Manini et al. 2011; Laurentino et al. 2012), although
increases in myofibrillar synthesis and elevated anabolic
signalling have also been reported with acute bouts of
low-intensity (30% 1RM) resistance training performed
to fatigue (Burd et al. 2010).
The observation of a consistent elevation in MFA
3–10 days following cessation of the BFR training protocol
along with no detectable pre to post change in MFA in
our control subjects indicates that cellular swelling was
unlikely to be the primary cause of the observed gains in
MFA during the later phase of training. Consequently,
elevated myofibrillar protein content seems to mainly
explain the gain in MFA observed following the relatively
short period (∼3 weeks) of BFR training.
In the present study individuals exposed to 19 days of
BFR resistance training demonstrated a marked increase
in Pax7+ cells illustrated by relative gains of ∼280% (Mid),
∼250% (Post3) and ∼140% (Post10), in both type I and
type II myofibres, while no changes were observed in the
controls performing a work-matched training protocol
without BFR. These changes in MSC number evoked by
BFR training were paralleled by increases in the number of
myonuclei per myofibre and MFA, respectively, resulting
in an unchanged MFA per myonuclei (e.g. unaltered
myonuclei domain). Previous studies have examined the
expression of selected MSC markers (NCAM/CD56, Pax7)
in human skeletal muscle within a number of different
contexts. Hitherto, the largest up-regulation in MSCs per
myofibre (157%) and in the MSC to myonuclei ratio
(112–192%) were reported between 24 and 96 h after acute
bouts of high-volume eccentric muscle contractions in
untrained young subjects (Crameri et al. 2004; Dreyer
et al. 2006). Following varying periods of heavy-load
resistance training in young men and woman, more
C 2012 The Authors. The Journal of Physiology
C 2012 The Physiological Society
4359
modest changes of 30–50% in MSCs per myofibre are
typically observed (Kadi & Thornell, 2000; Kadi et al.
2004; Olsen et al. 2006). Interestingly, 12 weeks of low-load
(15.5% 1RM) resistance training without BFR showed a
minor, yet significant change in MSCs per fibre of 18%
(Mackey et al. 2010). Notably, Mackey et al. did not find
any difference between low-load and high-load training
which in combination with the present data suggests that
both active muscle contractions and vascular occlusion
are required to obtain highly amplified elevations in MSC
proliferation and differentiation.
As the main and novel finding in the present
study, short-term high-frequency BFR training using
low external loading appears effective for inducing a
marked proliferation of MSCs compared to other training
modalities. Previous human studies have demonstrated
significant proliferation and differentiation of MSCs
during muscle regeneration or when myonuclei are needed
during myofibre hypertrophy (Dreyer et al. 2006; Petrella
et al. 2008). The unusually large increase in Pax7+ cells
observed in the present study could reflect both myofibre
regeneration and/or a need for myonuclei addition. The
latter notion was supported by the present finding of a
very large increase in MFA. Thus, it has been hypothesized
that the number of myonuclei per fibre sets the ceiling for
myofibre hypertrophy, when the volume of cytoplasm per
myonuclei exceeds the myonuclei’s ability to transcribe
mRNA. Most human studies support this notion, since
myonuclei addition has been observed concomitantly
with an increase in MFA greater than 25% and/or a
myonuclei domain of or greater than 2000 µm2 (Kadi
et al. 2004; Petrella et al. 2008). Interestingly, the initial
increase in MFA observed in the control subjects was not
paralleled by signs of increased MSC number or myonuclei addition, which implies that MSC proliferation and
differentiation are not per se sensitive to changes in myofibre area/myonuclei volume, but may need more stable
(i.e. non-transient) changes to emerge.
In the present study myonuclei domain remained
unchanged or a little reduced throughout the experimental
period (Fig. 5C and D) and a positive relationship
(r = 0.51; P < 0.01) between the relative change in myonuclei per fibre and MFA was found in the BFR subjects. These findings indicate that MSC proliferation and
myonuclei addition are at least in part responsible for
an amplified transcription rate and myofibrillar protein
synthesis with BFR training.
Furthermore, the increase in myonuclei addition might
represent an important mechanism for the maintenance
of muscle mass following cessation of training. Significant
myofibre type II atrophy was reported already 10 days
after cessation of a 90 day heavy-resistance training intervention (Jespersen et al. 2011), while no myofibre atrophy
was observed 3 or 10 days post-training in the present
study. Interestingly, a lack of myonuclei addition was
4360
J. L. Nielsen and others
noted in the study by Jespersen and colleagues (Kadi
et al. 2004), which potentially could explain the disparate
time courses of myofibre atrophy despite a similar period
of detraining (10 days). This suggestion of an atrophy
protective effect achieved by training-induced myonuclei
addition is supported by recent animal data, where
overload-induced myonuclei addition in mice partially
protected the affected muscle from subsequent atrophy
(Bruusgaard et al. 2010).
Although differentiation of MSCs was evident following
BFR training as reflected by an increase in the
myonuclei-to-fibre ratio, the present data suggest an
unusually high proliferation rate of MSCs. Increases
in cyclin-dependent kinase inhibitor-1 and myoblast
determination protein-1 mRNA have been observed
3 h after an acute bout of low intensity BFR exercise
(Drummond et al. 2008), which indirectly supports the
presence of MSC activation and proliferation with this
type of exercise. Corresponding changes were observed
in control subjects performing similar exercise without
BFR; however, it cannot be excluded that differential MSC
responses might have been observed if evaluated at later
time points (>3 h). Interestingly, a reduced myostatin
expression in the exercised muscle tissue recently was
reported 48 h after acute bouts of BFR exercise (Laurentino
et al. 2012), which indicates a potential regulating role
for myostatin, since it is known to regulate both MSC
proliferation and differentiation negatively.
Previously, the largest reported changes in MSC content
have been observed after unaccustomed high-volume
eccentric exercise, where signs of microfocal damage
and cellular regeneration may be evident (Crameri et al.
2004; Dreyer et al. 2006). With BFR resistance exercise
it is possible that the muscle cell membrane undergoes
microfocal damage followed by subsequent regeneration
(Grembowicz et al. 1999) due to external loading, hypoxia
or a combination of both. However, in terms of potential
microfocal damage, we did not find any visible signs of
damage to the basal lamina as indicated in our laminin
stainings.
Another possible explanation for the high degree
of MSC proliferation with BFR training could be
the role of hypoxia in combination with stretch
and/or contraction on different MSC mediators. Thus,
activation and proliferation of MSCs may be stimulated
acutely by BFR-induced stretch-, hypoxia- and/or
contraction-induced nitric oxide (NO) secretion (Blitzer
et al. 1996; Pattwell et al. 2004; Tatsumi et al. 2006).
In support of this notion, NO has been demonstrated
to release hepatocyte growth-factor, which has been
identified to activate MSCs directly in vivo (Tatsumi et al.
2006). Furthermore, inhibition of NO secretion has been
shown to inhibit MSC proliferation (Tatsumi et al. 2006),
which underlines a potential role of NO in MSC activation
that should be investigated in future studies.
J Physiol 590.17
In summary, the present study reports a rapid and
pronounced increase in myofibre area concomitantly with
marked increases in the proliferation and differentiation
of MSCs during and following 3 weeks of BFR muscle
exercise. The increase in MFA was accompanied by
corresponding increases in contractile function (elevated
MVC). These data indicate that the unusually large
increase in MFA observed with BFR training, at least in
part, may rely on an increase in MSC proliferation and
differentiation that results in the donation of additional
myonuclei to the myofibres. In turn, this incorporation of
MSC-derived myonuclei provides an improved capacity
for myofibrillar gene transcription, which is likely to
contribute to an enhanced activity of cellular protein
synthesis.
The present findings may have useful clinical implications. Thus, an amplified proliferation, differentiation
and self-renewal of MSCs by means of BFR training
potentially could benefit clinical patients with loss in
skeletal muscle mass and elderly sarcopaenic individuals.
Future studies should address these important aspects
of skeletal muscle homeostasis in selected patient
populations.
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Author contributions
The project was performed at the Institute of Sports Science
and Clinical Biomechanics, University of Southern Denmark,
Denmark. The contributions of the authors were as follows:
conception and design of the study: J.N., P.A., M.B. and U.F.;
collection, analysis and interpretation of data: J.N., P.A., R.B.,
T.B., L.G., C.S. and U.F.; drafting the article or revising it critically
for important intellectual content: J.N., P.A., R.B., T.B., M.B.,
L.G., C.S. and U.F. All authors have approved the final version
of the manuscript.
Acknowledgements
We wish to express our gratitude to all the subjects who
volunteered to participate in the study. Also, we thank the Danish
Rheumatism Association (Gigtforeningen) and the Foundation
of A.P. Møller and Wife Chastine Mc-Kinney Møller for their
support. None of the authors declare any conflict of interest.