Acta Physiol 2014, 210, 600611

Substantial skeletal muscle loss occurs during only 5 days

of disuse

B. T. Wall,1 M. L. Dirks,1 T. Snijders,1 J. M. G. Senden,1 J. Dolmans2 and L. J. C. van Loon1

1 Department of Human Movement Sciences, NUTRIM School for Nutrition, Toxicology and Metabolism Maastricht University
Medical Centre, Maastricht, the Netherlands
2 Department of Surgery, Maastricht University Medical Centre, Maastricht, the Netherlands

Received 15 July 2013, Abstract

revision requested 31 July 2013,
revision received 2 September
2013,
accepted 24 October 2013
Correspondence: L. J. C. van
Loon, PhD, Department of
Human Movement Sciences,
Maastricht University Medical
Centre, PO Box 616, Maastricht
6200 MD, the Netherlands.
E-mail: L.vanLoon@maastrichtuni-
versity.nl
Aim: The impact of disuse on the loss of skeletal muscle mass and
strength has been well documented. Given that most studies have investi-
gated muscle atrophy after more than 2 weeks of disuse, few data are
available on the impact of shorter periods of disuse. We assessed the
impact of 5 and 14 days of disuse on skeletal muscle mass, strength and
associated intramuscular molecular signalling responses.
Methods: Twenty-four healthy, young (23  1 year) males were subjected
to either 5 (n = 12) or 14 (n = 12) days of one-legged knee immobilization
using a full leg cast. Before and immediately after the immobilization period,
quadriceps muscle cross-sectional area (CSA), leg lean mass and muscle
strength were assessed, and biopsies were collected from the vastus lateralis.
Results: Quadriceps muscle CSA declined from baseline by 3.5  0.5
(P < 0.0001) and 8.4  2.8% (P < 0.001), leg lean mass was reduced by
1.4  0.7 (P = 0.07) and 3.1  0.7% (P < 0.01) and strength was
decreased by 9.0  2.3 (P < 0.0001) and 22.9  2.6% (P < 0.001) fol-
lowing 5 and 14 days of immobilization respectively. Muscle myostatin
mRNA expression doubled following immobilization (P < 0.05) in both
groups, while the myostatin precursor isoform protein content decreased
after 14 days only (P < 0.05). Muscle MAFBx mRNA expression
increased from baseline by a similar magnitude following either 5 or
14 days of disuse, whereas MuRF1 mRNA expression had increased
significantly only after 5 days.
Conclusion: We conclude that even short periods of muscle disuse can
cause substantial loss of skeletal muscle mass and strength and are accom-
panied by an early catabolic molecular signalling response.
Keywords disuse atrophy, immobilization, myostatin, skeletal muscle.

The recovery from illness or injury often requires
otherwise healthy humans to undergo a period of
muscle disuse (e.g. bed rest or limb immobilization).
A major consequence of disuse is skeletal muscle atro-
phy (Deitrick 1948, Ingemann-Hansen & Halkjaer-
Kristensen 1980, Gibson et al. 1987). The ensuing
impairments in muscle function (Deitrick 1948, Inge-
mann-Hansen & Halkjaer-Kristensen 1980, White
et al. 1984, Gibson et al. 1987, LeBlanc et al. 1992),
metabolic rate (Tzankoff & Norris 1977, Haruna
et al. 1994) and insulin sensitivity (Stuart et al. 1988),
and accrual of body fat mass (Ferrando et al. 1996,
1997, Brooks et al. 2008) following two or more
weeks of muscle disuse have been well documented.
Studies investigating muscle disuse atrophy gener-
ally employ relatively long experimental periods, rang-
ing from two to as long as 17 weeks of bed rest or
limb immobilization (e.g. Gibson et al. 1987, LeBlanc

600 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190
Acta Physiol 2014, 210, 600611
et al. 1992, Jones et al. 2004, Paddon-Jones et al.
2004). However, over the last decade, efforts have
been made within healthcare systems to reduce the
duration of bed rest/immobilization that patients
endure due to illness or injury. At present, the average
length of hospitalization for elderly patients admitted
with acute illness is only 56 days (Fisher et al. 2010).
Moreover, periods of illness and minor injury that do
not require hospitalization but necessitate inactive
recovery at home generally last only a few days. Recent
work has reported that limb immobilization for only
4 days already causes a decline in functional strength in
older adults (Suetta et al. 2012, Hvid et al. 2013).
However, currently, no data exist on the impact of such
short periods of disuse (i.e. <7 days) on skeletal muscle
mass. Such information is of important clinical rele-
vance as it has been hypothesized that successive bouts
of muscle loss during short periods of disuse accumulate
throughout the lifespan and may be responsible for
much of the loss of muscle mass typically observed in
the ageing population (English & Paddon-Jones 2010,
Wall & van Loon 2012, Wall et al. 2013). Accordingly,
we hypothesize that even short periods of muscle disuse
can lead to substantial loss of muscle tissue. Therefore,
in the present study, we compared the impact of a short
(i.e. 5 days) vs. more prolonged (i.e. 14 days) period of
immobilization on skeletal muscle mass and functional
strength.
Skeletal muscle atrophy during more prolonged dis-
use (i.e. >10 days) is attributed to declines in both the
rate of post-absorptive and post-prandial muscle pro-
tein synthesis, without any apparent changes in muscle
protein breakdown rates (Gibson et al. 1987, Ferran-
do et al. 1996, Glover et al. 2008, Wall & van Loon
2012). We (Wall & van Loon 2012, Wall et al.
2013), and others (Phillips et al. 2009, Murton &
Greenhaff 2010, Marimuthu et al. 2011, Suetta et al.
2012), have argued that elevated muscle protein
breakdown may also contribute to muscle loss during
the first few days of disuse. Myostatin has been dem-
onstrated to be a key negative regulator of muscle
mass in animals (McPherron & Lee 1997, Mosher
et al. 2007) and humans (Schuelke et al. 2004). Aside
from its documented role in the regulation of muscle
protein synthesis (Rodriguez et al. 2011) and myogen-
esis (Amthor et al. 2002, Muroya et al. 2009), myost-
atin has also been suggested as a regulator of muscle
protein breakdown (McFarlane et al. 2006). There-
fore, in the present study, we also assessed the impact
of 5 and 14 days of limb immobilization on the skele-
tal muscle mRNA and protein expression of myostatin
and associated genes.
In this study, we selected 24 healthy, young men
who were subjected to either 5 (n = 12) or 14
(n = 12) days of one-legged knee immobilization.
2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190
B T Wall et al. Short-term muscle disuse atrophy
Skeletal muscle mass and function were assessed
before and after immobilization, and muscle biopsies
were collected to assess the associated molecular sig-
nalling responses. This is the first study to report sub-
stantial muscle atrophy after merely 5 days of muscle
disuse in vivo in humans.
Methods
Subjects
Twenty-four healthy, young (23  1 year) men volun-
teered to participate in the present study. All subjects
were fully informed of the nature and possible risks of
the experimental procedures, before their written
informed consent was obtained. Subjects were
screened to exclude any person with lower limb and/
or back injuries sustained within a year prior to
beginning the study, a (family) history of thrombosis/
cardiovascular disease, use of anticoagulants, musculo-
skeletal/orthopaedic/haemostatic disorders or partici-
pation in any regular resistance training programme
within 6 months of beginning the study. During
screening, all subjects were instructed and familiarized
with safe lifting technique for the leg extension exer-
cise. Maximum strength was assessed using the multi-
ple repetitions testing procedure (Mayhew et al. 1995)
for each leg separately. The study was approved by
the Medical Ethics Committee of the Maastricht
University Medical Centre+, Maastricht, the Nether-
lands. The present study is part of a greater project
investigating muscle disuse atrophy in humans.
Experimental design
In this study, a parallel design was applied with a sub-
ject cohort being assigned to an experimental treat-
ment in which subjects underwent either 5 or 14 days
of one-legged knee immobilization by means of a full
leg cast. Before and after the immobilization period,
computed tomography (CT) scans, dual-energy x-ray
absorptiometry (DEXA) scans and 1 repetition max
(1-RM) tests were performed to determine changes in
muscle mass and strength, and muscle biopsies were
collected to assess changes in muscle fibre characteris-
tics and the mRNA and protein expression of key
genes associated with the regulation of muscle mass.
Diet and physical activity
All subjects received the same standardized meal the
evening prior to the experimental test days (33 
2 kJ kg 1 body weight, providing 44 energy% (En%)
carbohydrate, 22 En% protein and 34 En% fat). All
volunteers were instructed to refrain from strenuous
601
Short-term muscle disuse atrophy B T Wall et al.
physical activity, avoid alcohol intake and keep their
diet as constant as possible for 2 days prior to the
experimental test day.
Experimental visits
Subjects participated in two identical experimental test
days, before and immediately after the immobilization
period. Approximately 2 days prior to the immobiliza-
tion period, subjects participated in the first test day.
During the visit, subjects arrived at the laboratory at
08.00 h in the fasted state, and body weight was mea-
sured with a digital balance with an accuracy of
0.1 kg (SECA GmbH, Hamburg, Germany). Thereaf-
ter, a single-slice CT scan (Philips Brilliance 64; Phi-
lips Medical Systems, Best, the Netherlands) was
performed to assess upper leg muscle cross-sectional
area (CSA). The scanning characteristics were as fol-
lows: 120 kV, 300 mA, rotation time of 0.75 s and a
field of view of 500 mm. With subjects lying supine
with their legs extended and feet secured, a 3-mm-
thick axial image was taken 15 cm proximal to the
top of the patella. The precise scan position was
marked with semipermanent ink for the duration of
the experimental protocol to ensure accurate repeat
measurements. Muscle area of the right leg was
selected between 0 and 100 Hounsfield units (Good-
paster et al. 2000), after which the quadriceps muscle
was selected by manual tracing using ImageJ software
(version 1.45d; National Institute of Health, Bethesda,
MD, USA) (Goodpaster et al. 2000, Strandberg et al.
2010). After CT scanning, body composition (fat, fat-
free mass and bone mineral content) was determined
by DXA scan (Hologic, Bedford, TX, USA). Lean
mass and per cent body fat were determined on a
whole body level and for specific regions (e.g. legs).
Thereafter, a muscle biopsy was collected from the
vastus lateralis muscle of the leg identified as the leg
to become immobilized (or the previously immobilized
leg in the case of the second visit). Muscle biopsy
samples were obtained from the middle region of the
vastus lateralis, approx. 13 cm below the level that
the CT scan was performed, and approx. 3 cm below
entry through the fascia, by using the percutaneous
needle biopsy technique (Bergstr om 1975). Muscle
samples were dissected carefully and freed from any
visible non-muscle material and were immediately fro-
zen in liquid nitrogen and stored at 80 C until
further analysis. Thereafter, subjects single-leg one
repetition maximum (1-RM) was assessed (Kraemer
& Fry 1995). After warming up, the load was set at
97.5% of the estimated 1-RM from the screening visit
and increased after each successful lift until failure.
Three min rest periods were allowed between lifts. A
repetition was considered valid when the subject used
602 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190
Acta Physiol 2014, 210, 600611
proper form and was able to complete the entire lift
in a controlled manner without assistance. Finally,
subjects were instructed on, and familiarized with, the
use of crutches. On the day of cast removal, all mea-
sures of muscle mass and strength were repeated in
the same manner, and a muscle biopsy was collected
prior to any weight bearing activity from a site
approx. 2 cm from the position of the first biopsy.
Limb immobilization
Approximately 48 h after the first test day, subjects
reported at 8.00 h at the Casting Room at Maastricht
University Medical Centre, to have a full leg cast fit-
ted. The application of the cast signified the first day
of either the 5 (n = 12) or 14 (n = 12) day immobili-
zation period. The circular leg cast extended from
10 cm above the ankle to approx. 25 cm above the
patella. The knee was casted at a 30 angle of flexion
to prevent subjects performing any weight bearing
activities with the casted leg. Subjects were provided
with crutches for proper ambulation. All subjects
were instructed to perform a series of simple ankle
exercises (i.e. plantar and dorsal flexion, and circular
movements of the entire foot) to keep the calf muscle
pump activated in the immobilized leg, thereby mini-
mizing the risk of developing deep vein thrombosis.
Subjects remained in the cast for the duration of the
period but those in the group immobilized for 14 days
were required to visit the Casting Room after 7 days
to have the cast tightened. At the end of the immobi-
lization period, subjects were collected from their
home by car and brought into the laboratory for the
second test day at 08.00 h. Prior to the start of the
second test day, subjects visited the Casting Room to
have the cast removed. Thereafter, subjects were
taken by wheelchair to the laboratory, so the muscle
biopsies could be collected prior to any weight
bearing exercise being performed. Following biopsy
collection, subjects were able to bear weight on the
immobilized leg for approx. 12 h prior to strength
testing to minimize deficits due to initial joint
stiffness.
Immunohistochemistry
Frozen muscle biopsies were cut into 5-lm-thick cryo-
sections using a cryostat at 20 C, and thaw
mounted on uncoated pre-cleaned glass slides. Samples
from pre- and post-immobilization were mounted
together on the same glass slide. Care was taken to
properly align the samples for cross-sectional fibre
analyses. Muscle biopsies were stained for muscle fibre
typing (FT). First antibodies were directed against
laminin (polyclonal rabbit antilaminin, dilution 1:50;
Acta Physiol 2014, 210, 600611
Sigma, Zwijndrecht, the Netherlands) and myosin
heavy chain (MHC)-I (A4.840, dilution 1:25; Devel-
opmental Studies Hybridoma Bank, Iowa City, IA,
USA). Appropriate secondary antibodies were applied:
goat anti-rabbit IgG AlexaFluor647 and goat anti-
mouse IgM AlexaFluor555 (dilution 1:400 and 1:500,
respectively; Molecular Probes, Invitrogen, Breda, the
Netherlands). All primary and secondary antibodies
were diluted in 0.1% bovine serum albumin (BSA) in
0.1% Tweenphosphate-buffered saline (PBS). All
incubations took place at room temperature, unless
otherwise stated. Staining procedures were as follows.
After fixation (5 min acetone), slides were air-dried
and incubated at room temperature for 30 min with
3% BSA in 0.1% TweenPBS. Slides were then
washed (5 min PBS). Thereafter, primary antibodies
against laminin and MHC-I were applied for 45 min.
Slides were then washed and incubated with the appro-
priate secondary antibodies. After a final washing step,
all slides were mounted with cover glasses using Mowi-
ol (Calbiochem, Amsterdam, the Netherlands).
Images were visualized and automatically captured
at 109 magnification with a fluorescent microscope
equipped with an automatic stage (IX81 motorized
inverted microscope; Olympus, Hamburg, Germany)
EXi Aqua CCD camera (Q Imaging, Surrey, Canada).
Micromanager 1.4 software was used for image acqui-
sition (Edelstein et al. 2010). Quantitative analyses
were carried out using Image J software package (ver-
sion 1.45d, National Institute of Health; Strandberg
et al. 2010). All image recordings and analyses were
performed by an investigator blinded to subject cod-
ing. Mean muscle fibre size was calculated for the type
I and type II muscle fibres separately. As a measure of
fibre circularity, form factors were calculated by using
the following formula: (4p CSA)/(perimeter)2. No dif-
ferences in fibre circularity were observed over time or
between groups (data not shown).
rtPCR
Total RNA was isolated from 10 to 20 mg of frozen
muscle tissue using TRIzol Reagent (Life Technolo-
gies, Invitrogen, Bleiswijk, the Netherlands), according
to the manufacturers protocol. Total RNA quantifica-
tion was carried out spectrophotometrically at
260 nm (NanoDrop ND-1000 Spectrophotometer;
Thermo Fisher Scientific, Waltham, MA, USA), and
RNA purity was determined as the ratio of readings
at 260/280 nm. Thereafter, first-strand cDNA was
synthesized from 1 lg RNA sample using iScriptTM
cDNA synthesis kit (BioRad, Veenendaal, the Nether-
lands; cat. 170-8891) in a reaction volume of 20 lL.
Taqman PCR was carried out using a 7300 real-time
PCR System (Applied Biosystems, Foster City, CA,
2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190
B T Wall et al. Short-term muscle disuse atrophy
USA), with 2 lL of cDNA, 12.5 lL TaqmanTM master
mix, 1.25 lL TaqmanTM probe and 9.25 lL H2O in a
25 lL final well volume. Each sample was run in
duplicate, together with a serial dilution standard
curve. The housekeeping gene 18S was used as an
internal control as this gene has been used previously
in similar human immobilization studies (Jones et al.
2004, Chen et al. 2007, Gustafsson et al. 2010) and
was unaffected by immobilization (i.e. mean Ct values
were unaffected over time during each assay: example,
Ct values pre- and post-immobilization were
8.99  0.08 and 9.04  0.05; P = 0.53, for the 5 day
group: 9.46  0.10 and 9.52  0.12; P = 0.56 for the
14 day group). Taqman primer/probe sets were
obtained from Applied Biosystems: myogenin, MyoD,
myostatin, MAFBx, MuRF1, FOXO1, mTOR,
P70S6K, citrate synthase, PGC1a, FAK, PAT1, LAT1
and 18S. The thermal cycling conditions used were as
follows: 2 min at 50 C, 10 min at 95 C, followed
by 40 cycles at 95 C for 15 s and 60 C for 1 min.
Ct values of the target genes were normalized to Ct
values of the internal control, and final results were
calculated as relative expression against the standard
curve. The Ct values of all genes of interest were
always within the lower and upper boundaries of the
standard curve.
Western blotting
A portion of each muscle sample frozen for biochemi-
cal analyses was homogenized in 14 volumes Tris buf-
fer (20 mM TrisHCL, 5 mM EDTA. Ten mill molar
Na-pyrophosphate, 100 mM NaF, 2 mM Na3VO4,
1% Nonident P-40; pH 7.4) supplemented with the
following protease and phosphatase inhibitors: aproti-
nin 10 lg mL 1, leupeptin 10 lg mL 1, benzamidin
3 mM and PMSF 1 mM. After homogenization, each
muscle extract was centrifuged for 5 min at 10 000 g
(4 C), and sample buffer was added to the superna-
tant to final concentrations of 60 mM Tris, 5% glyc-
erol, 20 mg mL 1 SDS, 0.1 mM DTT, 20 lg mL 1
bromophenol blue. The supernatant was then heated
for 5 min at 100 C and immediately placed on ice.
Immediately before analyses, the muscle extraction
sample was warmed to 50 C and centrifuged for
1 min at 3000 g (RT). Total amount of sample loaded
on the gel was based on weight (1.0 mg per lane).
Protein samples were run on a criterion any kDa gel
(Biorad Order No. 567-1124) for 10 min at 50 V
(constant voltage) and  90 min at 150 V (constant
voltage) and transferred onto a Trans-blot Turbo
0.2 lm nitrocellulose membrane (Biorad Order No.
170-4159) in 7 min. at 2.5 A and 25 V. Specific pro-
teins were detected by overnight incubation at 4 C
on a shaker with specific antibodies in 50% in PBS
603
Short-term muscle disuse atrophy B T Wall et al.
Odessey blocking buffer (Li-Cor Biosciences Part No.
927-40000) after blocking for 60 min at RT in 50%
in PBS Odessey blocking buffer. The antibodies used
in this study were antimyostatin (52 and 26 kDa; rab-
bit polyclonal IgG, (Santa Cruz Biotechnology, Dallas,
TX, USA), sc-6885-R; dilution 1/500 in 50% Odessey
blocking buffer), anti-myoD (37 kDa; rabbit poly-
clonal IgG, Santa Cruz, sc-760; dilution 1/1000 in
50% Odessey blocking buffer), antimyogenin
(34 kDa; rabbit polyclonal IgG, Santa Cruz, sc-576;
dilution 1/500 in 50% Odessey blocking buffer) and
anti-a-actin (42 kDa; mouse monoclonal IgM, Sigma
A2172; dilution 1/10 000 in 50% Odessey blocking
buffer). Following incubation membranes were washed
three times 10 min in 0.1% PBSTween-20 and 1 time
10 min. with PBS. Next, samples were incubated (1 h
at RT) with infrared secondary antibodies, donkey anti-
rabbit IRDYE 680 (Li-Cor, Cat. No. 926-32223, dilu-
tion 1:10000) and donkey anti-mouse IRDYE 800CW
(Li-Cor, Cat. No. 926-32212, dilution 1:10000) dis-
solved in 50% in PBS Odessey blocking buffer. After a
final wash step (3 9 5 min) in 0.1% Tween-20PBS
and 1 time 10 min. with PBS, protein quantification
was performed by scanning on an Odyssey Infrared
Imaging System (LI-COR Biotechnology, Lincoln, NE,
USA). Values for target proteins were adjusted for total
a-actin content as the protein levels of this housekeeper
were not altered by either 5 (from 20.24  3.06 to
17.30  2.18 arbitrary units; P = 0.39) or 14 days of
immobilization (from 15.69  3.36 to 15.98  3.17
arbitrary units; P = 0.90).
Statistics
All data are expressed as means  SEM. A two-way
ANOVA with time (pre and post) and treatment (5 and
14 days) as factors was used to compare differences in
all time-dependent parameters. When a significant
main effect was detected, a Bonferonni post hoc test
was applied to locate these differences. For non-time-
dependent variables, a paired t-test was used to com-
pare means. Statistical significance was set at
P < 0.05. All calculations were performed by using
GraphPad Prism version 5.0 for Windows (GraphPad
Software, San Diego, CA, USA).
Results
Participants
Subjects characteristics in the 5 day immobilization
group (body mass; 76.0  4.2 kg, BMI; 22.5 
1.2 kg m 2, body fat; 15.6  1.4%, lean body mass;
60.0  2.7 kg and blood HbA1c; 5.1  0.1%) did not
differ from those in the 14 day immobilization group
604 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190
Acta Physiol 2014, 210, 600611
(body mass 82.5  3.1 kg, BMI; 25.0  1.1 kg m 2,
body fat; 20.0  2.1%, lean body mass; 63.5  2.7 kg,
and blood HbA1c; 5.7  0.3%). No changes in any of
these parameters were observed during the experimental
periods (data not shown).
Muscle mass, strength and muscle fibre cross-sectional
area
Measurements of muscle mass and strength are dis-
played in Figure 1. Quadriceps cross-sectional area
(CSA) determined by CT scan (a) did not differ between
groups at baseline, but significantly declined by
3.5  0.5% (from 7504  342 to 7238  324 mm2;
P < 0.001) and 8.4  2.8% (from 7666  382 to
7022  375 mm2; P < 0.001) following 5 and 14 days
of immobilization respectively. A significantly greater
amount of muscle mass was lost following 14 days when
compared with 5 days of immobilization (significant
interaction; P < 0.05). Interestingly, when taking the m
rectus femoris as an individual muscle, no atrophy was
present in either the 5 (from 745  55 to 737 
52 mm2; P > 0.05) or 14 (from 598  34 to 659  56
mm2; P > 0.05) day groups. Leg lean mass, as deter-
mined by DEXA (b), did not differ between groups at
baseline and declined over time in both groups (effect of
time; P < 0.001) by 1.4  0.7 (from 10133  425 to
9986  411 g; P = 0.66) and 3.1  0.7% (1187 
452 to 10841  450 g; P < 0.01) following 5 and
14 days of immobilization respectively. Whole body
lean and fat mass did not change throughout the experi-
ment in either group.
Muscle strength (c) did not differ between groups at
baseline, but declines of 9.0  2.3 (from 77.9  3.9
to 71.1  4.1 kg; P < 0.001) and 22.9  2.6% (from
73.8  4.2 to 55.9  3.6 kg; P < 0.0001) were
observed following 5 and 14 days of immobilization
respectively (effect of time; P < 0.001). A significantly
greater loss in muscle strength was experienced by
volunteers following 14 vs. 5 days of immobilization
(interaction effect; P < 0.001).
Type I and II muscle fibre CSA of biopsies collected
before and after immobilization in both groups are
displayed in Figure 2. Type I muscle fibre CSA was
significantly greater at baseline in the 14 day com-
pared with 5 day immobilization group (P < 0.05).
Limb immobilization resulted in a significant decrease
in type I muscle fibre CSA after 14 days of immobili-
zation (7  3% decline; P < 0.05) but not after
5 days of muscle disuse (pre: 5259  328 lm2, post:
5404  404 lm2; P > 0.05). Type II muscle fibre CSA
did not differ between groups at baseline (P = 0.11)
and declined over time in both groups (significant
effect of time; P < 0.05), reaching statistical signifi-
cance after 14 days of immobilization only
Acta Physiol 2014, 210, 600611 B T Wall et al. Short-term muscle disuse atrophy
(a) 0 10 000 (a)

Type I muscle fiber CSA (m2)

loss of quadriceps CSA (%)

8000
*
5 ***
6000

4000
10
*** 2000

0
15

(b) 0 10 000
(b)

Type II muscle fiber CSA (m2)

loss of leg lean mass (%)

8000
1
*
6000
2

4000
3

2000
4
**
0
Pre Post Pre Post
5
5 days 14 days
(c) 0
Figure 2 Mean ( SEM) type I (a) and type II (b) muscle
fibre cross-sectional area (CSA) before (pre) and after (post)
loss of strength (%)

either 5 (n = 12) or 14 days (n = 12) of one-legged knee

10 immobilization in healthy, young men. Data were analysed
*** using with a two-way ANOVA (time 9 treatment). In case of a
significant main effect, Bonferroni post hoc tests were applied
to locate the individual differences. (a) Significant treatment
20
(P < 0.01) effect. (b) Significant time (P < 0.01) effect.
5 days *P < 0.05 compared with corresponding pre-immobilization
14 days *** values.
30

Figure 1 Mean ( SEM) percentage loss of quadriceps cross-

sectional area (CSA; a), lean leg mass (b) and single-leg 1 repe-
tition maximum strength (1-RM; c) following either 5 (n = 12)
or 14 days (n = 12) of one-legged knee immobilization in
healthy, young men. Data were analysed using absolute values
pre- and post-immobilization in both groups with a two-way
ANOVA (time 9 treatment). In case of a significant interaction
effect, Bonferroni post hoc tests were applied to locate the indi-
vidual differences. (a) Significant time (P < 0.001) and interac-
tion (P < 0.05) effects. (b) Significant time (P < 0.001) effect.
(c) Significant time (P < 0.001) and interaction (P < 0.001)
effects. ** P < 0.01 compared with pre-immobilization and
***P < 0.001 compared with pre-immobilization.
(7735  463 to 6619  311; 13  4% decline;
P < 0.05).
At baseline, muscle fibre type composition showed
43  3 and 57  3% type I and II muscle fibre,
respectively, in the 5 day group, and 35  3 and
65  3%, respectively, in the 14-day group. No dif-
ferences were observed between groups. There was a
significant time effect (P < 0.01) for a greater type I
muscle fibre proportion following immobilization with
10  7% (P = 0.10) and 26  12% (P < 0.05) more
type I muscle fibres following 5 and 14 days of immo-
bilization, respectively.
Skeletal muscle mRNA expression and protein content
Figure 3 depicts the skeletal muscle mRNA expression
and protein content of myostatin, myogenin and
MyoD. Muscle myostatin mRNA expression (a)
increased significantly by 68 and 54% after 5
(P < 0.05) or 14 (P < 0.01) days of immobilization,
respectively. While myostatin (52 kD isoform) protein
content (b) remained unaltered in the 5-day group,
14 days of limb immobilization led to a 32% reduc-
tion (P < 0.05). No significant differences were
observed between groups or over time in muscle
protein content of the 26 kD isoform of myostatin (b;
inset). Muscle myogenin mRNA expression (c)

2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190 605
Short-term muscle disuse atrophy B T Wall et al. Acta Physiol 2014, 210, 600611

Myostatin Myogenin MyoD

Relative mRNA expression

3 15 20
(a) (c) (e)
**
15
2 10 ** *
*
10

1 5
5

0 0 0

0.8
PRE POST PRE POST
2.5 0.8 PRE POST 0.4 (f) 5 days
(b) 52 KD
(d)
Protein content (AU)

0.6
Protein content (AU)

0.4
14 days
2.0
26 KD
0.2 0.6 *** 0.3
0.0
1.5 PRE POST PRE POST

0.4 0.2
1.0
*
0.2 0.1
0.5

0.0 0.0 0.0

PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST

Figure 3 Skeletal muscle mRNA expression and protein content of myostatin (a and b, respectively), myogenin (c and d, respec-
tively) and myoD (e and f, respectively) before (PRE) and after (POST) either 5 (n = 12) or 14 days (n = 12) of one-legged knee
immobilization in healthy, young men. (b) represents the 52 (main) and 26 kD (inset) isoforms of myostatin. Data were analysed
with a two-way ANOVA (time 9 treatment). In case of a significant main effect, Bonferroni post hoc tests were applied to locate
the individual differences. (a) Significant time (P < 0.001) and treatment (P < 0.05) effects. (b) Significant interaction (P < 0.05)
effect. (c) Significant time (P < 0.001) effect. (d) Significant time (P < 0.001) and interaction (P < 0.01) effects. (e) Significant
treatment (P < 0.05) effect. (f) No significant effects were detected. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with
corresponding PRE value.

increased significantly by 96 and 64% following 5

Discussion
(P < 0.01) or 14 (P < 0.05) days of immobilization,
respectively. However, the muscle protein content of The principal novel finding of the present study is that
myogenin (d) increased only in the 14-day group by skeletal muscle mass already declines substantially
51% (P < 0.001) and did not change in the 5 day after only 5 days of limb immobilization. Moreover,
group. Muscle myoD mRNA expression (e) and pro- we provide evidence for a divergent molecular signal-
tein content (f) did not differ between groups and ling response within skeletal muscle tissue regarding
seemed to remain unaffected by either duration of the expression of myostatin and components of the
immobilization. ubiquitinproteasome pathway during the early
The skeletal muscle mRNA expression data for (5 days) compared with the later stages (14 days) of
selected genes implicated in the regulation of muscle muscle disuse. These findings underline the clinical
mass, amino acid metabolism or oxidative metabolism relevance of striving to prevent or minimize muscle
are displayed in Figure 4. Muscle MAFBx mRNA loss even during short periods of bed rest or immobili-
expression (a) increased by 48 and 40% following 5 zation due to illness or injury.
(P < 0.01) or 14 (P < 0.05) days of immobilization, The multitude of negative consequences that are
respectively, while muscle MuRF1 (b) mRNA expres- brought about by the loss of skeletal muscle tissue fol-
sion increased (56%) only in the 5-day group. Muscle lowing a period of bed rest or limb immobilization
mRNA expressions of FOXO1 (c), mTOR (d), LAT1/ (Wall & van Loon 2012) make muscle disuse atrophy
SLC (f), PAT1 (g) and FAK (j) were not altered by an important area for scientific research and clinical
either duration of immobilization. Muscle mRNA evaluation. Previous studies have demonstrated that
expression of P70S6K (e) increased modestly (18%; muscle loss during limb immobilization generally
P < 0.05) following 5 but not 14 days of immobiliza- occurs at a rate of 0.50.6% per day (Wall & van Loon
tion. PGC1a (h) and citrate synthase (i) muscle mRNA 2012). In keeping with this, in the present study, we
expression declined by 73 (P < 0.01) and 33% report that 14 days of limb immobilization led to an
(P < 0.01) in the 5-day group, and 72 (P < 0.01) and approx. 8.5% loss of quadriceps cross-sectional area
31% (P < 0.01) in the 14-day group, both respectively. (CSA; Fig. 1a), representing approx. 350 g lean tissue

606 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190
Acta Physiol 2014, 210, 600611 B T Wall et al. Short-term muscle disuse atrophy
MAFBx MuRF1 FOXO1 mTOR P70S6K
Relative mRNA expression

2 5 days (a) 5 (b) 6 (c) 2 (d) 1.0 (e)

14 days
4 * 0.8 *
** 4
* 3 0.6
1 1
2 0.4
2
1 0.2

0 0 0 0 0.0
LAT1/SLC PAT1 PGC1 Citrate synthase FAK
Relative mRNA expression

5 (f) 3 (g) 3 (h) 1.5 (i) 5 (j)

4 4
2 2 1.0
3 3
** **
2 2
1 1 0.5
1 ** ** 1

0 0 0 0.0 0
PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST

Figure 4 Skeletal muscle mRNA expression of MAFBx (a), MuRF1 (b), FOXO1 (c), mTOR (d), P70S6K (e), LAT1/SLC (f),
PAT1 (g), PGC1a (h), citrate synthase (i) and FAK (j) before (PRE) and after (POST) either 5 (n = 12) or 14 days (n = 12) of
one-legged knee immobilization in healthy, young men. Data were analysed with a two-way ANOVA (time x treatment). In case
of a significant main effect, Bonferroni post hoc tests were applied to locate the individual differences. (a) Significant time
(P < 0.001) effect. (b) Significant treatment (P < 0.01) effect. (c) No significant effects. (d) No significant effects. (e) Significant
time (P < 0.05) effect. (f) No significant effects. (g) No significant effects. (h) Significant time (P < 0.001) effect. (i) Significant
time (P < 0.001) effect. (j) Significant time (P < 0.05) effect. *P < 0.05, **P < 0.01.

lost (Fig. 1b). This considerable muscle atrophy was mass lost from a single leg after merely 5 days of dis-
accompanied by an approx. 25% loss of muscle use (Fig. 1b). Our data are in line with a previous
strength (Fig. 1c) following 14 days of immobilization, short-term disuse study which reported a 3% loss of
underlining the detrimental impact of prolonged disuse thigh muscle mass assessed by MRI after 7 days of
on functional capacity. On a muscle fibre level, we bed rest in young men (Ferrando et al. 1995). We
report that 14 days of immobilization led to a clear were unable to detect any significant changes in mus-
atrophy of both type I and type II muscle fibres (Fig. 2), cle fibre size from the collected muscle biopsies fol-
with the latter being quantitatively most affected. These lowing 5 days of immobilization. This is not
findings are in line with previous studies demonstrating surprising given the considerable inter- and intra-sub-
disuse atrophy in both fibre types (Veldhuizen et al. ject variation in muscle fibre size (Nilwik et al. 2013).
1993, Bamman et al. 1998, Yasuda et al. 2005, Hvid In fact, decreases in muscle mass likely need to exceed
et al. 2010), with type II muscle fibres being the most approx. 6% before specific muscle fibre atrophy can
susceptible (Hvid et al. 2010). be detected using immunohistochemistry in combina-
Innovations within healthcare systems have reduced tion with fluorescence microscopy (Nilwik et al.
the duration that patients typically remain hospital- 2013). It should be noted that a recent study was able
ized, such that 56 days are now the average stay for to show an approx. 8 and 12% decline in type I and
an elderly person admitted with acute illness (Fisher II muscle fibre CSA, respectively, following only
et al. 2010). However, to what extent such a short 4 days of immobilization (Suetta et al. 2012). The rea-
period of disuse may impact upon skeletal muscle son(s) for the apparent discrepancy between this and
mass has not yet been evaluated. This is the first study the present study is unclear, but could relate to a dif-
to show that only 5 days of limb immobilization fering immobilization protocol (knee brace vs. casting)
already induces a 3.5% decline in quadriceps muscle or less variability in the data. Importantly, the loss of
CSA in vivo in humans (Fig. 1a). We assessed muscle muscle mass following 5 days of immobilization led
mass of the quadriceps as an entire unit due to its to an even greater (relative) decline in leg muscle
important role in numerous daily functions. However, strength of approx. 9% (Fig. 1c), suggesting that
it is also true that the 3.5% observable atrophy may already compromised patients could easily fall below
represent an underestimate of the muscles more spe- a critical threshold with respect to carrying out nor-
cific to knee extension, as the m rectus femoris did mal daily activities following only a few days of illness
not atrophy in either group. The lack of atrophy in or injury. A striking observation of the present study
this specific muscle likely relates to its contribution to was that a substantial amount of the atrophy and loss
hip extension which may have still occurred to a cer- of strength observed after 14 days was already present
tain extent during the immobilization period. Regard- after only 5 days of limb immobilization. As losses of
less, the decline in muscle CSA was accompanied by strength over a more prolonged period seem mostly
our observation of as much as approx. 150 g lean attributable to continued loss of muscle mass (Camp-

2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190 607
Short-term muscle disuse atrophy B T Wall et al.
bell et al. 2013), this underlines the importance of
intervening as early as possible when a period of dis-
use is unavoidable.
While muscle atrophy during prolonged disuse is
generally attributed to declines in muscle protein syn-
thesis rates (Gibson et al. 1987, Ferrando et al. 1996,
Glover et al. 2008, Wall & van Loon 2012), evidence
also suggests that a rapid increase in muscle protein
breakdown during the first few days of disuse may also
contribute to overall muscle loss (Urso et al. 2006,
Chen et al. 2007, Tesch et al. 2008, Gustafsson et al.
2010, Wall & van Loon 2012, Wall et al. 2013).
Myostatin, a proposed master negative regulator of
skeletal muscle mass in animals (McPherron & Lee
1997, Mosher et al. 2007) and humans (Schuelke et al.
2004), acts via the inhibition of mammalian target of
rapamycin (mTOR) muscle protein synthesis signalling
(Rodriguez et al. 2011) and by initiating muscle protein
breakdown through the ubiquitinproteasome system
(McFarlane et al. 2006). Consistent with previous ani-
mal (Murphy et al. 2011) and human (Jones et al.
2004, Gustafsson et al. 2010, Bunn et al. 2011) disuse
studies, we report a clear up-regulation of myostatin
gene expression in response to 5 and 14 days of limb
immobilization (Fig. 3). In parallel, we observed a pro-
found increase in gene expression of the two primary
ubiquitin ligases, MAFBx and MuRF1 (Murton et al.
2008), after 5 days of immobilization, which became
less striking after 14 days (Fig. 4). These data extend
on previous findings (Urso et al. 2006, Chen et al.
2007, Abadi et al. 2009, Glover et al. 2010, Gustafs-
son et al. 2010) by suggesting that an early and tran-
sient increase in ubiquitinproteasome-mediated
muscle protein breakdown, possibly mediated via
increased myostatin transcription, contributes to mus-
cle disuse atrophy during the first few days of disuse. In
support, it has previously been shown that antibody
directed inhibition of endogenous myostatin is capable
of attenuating muscle atrophy in the short- but not
long-term phase of hind-limb unloading in rodents
(Murphy et al. 2011). However, it should be noted that
we were unable to demonstrate that increased myosta-
tin transcription led to the successful translation of
either the (52 kD) precursor or purported biologically
active (26 kD) isoforms of the protein. Clearly, more
research is warranted to directly assess muscle protein
breakdown rates in combination with myostatin signal-
ling during (short-term) disuse. Such information will
allow us to better define their contribution to muscle
disuse atrophy and assess whether myostatin (Elliott
et al. 2012) and/or proteasome inhibition (Hollinger &
Selsby 2013) are viable future targets for attenuating
muscle disuse atrophy in humans.
Myostatin also plays a key role in myogenesis
through the inhibition of the myogenic regulatory fac-
608 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12190
Acta Physiol 2014, 210, 600611
tors, most notably myogenin and MyoD (Amthor
et al. 2002, Muroya et al. 2009, Krause et al. 2013).
Here, we provide novel evidence that the up-regula-
tion of myostatin transcription does not translate into
increased protein levels during limb immobilization.
In fact, after 14 days of limb immobilization, myosta-
tin protein content (52 kD isoform) had significantly
declined by 32% (Fig. 3). We extend on previous
work with the observation that myogenin mRNA
expression, considered mandatory for satellite cell dif-
ferentiation and therefore myogenesis, increased after
5 and 14 days of immobilization, which translated
into greater protein content after 14 days of immobili-
zation (Fig. 3). The present findings imply that pro-
longed disuse down-regulates myostatin at the
functional level (i.e. protein) and thereby facilitates
the successful translation of myogenin, possibly in
anticipation of a forthcoming anabolic stimulus (i.e.
re-ambulation). Therefore, it could be speculated that
the regulation of myogenesis/satellite cells via myosta-
tin and the myogenic regulatory factors may be of
more physiological relevance in the regeneration of
muscle mass (rather than in muscle atrophy) following
more prolonged periods of disuse. As such, a disuse
induced priming of the myogenic machinery for an
anabolic stimulus may explain the rapid rate of mus-
cle growth generally observed in healthy individuals
recovering from disuse atrophy (Hespel et al. 2001,
Jones et al. 2004). This has important clinical rele-
vance when considering the design of rehabilitative
programmes to improve muscle mass and function
following disuse in various populations.
Concerning our measures of gene expression, an
interesting consideration is the impact of disuse on
total ribosome content and RNA abundance. It has
been shown that limb disuse (Gamrin et al. 1998) and
denervation (Machida et al. 2012) can result in a
reduction in both de novo ribosomal RNA synthesis
and global (ribosomal) RNA content. Although we
did not make these measurements in the present
study, we did not observe any differences in total
RNA quality obtained from samples pre- and post-
immobilization nor did we see any changes over time
in our housekeeping gene (18S). Moreover, any such
changes in global RNA abundance would only make
the elevated expression of the muscle atrogenes, myo-
statin and myogenic regulatory factors all the more
striking.
The present study illustrates the impact of only a few
days of disuse on skeletal muscle mass and strength.
Extending on this, we also report a decline in citrate syn-
thase and PGC1a gene expression after only 5 days of
immobilization (Fig. 4), suggesting that a decline in oxi-
dative capacity and/or insulin sensitivity may also occur
after only a few days of disuse. Thus, in clinically
Acta Physiol 2014, 210, 600611
compromised populations (e.g. hospitalized elderly, crit-
ically ill, hip-fracture or acute illness patients), just a
few days of bed rest or immobilization can likely already
compromise functional capacity and metabolic health.
Such patient groups are particularly vulnerable to atro-
phy during disuse as the presence of illness-related stress
responses is known to accelerate muscle loss during dis-
use (Ferrando et al. 1999, Fitts et al. 2007). Further-
more, short successive periods of muscle disuse atrophy
throughout the lifespan will contribute significantly to
the age-related loss of muscle mass and strength, and
muscle quality (English & Paddon-Jones 2010, Mithal
et al. 2012, Wall & van Loon 2012, Wall et al. 2013).
Together with the observation that we seem to lose our
ability to regain lost muscle tissue as we age (Hvid et al.
2010), these data support the concept that successive
short periods of bed rest or immobilization throughout
the lifespan can contribute substantially to the develop-
ment of sarcopenia and decline in metabolic health. As a
whole, our data underline the importance of understand-
ing the response to muscle disuse, both in terms of
molecular signalling as well as physiological/functional
changes, if we are to develop effective nutritional, exer-
cise and/or pharmacological interventions aimed at
attenuating muscle disuse atrophy during short periods
of bed rest or immobilization.
In conclusion, limb immobilization leads to a rapid
loss of skeletal muscle mass and strength, with sub-
stantial atrophy already occurring during the first
5 days of muscle disuse. These data imply that succes-
sive short periods of muscle disuse, due to illness or
injury, can be of important clinical relevance and,
therefore, effective interventional strategies should be
designed to prevent muscle atrophy during such short
periods of muscle disuse.
Conflict of interest
None of the authors had a personal or financial con-
flict of interest.
BTW and LJCvL designed the study. BTW, MLD and TS
organized and carried out the clinical experiments. BTW and
JMGS performed the Western blot analyses. BTW performed
the real-time PCR analyses. JD applied the leg casts, and
BTW performed the statistical analysis of the data and wrote
the manuscript together with LJCvL. All the authors contrib-
uted to the interpretation of the data and approved the final
version of the manuscript.
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