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J Physiol 591.15 (2013) pp 3789–3804
Ageing is associated with diminished muscle re-growth
and myogenic precursor cell expansion early after
immobility-induced atrophy in human skeletal muscle
C. Suetta1,2 , U. Frandsen3 , A. L. Mackey1 , L. Jensen3 , L. G. Hvid3 , M. L. Bayer1 , S. J. Petersson4 ,
H. D. Schrøder4 , J. L. Andersen1 , P. Aagaard3 , P. Schjerling1 and M. Kjaer1
1
The Journal of Physiology
Institute of Sports Medicine and Center of Healthy Ageing, Faculty of Health and Medical Sciences, University of Copenhagen, Bispebjerg Hospital,
Denmark
2
Department of Clinical Physiology and Nuclear Medicine, Glostrup Hospital, University of Copenhagen, Denmark
3
Institute of Exercise Physiology and Clinical Biomechanics, University of Southern Denmark, Denmark
4
Department of Pathology, Odense University Hospital, Odense, Denmark
Key points
• Elderly individuals require a prolonged recovery phase in order to return to initial muscle mass
levels following short-term immobilisation.
• The cellular mechanisms responsible for the attenuated re-growth and associated molecular
signalling processes in ageing human skeletal muscle are not fully understood.
• The main study finding was the observation of a less marked muscle mass recovery after
immobilisation in elderly compared to young individuals that was paralleled by an elevation
in myogenic precursor cell content in young individuals only, whereas the elderly failed to
demonstrate any change in myogenic precursor cells.
• No age-related differences were observed in the expression of major myogenic regulating factors
known to promote skeletal muscle hypertrophy or satellite cell proliferation (IGF-1Ea, MGF,
MyoD1, myogenin, HGF gene products).
• In contrast, the expression of myostatin demonstrated a more pronounced up-regulation
following immobilisation along with an attenuated down-regulation in response to reloading
in older compared to young individuals, which may have contributed to the present lack of
satellite cell proliferation in ageing muscle.
Abstract Recovery of skeletal muscle mass from immobilisation-induced atrophy is faster in
young than older individuals, yet the cellular mechanisms remain unknown. We examined the
cellular and molecular regulation of muscle recovery in young and older human subjects subsequent to 2 weeks of immobility-induced muscle atrophy. Retraining consisted of 4 weeks of
supervised resistive exercise in 9 older (OM: mean age) 67.3, range 61–74 yrs) and 11 young
(YM: mean age 24.4, range 21–30 yrs) males. Measures of myofibre area (MFA), Pax7-positive
satellite cells (SCs) associated with type I and type II muscle fibres, as well as gene expression
analysis of key growth and transcription factors associated with local skeletal muscle milieu, were
performed after 2 weeks immobility (Imm) and following 3 days (+3d) and 4 weeks (+4wks)
of retraining. OM demonstrated no detectable gains in MFA (vastus lateralis muscle) and no
increases in number of Pax7-positive SCs following 4wks retraining, whereas YM increased their
MFA (P < 0.05), number of Pax7-positive cells, and had more Pax7-positive cells per type II fibre
than OM at +3d and +4wks (P < 0.05). No age-related differences were observed in mRNA
C 2013 The Authors. The Journal of Physiology
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DOI: 10.1113/jphysiol.2013.257121
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C. Suetta and others
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expression of IGF-1Ea, MGF, MyoD1 and HGF with retraining, whereas myostatin expression
levels were more down-regulated in YM compared to OM at +3d (P < 0.05). In conclusion, the
diminished muscle re-growth after immobilisation in elderly humans was associated with a lesser
response in satellite cell proliferation in combination with an age-specific regulation of myostatin.
In contrast, expression of local growth factors did not seem to explain the age-related difference
in muscle mass recovery.
(Resubmitted 16 April 2013; accepted after revision 29 May 2013; first published online 3 June 2013)
Corresponding author C. Suetta: Department of Clinical Physiology and Nuclear Medicine, Glostrup Hospital,
University of Copenhagen, Ndr. Ringvej 57, 2600 Glostrup, Denmark. Email: csuetta@gmail.com
Abbreviations CDKN1A (p21), cyclin-dependant kinase inhibitor 1A; CDKN1B (p27), cyclin-dependant kinase
inhibitor 1B; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HGF, hepatocyte growth factor;
IGF-1Ea, insulin-like growth factor-1Ea; MFA, mean fibre area; MGF, mechano growth factor; MFA, myofibre area;
OM, older males; RM, repetition maximum; SC, satellite cell; YM, young males.
Introduction
Human skeletal muscle is a highly plastic tissue, which
is reflected by its ability to rapidly adapt to short-term
changes in habitual loading intensity (Hespel et al. 2001;
Jones et al. 2004; Hvid et al. 2011) and it has been
demonstrated that elderly individuals require a prolonged
recovery phase in order to return to initial muscle mass
levels following short-term immobilisation (Suetta et al.
2009; Hvid et al. 2010). Yet, there is a paucity of studies
examining the cellular mechanisms responsible for the
attenuated re-growth and associated molecular signalling
processes in ageing human skeletal muscle.
The regulation of muscle growth and maintenance of
muscle mass are known to be influenced by a unique
population of muscle resident stem cells referred to
as satellite cells (SCs) or myogenic stem cells (Mauro,
1961; Moss & Leblond, 1970; Heslop et al. 2001).
Notably, an impaired response to muscle damage has
been documented as a consequence of ageing in mice
(Conboy et al. 2003) and recently also demonstrated in
human individuals when examining a subpopulation of
individuals from the present intervention (Carlson et al.
2009). As suggested by the latter data, the age-related
impairment in muscle re-growth following disuse could,
at least in part, reside in an impaired capacity for myogenic stem cell proliferation and activation in aged myofibres (Carlson et al. 2009), but it is not known whether
such changes are related to muscle fibre phenotype (type
I vs. type II fibres). Further, systemic factors appear
to play an important role in explaining the impaired
proliferative capacity of SCs cultured from old vs. young
human adults (Carlson et al. 2009) in close accordance
with previous findings using the murine model (Conboy
et al. 2005). There is, however, also evidence of local
mechanisms influencing satellite cell activation (Sheehan
et al. 2000; Horsley & Pavlath, 2003; Lorenzon et al.
2004; Mitchell & Pavlath, 2004) and recent data suggest
a close relation between various systemic and local factors
in the regulation of SC function in vivo (Chakkalakal
et al. 2012). Furthermore, myogenic regulatory factors
such as MyoD and myogenin, the growth and
differentiation factor myostatin, as well as growth factors
like hepatocyte growth factor (HGF), fibroblast growth
factor (FGF) and insulin-like growth factor (IGF-I)
have been shown to be involved in the regulation of
muscle mass with changes in mechanical muscle loading
while also affecting satellite cell activation, proliferation
and differentiation (Mezzogiorno et al. 1993; Adams &
Haddad, 1996; McPherron et al. 1997; McCroskery et al.
2003; Gopinath & Rando, 2008). However, it is not known
to what extent the expression of these factors are associated
with any age-related differences in recovery of muscle
mass after a period of muscle immobilisation. Based on
the previous findings, we hypothesised that satellite cell
proliferation would be impaired especially in relation
to type II myofibres along with a reduced expression
of key anabolic genes in elderly compared to young
individuals during rehabilitation after immobilisation of
skeletal muscle.
Methods
Subjects
Twenty healthy males, 11 young males (YM; 24.4 years,
range 21–30 years) and 9 older males (OM; 67.3 years,
range 61–74 years) volunteered to participate in the
study. Prior to inclusion all subjects were screened by
a physician to exclude individuals with cardiovascular
disease, diabetes, neural or musculoskeletal disease,
inflammatory or pulmonary disorders or any known predisposition to deep venous thrombosis. The local ethics
committee of Copenhagen and Frederiksberg approved
the conditions of the study (KF01-322606) and all
experimental procedures were performed in accordance
with the Declaration of Helsinki. Written informed consent
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Ageing affects human skeletal muscle recovery
was obtained from all participants before inclusion in the
study.
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and the other directly frozen in liquid nitrogen and stored
at −80◦ C until further analyses.
ATPase staining and muscle fibre area
Intervention procedures
The intervention protocol along with data on changes in
muscle contractile function and morphology have been
described previously (Suetta et al. 2009; Hvid et al. 2010).
In brief, immobility was accomplished by 2 weeks of
randomised unilateral whole-leg casting using a lightweight whole-leg fibre cast extending from the malleoli
to the proximal groin region. The retraining protocol
comprised 4 weeks of surveyed and supervised unilateral
strength training for the immobilised leg, with three
sessions performed per week using a protocol consistently
proven effective for inducing substantial gains in muscle
size in elderly individuals with 12 weeks of training in our
laboratory (Esmarck et al. 2001; Lange et al. 2002; Suetta
et al. 2004a,b). After a 5–8 min warm-up on a stationary
bike, subjects performed isolated knee extension and
flexion, and leg press exercises. Each exercise was
performed in 3–4 sets × 12 repetitions (reps) (at 15 rep
maximum (RM)) in week 1, followed by 5 sets × 10 reps
(at 12RM) in weeks 2 and 3, and 4 sets × 10 reps (at 12RM)
in week 4. Training loads were determined at baseline and
loads were progressively adjusted on a weekly basis by use
of 5RM testing.
Muscle biopsy sampling
Muscle biopsies were obtained at ∼1 week prior to
the immobilisation (Pre), immediately after 2 weeks of
immobilisation (Imm), following 3 days of retraining
(+3d) and finally after 28 days of retraining (+4wks). Subjects were prohibited from exercise for at least 2 days before
the first biopsy and were carefully instructed only to eat a
light meal in the morning of the day of biopsy sampling.
Further, to minimise the influence of diurnal variation, all
subjects were biopsied at the same time of the day (±1 h)
at successive test sessions.
As described in detail elsewhere (Suetta et al. 2008;
Hvid et al. 2010) biopsies were obtained from the
middle portion of vastus lateralis muscle utilising the
percutaneous needle biopsy technique of Bergström
(Bergström, 1962), and careful efforts were made to extract
tissue from the same depth and within ∼2–3 cm distance
between each biopsy, to avoid the potential influence
of muscle damage from repeated biopsies (Guerra
et al. 2011). After dissecting the muscle samples of visible
blood, adipose and connective tissue, samples were divided
into two separate pieces, one oriented in embedding
medium (Tissue-Tek, Sakura Finetek, USA) frozen in isopentane cooled with liquid nitrogen and stored at −80◦ C
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Subsequently serial transverse sections (10 μm) were cut
in a cryotome at −20◦ C and stained for myofibrillar
ATPase at pH 9.4 after both alkaline (pH 10.3) and acid
(pH 4.3 and 4.6) preincubations (Brooke & Kaiser, 1970).
All samples of each individual person were stained in the
same batch to avoid interassay variation. Based on the
ATPase staining pattern muscle fibres were characterised
as type I and type II and an average of 213 ± 39 fibres
were analysed in each biopsy. For the determination of
muscle fibre size only truly horizontal fibres were used,
with a minimum of 50 fibres included for the analysis.
A videoscope consisting of a microscope (Olympus BX
50) and colour video camera (Sanyo high resolution
CCD) in combination with Tema Image Analysis System
(Scanbeam, Denmark) were used to determine the mean
fibre area of the muscle fibres.
Satellite cell analysis
SC analysis was carried out by microscopic evaluation
of cryosections that had been immunohistochemically
stained for Pax7, as previously described in detail (Mackey
et al. 2010). A combination of immunoenzymatic and
immunofluorescence methods was employed to allow
the staining of Pax7, type I myosin and laminin on the
same section. Sections were fixed for 5 min with a 5%
formaldehyde solution (Histofix, Histolab, Gothenburg,
Sweden), followed by incubation for 1 h with blocking
buffer (0.05 M Tris-buffered saline (TBS) containing
0.01% Triton X-100, 1% bovine serum albumin, 1%
skimmed milk powder and 0.1% sodium azide). Satellite
cells were labelled with a mouse anti-Pax7 antibody (cat.
no. MO15020; Neuromics, Edina, MN, USA), diluted
1:500 in the blocking buffer, and incubated overnight
at 4◦ C. The next day, the slides were washed in two
changes of TBS containing 0.01% Triton. A biotinylated
goat anti-mouse secondary antibody (cat. no. E0433;
Dako Denmark, Glostrup, Denmark) was then applied,
followed by Vector Elite ABC kit (cat. no. PK6100;
Vector Laboratories, Peterborough, UK). Horseradish
peroxidase activity was visualised with the ImmPACT
diaminobenzidine substrate (cat. no. SK-4105; Vector
yes Laboratories). The sections were then incubated for
2 h at room temperature with a mixture of the two
primary antibodies, mouse anti-A4.951 (cat. no. A4.951;
Developmental Studies Hybridoma Bank, Iowa, IA, USA)
and rabbit anti-laminin (cat. no. Z0098; Dako, Denmark),
for visualisation of type I myosin and laminin, respectively.
A mixture of Alexa Fluor 488 goat anti-rabbit (Molecular
Probes cat. no. A11034; Invitrogen, Taastrup, Denmark)
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C. Suetta and others
and Alexa Fluor 568 goat anti-mouse secondary antibodies (Molecular Probes, cat. no. A11031) was applied
for 45 min. Washing in two changes of TBS was carried
out between all steps, except for between the blocking and
Pax7 incubation steps, where no washing was performed.
4′ ,6-Diamidino-2-phenylindole (DAPI) in the mounting
medium (Molecular Probes ProLong Gold anti-fade
reagent, cat. no. P36935) stained the nuclei, rendering
nuclei blue, type I myosin red and laminin green. Sites
of Pax7 antigenicity were stained brown, visible by light
microscopy. The number of Pax7 cells associated with
type I (A4.951-positive) or type II (A4.951-negative)
fibres was counted separately and expressed relative to
the total number of type I or type II fibres included in
the assessment, as described in detail (Mackey et al. 2010;
Fig. 2).
J Physiol 591.15
quality-check Ct values, assess triplicates, exclude runs
when the difference among triplicates exceeded 0.5Ct and
finally to normalise data to RPLP0 using the 2Ct method
(Livak & Schmittgen, 2001).
Correlation analyses
Correlation analyses were performed at post
immobilisation (Imm) to examine the relationship
between myofibre area (fibre type I and type II) and
number of Pax7-positive (+) cells (total, fibre type I
and type II), for young and older individuals combined.
Furthermore, correlation analyses were performed
between changes during the retraining (4wks relative to
Imm) in myofibre area (MFA, fibre type I and type II)
and number of Pax7+ cells (total, fibre type I and type II)
for young and older individuals combined.
RNA purification
Total RNA was isolated from ∼20 mg of frozen muscle
biopsy by phenol extraction (TriReagent; Molecular
Research Center, OH, USA) as previously described (Kadi
et al. 2004b). Intact RNA was confirmed by denaturing
agarose gel electrophoresis.
Real-time PCR
The mRNA expression of IGF-1Ea, mechano growth
factor (MGF, also known as IGF-1Ec) and RPLP0 was
analysed by real-time PCR as described previously (Suetta
et al. 2012). Total RNA (500 ng) was converted into
cDNA in 20 μl using the OmniScript reverse transcriptase
(Qiagen, CA, USA) according to the manufacturer’s
protocol. For each of the mRNA targets, 0.25 μl cDNA was
amplified in a 25 μl SYBR Green PCR reaction containing
1 × Quantitect SYBR Green Master Mix (Qiagen) and
100 nM of each primer (Table 1A). The amplification was
monitored real-time using the MX3000P real-time PCR
machine (Stratagene, CA, USA). The threshold cycle (Ct)
values were related to a standard curve made with the
cloned PCR products and specificity ensured by melting
curves analysis; the quantities were normalised to RPLP0.
Quantitative real-time PCR of myostatin, Pax7, MyoD1,
myogenin, FGF2, fibroblast growth factor receptor 1
(FGFR1), HGF, c-Met, CDKN1A (p21), CDKN1B (p27)
and RPLP0 mRNA were performed in the ABI Prism
7900HT Sequence Detection System (Applied Biosystems,
UK) using ABI TaqMan Low Density Arrays (Applied
Biosystems; Table 1B). Each sample was run in triplicate
with four samples per card. cDNA, 250 ng, was mixed with
100 μl TaqMan Gene Expression Mastermix and loaded
into two ports (2.5 ng cDNA per reaction). Raw data were
extracted and analysed using the SDS 2.1 software (Applied
Biosystems, UK) and qBasePlus (Biogazelle) was used to
Statistical analyses
Statistical analyses were performed with SigmaPlot v11.0.
Interaction between Age and Time was tested with a
two-way repeated measures ANOVA. Differences over
time were tested with one-way repeated measures ANOVA
for Young and Old separately. If an overall time difference
was found, the different time points were compared
using Student–Newman–Keuls post hoc test. Pair-wise
comparisons between Young and Old at each time point
were obtained from the two-way repeated measures
ANOVA (to include global variance) and Bonferroni
corrected.
Non-parametric statistical analysis was used to evaluate
changes in muscle fibre cross-sectional area, since not all
data were normally distributed. To evaluate the effect of
intervention over time, the Friedmann two-way analysis
of variance by ranks of related samples was used with
subsequent analysis using the Wilcoxon signed rank test
for paired samples. Intergroup differences were evaluated
using the Kruskal–Wallis signed rank test. Correlation
analysis was performed using the Spearman’s rho (r s )
method. Data are presented as mean values ± SEM or for
mRNA data geometric means ± back-transformed SEM.
A P value of less than 0.05 was considered significant.
Results
Muscle fibre cross-sectional area
Young individuals showed a 21.3% increase in type I
fibre area (4440.4 ± 499.9 vs. 5386.3 ± 508.9 μm2 ,
P < 0.05) along with a 35.5% increase in type II
myofibre area (4347.5 ± 419.5 vs. 5904.4 ± 483.3 μm2 ,
P < 0.05) after retraining (+4wks; Fig. 1). In contrast,
no increases in type I fibre area (4830.3 ± 517.5 vs.
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Table 1. Primers for qRT-PCR and TaqMan low density array (LDA) assay ID
A. Primers for qRT-PCR using SYBR Green assay
Gene products
MGF
IGF-1Ea
GAPDH
RPLP0
Sense primer
Antisense primer
GCCCCCATCTACCAACAAGAACAC
GACATGCCCAAGACCCAGAAGGA
CCTCCTGCACCACCAACTGCTT
GGAAACTCTGCATTCTCGCTTCCT
CGGTGGCATGTCACTCTTCACTC
CGGTGGCATGTCACTCTTCACTC
GAGGGGCCATCCACAGTCTTCT
CCAGGACTCGTTTGTACCCGTTG
B. Applied Biosystems TaqMan Low Density Array assay ID
Gene products
Assay ID
MyoD
Myogenin
HGF
c-Met
FGF2
FGFR1
Pax7
Myostatin
CDKN1A (p21)
CDKN1B (p27)
RPLP0
Hs02330075_g1
Hs01072232_m1
Hs00300159_m1
Hs01565581_m1
Hs00266645_m1
Hs01552926_m1
Hs00242962_m1
Hs00193363_m1
Hs01121172_m1
Hs00153277_m1
Hs99999902_m1
A. mRNA expression of MGF (IGF-1Ec), IGF-1Ea and RPLP0 was analysed by SYBR Green-based quantitative real-time RT-PCR. B.
TaqMan-based quantitative real-time RT-PCRs of MyoD1, myogenin, HGF, c-Met, FGF2, FGFR1, Pax7, myostatin, CDKN1A, CDKN1B and
RPLP0 mRNA were performed using ABI TaqMan Low Density Arrays.
4848.2 ± 336.9 μm2 ) or type II fibre area (4004.3 ± 467.4
vs. 4225.1 ± 276.2 μm2 ) were observed in the older
individuals. Furthermore type II fibre area was restored
with retraining in young individuals to reach
higher levels than observed in older individuals
(YM: 5386.3 ± 508.9 μm2 , OM: 4225.1 ± 276.2 μm2 ,
P < 0.05). No difference was observed in fibre-type
distribution between YM (type I: 55.5%; type II: 44.5%)
and OM (type I: 56.1%; type II: 43.9%) at Pre or following
the interventions (Hvid et al. 2010).
Satellite cells: association with fibre type
To analyse for satellite cells, Pax7+ cells were assessed for
type I and II fibres separately, as illustrated in Fig. 2. An
overall effect of age was found for Pax7+ cells in relation
to type II (P < 0.01), but not type I fibres (Fig. 3). The
number of Pax7+ cells in the young subjects increased
compared to Pre in both type I and II fibres at all time
points. No changes in the elderly individuals were seen
over time and in the type II fibres at +3d and +4wks; this
was significantly different to the young individuals.
mRNA expression levels at baseline
At baseline (Pre) there was no age-related differences in
the expression levels of IGF-1Ea, MGF, MyoD1, myogenin, HGF, c-Met, FGF2, FGFR1, Pax7, CDKN1A (p21)
or CDKN1B (p27) whereas the expression levels of myo
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statin mRNA and GAPDH were lower in old compared to
young (Fig. 4, P < 0.05).
IGF-1Ea and MGF
Expression levels of IGF-1Ea and MGF (IGF-1Ec) mRNA
increased in young individuals with immobilisation
(Fig. 5A and B). Expression levels of IGF-1Ea
demonstrated a subsequent marked decrease after 3 days
of retraining in young individuals and after 4 weeks
of retraining mRNA expression levels were increased
compared to baseline levels as well as post immobilisation
(Imm) in both young and old (Fig. 5A, P < 0.05).
Expression levels of MGF mRNA remained elevated
after 3 days of retraining in the young (P < 0.05), and
after 4 weeks of retraining mRNA expression levels
were further increased compared to baseline and post
immobilisation (Imm) levels, respectively, in the young
(Fig. 5B, P < 0.05).
MyoD1 and myogenin
MyoD1 expression increased in both young and older
males with immobilisation, and in addition showed a
marked decrease following 3 days of retraining in both
young and old (P < 0.05, Fig. 5C). After 4 weeks of
retraining MyoD1 mRNA levels returned to baseline
levels in both young and old (Fig. 5C). Myogenin mRNA
expression increased markedly in both young and old,
while decreasing after 3 days of retraining in both young
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and old (Fig. 5D, P < 0.05). After 4 weeks of retraining
myogenin expression levels remained reduced compared
to post immobilisation levels (Fig. 5D).
HGF and c-Met
Expression levels of HGF and c-Met mRNA increased in
young individuals in the early phase of retraining (+3d,
P < 0.05; Fig. 5E and F) but remained unaltered after
immobilisation and more prolonged retraining. In old
individuals no change in expression levels of HGF and
c-Met was observed at any time point.
FGF2 and FGFR1
FGF2 mRNA levels remained unaltered at all time points
in young as well as older individuals (P < 0.05, Fig. 5G).
Conversely, FGFR1 mRNA expression increased in both
young and old following immobilisation, followed by
marked decreases after 3 days of retraining in both young
Type I fiber area
A
Type I fiber area (µm2)
7000
#
6000
Young
Old
*
5000
4000
3000
0
Pre
Imm
Type II fiber area
Type I fiber area (µm2)
#
6000
Young
Old
* *
5000
4000
3000
0
Pre
Imm
and old (+3d; Fig. 5H). After 4 weeks of retraining, FGFR1
mRNA expression increased compared to baseline and the
initial training phase (+3d) approaching values similar to
the expression levels observed following immobilisation
(Fig. 5H).
Pax7
The expression of Pax7 mRNA following immobilisation
was higher in the older males compared to the young males
(Fig. 5I). Although there was an interaction (Age × Time,
P < 0.05), it was not possible to pinpoint whether this
difference was due to an increase in the elderly or a
decrease in the young, or both. No differences with time
or age were detected at the retraining time points, except
for a lower level at +3d compared to +4wks in the
young.
Myostatin
Myostatin mRNA expression increased in the elderly
with immobilisation, and showed a marked decrease
after 3 days of retraining (+3d) in both young and old
(P < 0.05, Fig. 5J). After 4 weeks of retraining, myostatin
expression levels returned to baseline levels in both young
and old. The temporal changes in myostatin mRNA
expression differed between young and old individuals,
with a less pronounced down-regulation of myostatin
expression levels in the initial phase of retraining (+3d)
in older individuals (P < 0.05, Fig. 5J).
CDKN1A (p21) and CDKN1B (p27)
+4wks
B
7000
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+4wks
Figure 1. Changes in myofibre cross-sectional area following
2 weeks of immobilisation and 4 weeks of retraining in young
and older human individuals
A, type I myofibre cross-sectional area. B, type II myofibre
cross-sectional area. Open bars represent young (n = 9) and grey
bars represent older individuals (n = 7). Pre, ∼1 week prior to the
immobilisation; Imm, after 2 weeks of immobilisation; +4wks, after
28 days of retraining. ∗ Time effect, P < 0.05, compared to Pre.
#Time effect, P < 0.05 compared to Imm. ∞Age effect, P < 0.05
young compared to old within time point. Group mean data ± SEM.
Expression levels of CDKN1A (p21) remained unchanged
with immobilisation (Imm) while decreasing in both
young and older males after 3 days of retraining (P < 0.05;
Fig. 5K ). After 4 weeks of retraining CDKN1A mRNA
expression returned to baseline levels in both young and
old (P < 0.05, Fig. 5K ). No change in the expression level
of CDKN1B (p27) was observed with immobilisation;
however, a marked decrease was observed in both young
and old after 3 days of retraining (P < 0.05, Fig. 5L).
After 4 weeks of retraining CDKN1B mRNA expression
levels returned to baseline levels in both young and older
individuals (Fig. 5L).
Correlation analysis
After muscle disuse, the number of Pax7+ cells was
positively related to myofibre area (r s = 0.712, P < 0.01;
n = 16; Fig. 6A). Comparison within fibre type showed
that this relationship was true for both type I (r s = 0.779;
P < 0.001; n = 16) and type II fibres (r s = 0.694; P < 0.01;
n = 16). Notably, analysing the retraining phase, relative
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Ageing affects human skeletal muscle recovery
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changes in MFA following 4 weeks retraining (relative
to post immobilisation) were positively associated
with the change in the total number of Pax7+
SCs (r s = 0.693, P < 0.01, n = 15; Fig. 6B). Comparison
within each fibre type revealed a strong correlation
for type I fibres (r s = 0.864, P < 0.001, n = 15), whereas
there was no significant correlation for type II fibres
(Fig. 6B).
Discussion
In the present study, we report measures of myofibre
area and myogenic stem cell number associated with
type I and type II muscle fibres in young and older
humans, in combination with transcriptional data from
regulatory signalling pathways related to skeletal muscle
re-growth, many of which have not been previously
studied in the immobilised and retrained ageing human
muscle. The main study finding was the observation of a
less marked muscle mass recovery after immobilisation
in elderly compared to young individuals that was
paralleled by an elevation in Pax7+ satellite cell content
in young individuals only, whereas the elderly failed to
demonstrate any change in Pax7+ cells. This potential
coupling of satellite cell proliferation and recovery in
myofibre area in young individuals occurred despite no
age-related differences in the expression of major myogenic regulatory factors known to promote skeletal muscle
hypertrophy or satellite cell proliferation gene products
(IGF-1Ea, MGF, MyoD1, myogenin, HGF). However, the
expression of myostatin demonstrated a more pronounced
up-regulation following immobilisation along with an
attenuated down-regulation in response to reloading in
older compared to young individuals, which may have
contributed to the present lack of satellite cell proliferation
in ageing muscle.
Changes in myofibre size with reloading
Reloading of disuse-induced skeletal muscle atrophy, by
means of resistive types of exercise, is known to restore
muscle mass in young individuals by increased myofibrillar protein synthesis and restoration of fibre-type area
(Hespel et al. 2001; Jones et al. 2004; Machida & Booth,
2004). In line with our previous data on whole quadriceps
muscle volume measured by magnetic resonance imaging
(Suetta et al. 2009), young men in the present study
responded positively to the reloading protocol, showing
robust increases in both type I and type II myofibre area
(Fig. 1). In contrast, no changes in type I or type II myofibre area emerged with 4 weeks of retraining in aged
individuals (Fig. 1). This demonstrates an attenuated
Figure 2. Pax7-positive cells associated with type I and II myofibres
Immunohistochemically detected Pax7+ cells, type I myosin and laminin on a muscle biopsy cross-section. Pax7+
cells (black arrows) are visualised by light microscopy (brown; upper left, ‘Pax7’), and type I myosin staining (red)
used to indicate Pax7+ cells associated with type I fibres (A4.951+; red) or type II fibres (unstained; upper right,
‘A4.951’). Laminin staining (green) was used to mark the myofibre basal membrane (lower left, ‘Laminin’). In the
merged image of the three stainings for Pax7, type I myosin (A4.951) and laminin, a Pax7+ cell was located within
a type I fibre while two Pax7+ cells were identified in type II fibres (lower right, ‘Merged’). Scale bar, 100 µm.
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3796
C. Suetta and others
response to short-term reloading in old compared to
young humans. However, due to the relatively short
observation period, we cannot conclude to what extent
the recovery of skeletal muscle mass in elderly undergoing
short-term immobilisation is impaired or just occurs
more slowly than in young counterparts. In support of
the second view, observations over more prolonged periods of reloading (e.g. 12 weeks) have demonstrated that
the elderly can fully recover whole muscle cross-sectional
area and myofibre area after long-term (months to years)
muscle disuse due to hip osteoarthritis and subsequent
elective hip-replacement surgery, but only if the reloading
phase comprises a systematic use of resistance-based
exercise (Suetta et al. 2004b, 2008).
Changes in myogenic progenitor cells with reloading
In skeletal muscle the myogenic stem cells, also referred
to as satellite cells (SCs), are known to play a key
role in the maintenance, growth and repair of myofibres (Mauro, 1961; Moss & Leblond, 1970; Heslop
et al. 2001; Pallafacchina et al. 2012). During the process
of load-induced muscle hypertrophy, satellite cells are
Figure 3. Pax7-positive cells pre- and post immobilisation and
following 4 weeks of retraining: association with fibre type
A, number of Pax7+ cells associated with fibre type I. B, number of
Pax7-positive satellite cells associated with fibre type II. +3d, after
3 days of retraining. ∗ Time effect, P < 0.05 compared to Pre. ∞Age
effect, P < 0.05 difference between young and old within time
point. Data are means ± SEM.
J Physiol 591.15
thought to proliferate, differentiate and eventually fuse
with existing myofibres (McCormick & Schultz, 1994).
The resulting donation of new myonuclei by the fusion of
SCs with existing myofibres is thought to ensure that myonuclear domain size stays within certain functional limits
in situations of marked myofibre hypertrophy (Kadi et al.
2004b; Petrella et al. 2008). In humans, skeletal muscle
SCs seems to be maintained into the seventh decade of
life (Roth et al. 2000; Petrella et al. 2006; Hikida, 2011),
with a decline in content and activation capabilities with
progressive ageing (Renault et al. 2002; Kadi et al. 2004a;
Verdijk et al. 2007) accompanied by a reduced migration
capacity of existing SCs in turn resulting in a reduced
regeneration potential after muscle injury and disuse
(Carlson & Faulkner, 1989; Mitchell & Pavlath, 2004;
Conboy et al. 2005; Carlson & Conboy, 2007). Despite
a mean age of only ∼70 years in our aged individuals signs
of impaired SC proliferation with immobilisation and subsequent retraining were observed compared to young subjects (∼25 years old), indicating an attenuated myogenic
response to changes in exercise pattern (Fig. 3A and B).
Interestingly, compared to the changes induced by 2 weeks
of disuse, more accentuated age-related differences in SC
activation were observed in the acute (+3d) as well as the
prolonged (+4wk) phase of reloading (Fig. 3A and B). The
latter trend is in line with Dreyer et al. (2006) reporting a
greater increase in SC content in young compared to aged
skeletal muscle within 24 h following 92 maximal eccentric
muscle contractions (Dreyer et al. 2006). The importance
of SC number in relation to muscle size across the age-span
was underlined by Kim and co-workers who demonstrated
a positive linear association between muscle size and SC
number in baseline muscle biopsies obtained from young
and older human individuals (Kim et al. 2005b). Extending
those data, here we report for the first time in human
individuals that a positive relationship exists between
SC number and myofibre area following disuse atrophy
(Fig. 5A) as well as in response to subsequent reloading
(Fig. 5B).
Since human ageing is associated with a preferential
reduction in muscle fibre type II size (Andersen, 2003;
Aagaard et al. 2010) it has been speculated that SC content
might decrease more in type II fibres compared to type I
fibres with ageing (Kim et al. 2005b). In young human
individuals, SC appears to be similar between type I and
type II muscle fibres (Kadi et al. 2006; Verdijk et al. 2007).
In contrast, age-related type II muscle fibre atrophy is
accompanied by a type II muscle fibre-specific reduction
in SC content (Verdijk et al. 2007; Verney et al. 2008).
In support of these observations, young and old adults
demonstrating marked exercise-induced gains in myofibre area (i.e. ‘hypertrophy responders’) are characterised
by a greater concurrent up-regulation in myogenic SCs
compared to individuals with a less robust hypertrophy
response (Petrella et al. 2008). However, although the
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J Physiol 591.15
Ageing affects human skeletal muscle recovery
3797
Changes in MyoD1 and myogenin expression with
reloading
regenerative capacity of human skeletal muscle seems to
decline at a more advanced age (reflected by a decline in
SC number and/or proliferative capacity), the impairment
in SC function does not seem to prevent a significant
capacity for muscle hypertrophy provided that the intervention period is sufficiently long (months), as reported
even at very old age (Thornell et al. 2003; Dedkov et al.
2003; Shefer et al. 2006).
In the present study MyoD1 and myogenin expression
were markedly up-regulated with immobilisation in both
age groups, whereas subsequent reloading led to both
an acute (+3d) and a sustained (+4wks) decrease in
MyoD1 and myogenin expression in young as well as aged
skeletal muscle. The rise in MyoD1 and myogenin mRNA
expression following immobilisation independently of
age may represent a compensatory signalling pathway
for partial muscle retention during periods of acute
muscle loss, while also observed in other atrophy
situations (Alway et al. 2001). Following resistance-type
exercise increased expression of MyoD1 and myogenin
mRNA has been observed in both young and older
adults (Psilander et al. 2003; Kim et al. 2005a; Kosek
et al. 2006; Raue et al. 2006; Costa et al. 2007; McKay
et al. 2008). In the present study, however, only a
modest up-regulation in myogenin expression compared
to the basal non-immobilised state was observed
following the reloading phase consisting of 4 weeks
of resistance training, suggesting that retraining after
short-term disuse atrophy may evoke different molecular
signalling stimuli compared to regular resistance exercise
intervention.
Changes in IGF-1 expression with reloading
Numerous growth factors are known to regulate satellite
cell activity, among which insulin-like growth factor 1
(IGF-1) is known to play an essential role in the process
of muscle hypertrophy (Rosenblatt et al. 1994; Adams
& Haddad, 1996; Suetta et al. 2010). The discovery of
distinct IGF-1 isoforms (mechano growth factor (MGF)
and IGF-1Ea) has suggested different roles for IGF-1,
namely that MGF mainly triggers satellite cell activation
and proliferation, while IGF-1Ea is thought to mainly
promote differentiation of proliferating SCs (Yang &
Goldspink, 2002). In line with those findings the present
study demonstrated a differentiated regulation in IGF-1Ea
and MGF expression, with both an acute and a sustained
up-regulation of MGF mRNA expression in response to
retraining (+3d and +4wks), whereas IGF-1Ea expression
was up-regulated in the later phase of reloading (+4wks)
only, suggesting a supportive role of paracrine/autocrine
IGF signalling in the process of human muscle hypertrophy, at least when recovering from short-term muscle
disuse. In contrast to earlier findings in rodent and human
skeletal muscle (Owino et al. 2001; Hameed et al. 2003)
the present up-regulation in MGF mRNA expression
with reloading (post 4 weeks resistance training) was not
attenuated in older vs. young individuals in the present
study.
Changes in HGF and c-Met expression with reloading
Hepatocyte growth factor (HGF) is generally considered
to be one of the most important growth factors involved
in organ regeneration (Zarnegar, 1995) as well as a
key regulator of satellite cell activity during muscle
regeneration (Jennische et al. 1993). The presence of HGF
transcripts in newly regenerated myotubes and in satellite
cells suggests that HGF activity is mediated primarily
through paracrine/autocrine mechanisms (Anastasi et al.
1997; Sheehan & Allen, 1999). Furthermore, HGF is a
mRNA pre-values relative to the young group
Young
Old
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GAPDH
CDKN1B (p27)
*
CDKN1A (p21)
Myostatin
Pax7
FGFR1
FGF2
c-Met
HGF
Myogenin
MyoD1
MGF
0.1
Figure 4. mRNA values relative to the
young group at baseline (Pre)
∗ Age effect, P < 0.05 young compared to
old. Data are geometric
means ± back-transformed SEM.
*
1
IGF-1Ea
Relative to meanYoung Pre
10
3798
C. Suetta and others
potent growth factor that has the ability to stimulate
quiescent satellite cells to enter the cell cycle early in
vitro as well as in vivo (Allen et al. 1995; Tatsumi et al.
1998) and is therefore considered most important during
E
IGF-1Ea
10
*
*# *#
Young
§
Old
§
*#
1
0.1
Pre
Imm
the early phase of re-growth (Tatsumi et al. 1998). In
support of this, we observed a significant increase in HGF
mRNA expression in young individuals in response to early
retraining (+3d) but not after more sustained retraining
+3d
Relative change from Pre
Relative change from Pre
A
J Physiol 591.15
HGF
10
1
0.1
+4wks
Pre
F
10
1
Pre
C
Relative change from Pre
*
Imm
+3d
MyoD1
Young
Old
* *
*#
#
#
§
#
1
0.1
Pre
Imm
+3d
+4wks
*#
1
Pre
10
* *
*#
#
#
#
§
1
Young
Old
Pre
Imm
+3d
+4wks
Young
Old
1
Pre
Imm
+3d
+4wks
FGFR1
10
* *
1
0.1
0.1
+3d
FGF2
0.1
Myogenin
Imm
10
H
D
+4wks
Young
Old
G
10
+3d
10
0.1
+4wks
Relative change from Pre
*
0.1
Relative change from Pre
Young
Old
§
Relative change from Pre
*#
Imm
c-Met
MGF
Relative change from Pre
Relative change from Pre
B
Young
Old
*
Pre
Imm
*§ *§
#
Young
Old
#
+3d
+4wks
+4wks
Figure 5. mRNA expression levels relative to baseline following 2 weeks of immobilisation and 4 weeks
of retraining
A, IGF-1Ea; B, MGF; C, MyoD1; D, myogenin; E, HGF; F, c-Met; G, FGF2; H, FGFR1; I, Pax7; J, myostatin; K,
CDKN1A (p21); L, CDKN1B (p27). ∗ Time effect, P < 0.05 compared to Pre. #Time effect, P < 0.05 compared
to Imm. §Time effect, P < 0.05 compared to +3d. ∞Age effect, P < 0.05 young compared to old. Data are
geometric means ± back-transformed SEM.
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Ageing affects human skeletal muscle recovery
J Physiol 591.15
(+4wks) (Fig. 5E). In contrast to HGF, a marked decrease
in the HGF receptor c-Met expression was observed in
response to early retraining (+3d) in young individuals,
which may support the hypothesis previously proposed
that increasing concentrations of HGF reduces c-Met
which forms a negative feedback mechanism inducing SC
quiescence in regenerating muscle (Tatsumi et al. 2009;
Yamada et al. 2010).
Changes in FGF2 and FGFR1 expression with reloading
In recent years, fibroblast growth factors (FGFs) and
their receptors (FGFRs) have gained increased focus as
major players in both embryonic development and skeletal
muscle tissue repair (Coutu & Galipeau, 2012). Moreover,
somatic stem cells have been suggested as major targets
of FGF signalling in both tissue homeostasis and repair
where FGFs appear to promote self-renewing proliferation
and inhibit cellular senescence in nearly all tissues tested
to date (Coutu & Galipeau, 2012). Fibroblast growth
factor 2 (FGF2) is a polypeptide growth factor that
stimulates SC proliferation in already activated SCs (Allen
& Boxhorn, 1989; Mezzogiorno et al. 1993). However,
despite our findings of marked SC proliferation with
reloading, expression levels of FGF2 remained unchanged
at all time points examined, suggesting that FGF may
be less important for SC proliferation in human skeletal
muscle, at least in relation to reloading subsequent to
immobilisation.
The observed differences in immunohistochemical Pax7+
cell content of the vastus lateralis muscle in young and
older individuals during immobilisation and subsequent
retraining (Fig. 3) were not associated with corresponding
changes in mRNA for Pax7. In fact, the expression level of
Pax7 was down-regulated in young and up-regulated in old
individuals following 2 weeks of immobilisation (Fig. 5I),
indicating that factors other than Pax7 mRNA levels may
influence the content of Pax7+ SCs during immobilisation
and retraining conditions in humans.
Changes in myostatin expression with reloading
Myostatin is a member of the transforming growth
factor-β superfamily and a strong negative regulator of
skeletal muscle mass, known to inhibit myogenic SC
activation (McPherron et al. 1997; Trendelenburg et al.
2009). Although the mechanisms are not fully understood,
myostatin is thought to modulate key regulators of the cell
cycle such as cyclin-dependent kinase inhibitors p21cip
and p27kip (Kim et al. 2005a), thereby inhibiting SC cycle
progression from G0 to S phase (McCroskery et al. 2003).
Down-regulated myostatin mRNA expression has been
observed in response to exercise training in both young
and elderly individuals (Roth et al. 2003; Kim et al. 2005a;
Raue et al. 2006; Costa et al. 2007), with some studies
reporting an attenuated response with ageing (Kim et al.
2005b; Haddad & Adams, 2006). In line with the latter
findings, McKay and colleagues recently demonstrated a
k
Pax7
Relative change from Pre
Changes in Pax7 expression with reloading
10
Relative change from Pre
I
Young
Old
Age*Time p<0.05
∞
§
1
0.1
Pre
Imm
+3d
CDKN1A (p21)
10
Young
Old
Pre
Imm
§
+3d
+4wks
l
CDKN1B (p27)
10
Young
Old
*
∞
*# *#
§
§
1
0.1
Pre
Imm
+3d
+4wks
Figure 5. Continued
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Relative change from Pre
Myostatin
Relative change from Pre
*#
#
1
0.1
+4wks
J
3799
10
Young
Old
*# *#
1
0.1
Pre
Imm
+3d
§
§
+4wks
C. Suetta and others
4000
2000
0
0.00
Relative change in MFA (%)
B
rs=0.712
p<0.01
Collectively, the present data suggest that significant
age-specific differences may exist for the ability of human
skeletal muscle to regenerate after immobility-induced
muscle atrophy. More specifically, our results indicate
an attenuated – or at least delayed – response in aged
individuals to active reloading subsequent to short-term
4000
2000
rs=0.779
p<0.01
0
100
80
60
40
20
-20
Conclusions
6000
0.05
0.10
0.15
0.20
Pax7+ cells per fibre at Imm
120
0
In line with the regulation in myostatin mRNA, expression
levels of cyclin-dependent kinase inhibitors CDKN1A
(p21) and CDKN1B (p27) decreased in response to acute
loading in the present study. Both CDKN1A and CDKN1B
are known to block cell cycle progression and induce
SC cell cycle withdrawal (Coqueret, 2003). Furthermore,
ectopic expression of CDKN1B has been shown to block
the IGF-I-induced increase in satellite cell proliferation
(Chakravarthy et al. 2000) and thus CDKN1B is considered
a key regulatory factor in the regulation of satellite cell cycle
progression (Machida et al. 2003).
rs=0.693
p<0.01
-40
-100 -50
0
50 100 150 200
Relative change in Pax7+ cells
per fibre (%)
8000
6000
4000
2000
0.00
0.05
0.10
0.15
0.20
Pax7+ cells per type I fibre at Imm
120
100
80
60
40
20
0
-20
rs=864
p<0.01
-40
-100 -50
0
50 100 150 200
Relative change in Pax7+ cells
per Type I fibre (%)
Relative change in type II area (%)
6000
Changes in cyclin-dependent kinase inhibitors with
reloading
8000
Type I fibre area at Imm (um2)
8000
Relative change in type I area (%)
A
MFA at Imm (um2)
markedly blunted myogenic response in older compared to
younger individuals (McKay et al. 2012). Although stem
cell-specific myostatin levels did not appear to differ at
baseline, acute resistance exercise (75% 1RM) was found
to evoke ∼70% greater content of myostatin-positive
type II-associated SCs in old versus young adults at 24 h
post exercise, suggesting that the greater co-localisation
of myostatin with SCs may provide a mechanism for
the impaired myogenic capacity of aged muscle (McKay
et al. 2012). Somewhat unexpectedly, the expression level
of myostatin mRNA was lower in old compared to young at
baseline in the present study, which might reflect the rather
high activity level (equal to that of young) of the present
group of elderly individuals. Despite this, an age-specific
difference in the regulation of myostatin was also observed
in the present study, manifested by a reduced suppression
with reloading in aged vs. young individuals (Fig. 5J). This
observation may explain, at least in part, the impaired
capacity for SC proliferation and re-growth in aged skeletal
muscle observed in the present study, although the interpretation of these data is limited if only assessing transcript
levels.
J Physiol 591.15
Type II fibre area at Imm (um2)
3800
rs=0.694
p<0.01
0
0.00 0.05 0.10 0.15 0.20 0.25
Pax7+ cells per type II fibre at Imm
120
100
80
60
40
20
0
-20
rs=0.304
p<0.01
-40
-100 -50
0
50 100 150 200
Relative change in Pax7+ cells
per Type II fibre (%)
Figure 6. Association between changes in myofibre area and number of Pax7+ cells
A, number of Pax7+ cells versus fibre area for all fibres collapsed or separated into type I or II fibres post
immobilisation (Imm). B, relative changes in myofibre area (MFA, type I or type II) following 4 weeks of retraining
(relative to post immobilisation) versus the change in number of Pax7+ cells (total, type I associated and type II
associated). Open triangles, young individuals; filled triangles, older individuals.
C 2013 The Authors. The Journal of Physiology
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J Physiol 591.15
Ageing affects human skeletal muscle recovery
disuse, as reflected by attenuated gains in myofibre area
and SC number despite no age-related differences being
observed in local growth factors responsible for promoting
skeletal muscle hypertrophy (IGF-1Ea, MGF, MyoD1,
myogenin, HGF). These disparate trends may partly reside
on a reduced cellular sensitivity to paracrine/autocrine
growth factors as basal MRF mRNA expression appears to
be chronically up-regulated in senescent muscle (Edström
& Ulfhake, 2005; Kim et al. 2005a; Kosek et al. 2006; Raue
et al. 2006). Our findings of an age-specific regulation in
myostatin expression levels may also have contributed to
the apparent lack of increase in SC number and myofibre area with ageing in response to reloading. Gaining
an improved understanding of the ability of human
skeletal muscle to recover from atrophy has important
implications for the development of effective molecular
and rehabilitative countermeasures against physical frailty
in the continuously growing population of elderly adults.
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Additional information
Competing interests
None.
3804
C. Suetta and others
J Physiol 591.15
Author contributions
Funding
The study was performed at the Institute of Sports Medicine
Copenhagen, Bispebjerg Hospital, Denmark. Conception and
design of the study: C.S., U.F., P.S., P.A. and M.K. Collection,
analysis and interpretation of data: C.S., U.F., A.L.M., L.J.,
L.G.H., M.L.B., S.J.P., H.D.S., J.L.A. and P.S. Writing or revising
the manuscript critically: C.S., U.F., A.L.M., P.S., P.A., L.J., L.G.H.
and M.K. All authors have approved the final version of the
manuscript.
This study was supported by grants from the Danish Medical
Research Council, the Danish Rheumatology Association,
Faculty of Health Sciences, University of Copenhagen, The
Danish Ministry of Culture, Lundbeck Foundation, EU 7th
framework programme ‘Myoage’, and Nordea Foundation
(Healthy Ageing Grant).
Acknowledgements
We wish to express our gratitude to the subjects who participated
in this study for their abundant efforts and contribution to this
work.
Translational perspective
We report measures of myofiber area and myogenic stem cell number (SC) associated with type
I and type II muscle fibres in young and older humans, in combination with transcriptional data
from regulatory pathways related to skeletal muscle re-growth in immobilised and re-trained aging
human muscle. The main study finding was a less marked muscle mass recovery after immobilisation
in elderly compared to young individuals that was paralleled by an elevation in SC content in young
only, whereas elderly failed to demonstrate any change in SC’s. This potential coupling of SC
proliferation and recovery in myofiber area in young individuals occurred despite no age related
differences in the expression of myogenic regulating genes normally known to promote skeletal
muscle hypertrophy or SC proliferation. However, expression of myostatin was more pronounced
after immobilisation along with an attenuated down-regulation in response to re-loading in older
compared to young individuals, which may have contributed to the lack of SC proliferation in
aging muscle. The age-specific regulation in myostatin expression may also have contributed to the
apparent lack of increase in SC number and myofiber area with aging in response to re-loading.
Collectively, the present findings underlines that elderly have an impaired ability to recover from
disuse muscle atrophy and thus, elderly may need longer time to recover from periods of disuse or
disease compared to younger ones.
Gaining insight in the ability to recover muscle from atrophy has implications for effective
molecular and rehabilitative countermeasures against frailty in the growing population of elderly.
C 2013 The Authors. The Journal of Physiology
C 2013 The Physiological Society