© 2015 John Wiley & Sons A/S.
Scand J Med Sci Sports 2015: ••: ••–••
doi: 10.1111/sms.12416
Published by John Wiley & Sons Ltd
Effects of loaded voluntary wheel exercise on performance and
muscle hypertrophy in young and old male C57Bl/6J mice
Z. Soffe1, H. G. Radley-Crabb1,2, C. McMahon3, M. D. Grounds1, T. Shavlakadze1,3
1
School of Anatomy, Physiology and Human Biology, the University of Western Australia, Nedlands, Western Australia, Australia,
School of Biomedical Sciences, CHIRI Biosciences Research Precinct, Curtin University, Bentley, Western Australia, Australia,
3
Developmental Biology Group, Agresearch Ltd, Hamilton, New Zealand
Corresponding author: Zoe Soffe, BSc (Hons), School of Anatomy, Physiology and Human Biology, the University of Western
Australia, 35 Stirling Highway, Crawley, Western Australia, 6009, Australia. Tel: (+61) 8 6488 3486, Fax: (+61) 8 6488 1051,
E-mail: [email protected]
2
Accepted for publication 23 December 2014
This study compared the capacity of young and old male
C57Bl/6J mice to exercise with increasing resistance over
10 weeks, and its impact on muscle mass. Young mice
(aged 15–25 weeks) were subjected to low (LR) and high
(HR) resistance exercise, whereas only LR was used for
old mice (107–117 weeks). Weekly patterns of voluntary
wheel activity, food consumption and body weights were
measured. Running patterns changed over time and with
age, with two peaks of activity detected for young, but
only one for old mice: speed and distance run was also
less for old mice. The mass for six limb muscles was
measured at the end of the experiment. The most pronounced increase in mass in response to exercise was for
the soleus in young and old mice, and also quadriceps and
gastrocnemius in young mice. Soleus and quadriceps
muscles were analyzed histologically for myofiber
number and size. A striking feature was the many small
myofibers in response to exercise in young (but not old)
soleus, whereas these were not present after exercise in
young or old quadriceps. Overall, there was a striking
difference in response to exercise between muscles and
this was influenced by age.
The age-related loss of skeletal muscle mass and function, called sarcopenia, adversely affects movement,
mobility, posture, and metabolism (Cruz-Jentoft et al.,
2010). The incidence of sarcopenia, reported as 14% by
65–69 years of age may reach greater than 50% by 80
years (Janssen, 2010) and the loss of muscle function
may occur before loss of muscle mass (Clark & Manini,
2008; Chan & Head, 2010). Sarcopenia results in diminished independence and frailty that increases the risk of
falls and fractures with escalating costs for the global
health system (Cruz-Jentoft et al., 2010; Sayer et al.,
2013). As the human population demographic is rapidly
ageing, there is a compelling need to better understand
the molecular mechanisms that cause sarcopenia, in
order to design and implement the best early intervention
strategies (Sayer et al., 2013).
Similar to humans, rodents (mice and rats) are
affected by sarcopenia (Brooks & Faulkner, 1988; Daw
et al., 1988; Holloszy et al., 1991; Blough & Linderman,
2000; Hamrick et al., 2006; Shavlakadze et al., 2010a;
Sheard & Anderson, 2012; Ibebunjo et al., 2013). In
C57Bl/6J mice, both males and females, sarcopenia is
conspicuous at 24 months (m) of age (roughly equivalent
to 70 years in humans (Flurkey et al., 2007), although
the severity of muscle loss varies between muscles
(Shavlakadze et al., 2010b; Sheard & Anderson, 2012)
and sarcopenia is more pronounced in older (27–29 m)
mice (Shavlakadze et al., 2010a).
Key features of sarcopenia in humans and animals,
among others, are decreased myofiber size (atrophy)
and myofiber death, remodeling of extracellular matrix
with deposition of connective tissue and functional
denervation of the ageing myofibers (Shavlakadze &
Grounds, 2003; MacIntosh et al., 2006; Chai et al., 2011;
Sayer et al., 2013). Recent comprehensive analyses
of gene expression changes throughout the life of
ageing rats (Ibebunjo et al., 2013) and mice (Barns et al.,
2014) emphasize progressive alterations associated
with myofiber denervation, metabolism, and extracellular matrix as well as the general dysregulation of
transcription.
Whether muscle ageing can be reversed is debated;
however, there is a consensus that sarcopenia can be
reduced by exercise (reviewed in Hunter et al., 2004;
Stewart et al., 2014). Exercise is widely recognized as a
relatively simple and accessible intervention to reduce
the decline of muscle mass and function and prolong
independence and healthy living (Buford et al., 2010;
Sayer et al., 2013). Human studies trialing progressive
resistance training (PRT), whereby participants exercise
against an increasing load, can maintain and increase
muscle mass with benefits that vary between young and
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Soffe et al.
old men and women. A standard 2–3 days/week PRT
protocol can result in significant myofiber hypertrophy
among elderly participants (≥ 60 years), with increases
ranging between 13 to 52% among men (Frontera et al.,
1988; Hikida et al., 2000; Trappe et al., 2000; Bamman
et al., 2003; Leenders et al., 2012), and 5% to 38%
among women (Charette et al., 1991; Häkkinen et al.,
2001; Trappe et al., 2001; Bamman et al., 2003;
Leenders et al., 2012) after 12 to 26 weeks of training. A
recent review also demonstrated that elderly men and
women (> 75 years) are capable of increasing muscle
size (1.5–15.6%) and strength after a variety of resistance training interventions (reviewed in Stewart et al.,
2014). Some studies suggest that elderly humans have a
tendency to exhibit smaller relative muscle hypertrophy,
or a blunted adaptive response to PRT than younger
individuals (Kosek et al., 2006; Mero et al., 2012).
In rodent models, the use of voluntary running
wheels paired with a progressive increase in resistance
is appropriate for increasing muscle mass and myofiber
size (Legerlotz et al., 2008). A number of different
exercise protocols, employing both high and low levels
of static resistance, have been used to assess the impact
of an increasing load on skeletal muscle adaptation.
Against a background of rapid postnatal growth, high
impact static wheel loading (up to 173% of total body
weight) increased muscle mass of young rats after only
6.5 weeks of training, with relative increases of 17%,
21%, 23%, and 29% observed in vastus lateralis, extensor carpi radialis longus and brevis, plantaris, and
soleus muscle groups, respectively (Legerlotz et al.,
2008). Similar increases in plantaris muscle mass (31%
relative increase) have also been observed in young rats
after 8 weeks of training, with a maximum load of 74%
total body mass (no information on other muscles
reported; Ishihara et al., 1998). Furthermore, administering a progressive wheel loading protocol in adult
mice (up to either 17% or 41% of total body mass),
while having no impact on plantaris, gastrocnemius or
tibialis anterior, was sufficient to increase soleus
muscle mass by ∼ 20% after 7 weeks of training
(Konhilas et al., 2005).
Muscles of ageing rodents are also capable of
exercise-induced muscle hypertrophy. Spontaneous
activity, such as voluntary wheel running (no resistance;
Brown et al., 1992; Gulve et al., 1993; McMahon et al.,
2014), in addition to forced exercise techniques
(weightlifting (Klitgaard et al., 1989) and treadmill
running (Daw et al., 1988)) and athletic environmental
enrichment (Brown et al., 2003) have shown that old
rodents are capable of adaptive increases in muscle mass
after continuous bouts of activity. As for humans, some
studies suggest that the hypertrophic integrity of ageing
rodent muscle in response to exercise is attenuated, with
some exercise protocols unable to reduce the rate of
atrophy associated with ageing (Farrar et al., 1981; Daw
et al., 1988; Klitgaard et al., 1989). The capacity of old
2
rodent muscle to hypertrophy in response to voluntary
resistance wheel running has not been investigated.
Thus, we subjected young (aged 15 weeks) and old
(aged 107 weeks) male C57Bl/6J mice to voluntary
wheel running with increasing resistance for 10 weeks to
characterize running patterns and determine the impact
of resistance exercise on skeletal muscle mass and phenotype at both ages. Histological and morphometric
analyses were performed on selected hind-limb muscles
(quadriceps and soleus) of young and old mice.
Materials and methods
Mice and voluntary resistance wheel running protocols
Young (13 weeks, n = 32) and old (105 weeks, n = 24) male
C57Bl/6J mice were obtained from the Animal Resources Centre,
Western Australia and housed at the University of Western Australia under pathogen-free conditions. All experiments were conducted in accordance with the guidelines of the National Health
and Medical Research Council, Australia and were approved
by the Animal Ethics Committee of the University of Western
Australia.
Mice were maintained on a 12-h light-dark cycle (lights turned
on at 07:00 h), at 22 °C, with free access to meat-free rat and
mouse diet (protein, 20%; total fat, 4.8%; total fiber, 28.8%; total
carbohydrate, 59.4%) fortified with vitamins and minerals (Specialty Feeds, Perth, WA, Australia) and drinking water. Mice were
acclimated for 2 weeks until they reached 15 weeks and 107 weeks
of age and were assigned to the following groups: (1) young
sedentary (SED; 15 weeks, n = 10); (2) young low resistance (LR;
15 weeks, n = 7); (3) young high resistance (HR; 15 weeks, n = 7);
(4) old sedentary (SED; 107 weeks, n = 9); (5) old low resistance
(LR; 107 weeks, n = 7). A group of n = 8 mice was also sampled at
105 weeks of age to obtain baseline data for old mice prior to
starting the exercise regime. Old mice were not subjected to HR
exercise because of limited numbers. An additional group (n = 4)
young mice (aged 12 weeks) were exercised for 1 week (without
resistance) and then sampled to test whether recent myogenesis
(indicative of necrosis and regeneration) had occurred after such
mild exercise. Where ages are also expressed as months, this
represents calendar months, so that 15, 25, 105, 107, and 117
weeks corresponds to approximately 4, 6, 24, 25, and 27 months,
respectively. The age of young mice at time of tissue collection
was 25 weeks (∼6 months) and for old mice 105 (∼24 months), and
117 weeks (∼27 months).
SED mice were housed individually in standard mouse cages
with transparent walls (19.5 cm × 28 cm) for the duration of the
experiment. Exercising mice were housed individually in Lafayette Mouse Activity Wheel Chambers (23.5 cm × 35.3 cm; Model
80820; Lafayette Instrument, IN, USA) equipped with a 12.7 cm
diameter exercise wheel with a 5.72 cm wide running surface
(Model 80820RW, Lafayette) and an adjustable servo-brake
(Model 86070-B1) to control resistance application and wheel
function. Each chamber was equipped with an activity wheel
counter (Model 86070A) to monitor wheel revolution, distance
travelled (set at 0.40 m/revolution) and speed (m/min). The Activity Wheel Monitoring (AWM) Software (Model 86065) was used
to record all data sets. Wheel loading was determined by hanging
known weights on each individual wheel and adjusting the brake
to hold each selected weight (per manufacturer’s instructions).
These wheels are considered to be low resistance, or free spinning
wheels, given that wheel inertia is very low (< 1 g).
Voluntary resistance exercise began at 15 weeks for young mice
and at 107 weeks for old mice and lasted for 10 weeks, until mice
reached 25 weeks and 117 weeks, respectively. Exercise protocols
for LR and HR groups are detailed in Fig. 1. LR exercise groups
Exercise and muscle hypertrophy in old mice
Image acquisition and morphometric analyses
Fig. 1. Experimental protocols for low (LR) and high (HR)
resistance voluntary wheel running over 10 weeks. Young mice
were exercised with LR and HR protocols from 15 weeks of age
until sacrifice at 25 weeks. Old mice (107 weeks) were exercised
with the same LR protocol and sacrificed at 117 weeks (see text
for details).
ran without resistance for the first 2 weeks, with a 1 g increase in
resistance commencing at the start of every 2 weeks (up to a
maximum of 4 g; Fig. 1). The HR group ran without resistance for
the first week, and was then subjected to a 1 g increase in resistance at the start of every week for the first 4 weeks (up to 3 g), and
a further increase of 1 g every 2 weeks for the remaining 6 weeks
(up to 6 g) (Fig. 1). Values for distance run and speed were
recorded every hour, for each mouse, throughout the duration of
the study by AWM Software and the data shown as an average
value over 5 days. Body weights and food consumption were
recorded for each mouse three times a week for 10 weeks. Food
consumption was calculated by weight of food remaining in the
feeding tray.
Tissue collection
Mice were killed by cervical dislocation while under terminal
anesthesia (2% v/v Attane isoflurane, Bomac, NSW, Australia,
400 mL NO2 and 1.5L O2). Lower and upper limb muscles [quadriceps femoris, gastrocnemius, tibialis anterior (TA), soleus, extensor digitorum longus (EDL) and triceps brachii were removed and
weighed. Soleus and quadriceps muscles were cut transversely in
the middle, mounted onto tragacanth gum (Sigma-Aldrich Pty Ltd,
Sydney, Australia) and frozen in liquid nitrogen cooled isopentane
for histological analyses. The epididymal fat pads were weighed
and the length of the tibial bones were measured and used for
normalizing wet muscle weights.
Haematoxylin and eosin (H&E) and nicotinamide adenine
dinucleotide nitro-blue tetrazolium (NADH-TR) staining
Transverse frozen muscle sections (8 μm) of soleus and quadriceps muscles were stained with H&E to assess general tissue
architecture. NADH-TR histochemical staining was used to identify fast and slow myofiber types in quadriceps transverse sections
(Shavlakadze et al., 2005).
Laminin immunostaining
Polyclonal rabbit anti-PAN laminin antibody (L9393, Sigma, Australia; dilution 1:300) was used to label myofiber basement membrane (Shavlakadze et al., 2005). The primary antibody was
detected by goat anti-rabbit ALEXA594 (A-11012, Invitrogen,
Molecular Probes, Oregon, USA; dilution 1:500).
Tiled images of transverse muscle sections stained with H&E,
NADH-TR and laminin were captured at ×10 magnification using
a Nikon Eclipse Ti inverter microscope equipped with Nikon
DS-Fi2 camera (Nikon Corporation, Tokyo, Japan) for bright field
imaging (H&E and NADH-TR) and CoolSNAP EZ camera
(Roper Scientific Photometrics, Ottobrunn, Germany) for fluorescence imaging (laminin). Images were captured using NISElements BR 4.1 software. Nontiled images of transverse muscle
sections were captured at 40× magnification using a Nikon 90i
microscope equipped with Nikon DS-Fi2 camera. Images were
captured using NIS-Elements AR 3.0 software (Laboratory
Imaging Ltd., Czechoslovakia, http://www.lim.cz/en/). Colour
enhancements on H&E and laminin images were performed using
Adobe Photoshop (Adobe Systems Incorporated) Version 7.
All morphometric analyses were carried out with ImagePro
Plus 4.5 (Media Cybernetics, MD, USA) software. The soleus was
selected for detailed analysis because it showed the most pronounced hypertrophic response to LR exercise at both ages. Tiled
images of entire soleus muscles stained with laminin were portioned into four equal quadrants and the cross-sectional area
(CSA) of 60 myofibers were measured in each quadrant, totaling
240 myofibers for each muscle section. Individual myofiber
numbers were counted on entire soleus cross sections stained with
laminin. The number of myofibers with displaced myonuclei were
counted on entire soleus cross sections stained with H&E.
The number and size of different myofiber types were quantified on tiled transverse sections of the quadriceps stained with
NADH-TR, that differentiates fast, intermediate, and slow-type
myofibers (Shavlakadze et al., 2005). Quadriceps was selected for
this analysis since it showed a loss of muscle mass with ageing and
a hypertrophic response to LR wheel exercise in young mice. The
quadriceps is predominantly a mixed fibre muscle and the percentage distribution of myofiber types differs between the four muscles
that comprise it (rectus femoris and vastus medius, lateralis and
intermedius). Tiled images of entire quadriceps muscle stained
with NADH-TR were portioned into two equal parts, deep (close
to the bone) and superficial (peripheral to the bone). The deep
region was used to measure slow-type myofibers and the superficial region to measure fast-type myofibers. Intermediate type
myofibers were excluded from the analyses. Two hundred
myofibers were measured each in deep and superficial layers.
Individual myofiber numbers were counted on entire quadriceps
cross sections stained with NADH-TR.
Statistical analyses
Longitudinal analyses for body weights, average daily food consumption, running distance, and speed were performed with a
repeated measure analysis of variance (ANOVA) using Genstat
v15 (VSN International Ltd., Hemel, Hempstead, UK), with all
experimental groups included in the treatment structure statement.
Post-hoc multiple comparisons were performed using Tukey’s
method (GenStat, 2003). The effect of age on SED mouse phenotype or muscle mass (15, 105, and 117 weeks) was analyzed using
a one-way ANOVA (Genstat v15). Post-hoc tests of least significant difference were used for direct mean comparisons. The same
treatment structure was used to analyze the effect of different
exercise protocols on young mice (SED, LR, and HR). Data comparisons between SED and LR exercising young and old mice
were analyzed with a two-way ANOVA (GenStat v15) using age
(young and old) and activity (SED or LR) as sources of variance.
If no interaction between age and activity was detected, post-hoc
tests of least significant difference were used for direct mean
comparisons. Where an interaction between factors (age and
activity) was detected, differences were determined by independent samples t-test (two-tailed). Any additional analyses by
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Soffe et al.
Table 1. Average daily distances (km) run by young (LR and HR) and old
mice (LR) after each successive week of exercise
Young
W1
W2
W3
W4
W5
W6
W7
W8
W9
W10
Old
LR (n = 7)
HR (n = 7)
LR (n = 7)
7.63 ± 0.65a
9.70 ± 0.62a
7.15 ± 1.18a
5.54 ± 1.06
4.41 ± 0.83a
2.84 ± 0.56
2.82 ± 0.29
2.64 ± 0.30a
1.67 ± 0.64
1.61 ± 0.32
6.55 ± 0.46a
9.75 ± 0.97a
5.40 ± 0.43ab
2.63 ± 0.54
1.40 ± 0.26b
2.50 ± 0.29
1.37 ± 0.24
1.02 ± 0.18b
0.56 ± 0.13
0.87 ± 0.52
3.10 ± 0.63b
3.47 ± 1.11b
2.63 ± 0.90b
3.03 ± 0.92
2.72 ± 1.02ab
2.78 ± 1.02
2.13 ± 0.58
1.50 ± 0.57ab
0.53 ± 0.13
0.70 ± 0.24
Values are means ± SE. Differing lower case letters indicate significant
differences between young and old mice at a particular week (P < 0.05).
HR, high resistance; LR, low resistance; n, number of mice; W, weeks.
young mice consumed more food (18%) compared with
old mice (P < 0.001) and there was a tendency (P < 0.1)
for the old LR group to consume more food (9%) compared with the old SED group (Fig. 2(b)).
Running distance
Fig. 2. Average weekly body weights (a) and food consumption
(b) in young and old mice over 10 weeks. The asterisks (*)
indicate significant differences between the young SED and the
average of the young LR and HR groups (a) and the average
amount of food consumed by young and old mice (b), respectively, for each week of the study. *P < 0.05; **P < 0.01;
***P < 0.001. For each weekly time point, data are
mean ± SEM. n = 7–10 mice per group.
independent samples t-test (two-tailed) are stated where necessary.
Differences were accepted as significant at P ≤ 0.05. Data are
means ± standard error of the mean (SEM).
Results
Body weights
At the start of the experiment (week 1; W1), young (15
weeks) mice were lighter than old (107 weeks) mice,
weighing on average 28.3 g and 31.6 g, respectively
(P < 0.001; Fig. 2(a)). For all young mice, body weights
increased progressively from 15 to 25 weeks of age
(23% for SED, 13% for the LR and 11% for the HR;
P < 0.001). Old SED mice did not change body weight
over the 10 weeks of study, and old LR mice did not alter
body weight significantly from SED controls. At the end
of the experiment (W10), young SED mice were heavier
than all other groups (P < 0.001; Fig. 2(a)).
Food consumption
Overall, the food consumed by all groups decreased
(17%) over 10 weeks of the study (P < 0.001). However,
4
Distances run by young mice increased from W1 to peak
at W2 (at least P < 0.05) before declining over the
remainder of the study (P < 0.001). The distance run by
old mice declined between W1 and W10 (P < 0.01) and
was significantly lower than that of young mice during
the first 3 weeks (Table 1).
Running speed
Average nightly speeds achieved by young mice either
increased (HR, P < 0.05), or tended to increase (LR,
P < 0.1) from W1 to peak at W2, before declining over
the remainder of the study (P < 0.001). Nightly speeds
among old mice also declined from W2 to W10
(P < 0.05) and were lower than that of the young mice
during the first three weeks of the study (Table 2).
Running patterns
Average hourly distances run by young and old (LR)
mice over 24 h throughout each fortnight are shown in
Fig. 3. Overall, young mice subjected to LR or HR exercise ran further than old mice given LR exercise
(P < 0.01). Mice are nocturnal and exercise mainly at
night. Accordingly, in both young and old mice, most of
the wheel activity coincided with the dark phase (19:00–
07:00 h). Young mice had two major intervals of activity.
The first occurred during the dark phase, where running
distance peaked 2 h after lights out, before progressively
declining throughout the remainder of the phase
(Fig. 3(a–e)). The second was initiated 2 h before the
Exercise and muscle hypertrophy in old mice
Table 2. Average nightly speeds (m/min) achieved by young (LR and HR)
and old mice (LR) after each successive week of exercise
Young
W1
W2
W3
W4
W5
W6
W7
W8
W9
W10
Old
LR
HR
LR
8.50 ± 0.94a
11.40 ± 1.00a
8.68 ± 1.65a
6.81 ± 1.57
6.03 ± 1.03
4.14 ± 0.67
4.16 ± 0.40
3.65 ± 0.40
1.91 ± 0.28
2.62 ± 0.44
8.10 ± 0.80a
13.03 ± 1.50a
7.72 ± 0.64ab
3.72 ± 0.69
2.20 ± 0.34
4.26 ± 0.39
2.31 ± 0.36
2.22 ± 0.46
1.11 ± 0.26
1.68 ± 0.87
4.10 ± 0.78b
5.10 ± 1.47b
3.95 ± 1.20b
4.48 ± 1.27
4.90 ± 1.64
4.07 ± 1.38
2.65 ± 0.73
2.40 ± 0.79
1.27 ± 0.41
1.29 ± 0.41
Values are means ± SE. Differing lower case letters indicate significant
differences between young and old mice for a particular week (P < 0.05).
n, number of mice; HR, high resistance; LR, low resistance; W, weeks.
light phase and peaked within 1 h after lights on. In
contrast, running activity in old mice peaked 2 h into the
dark phase, but there was no second phase of activity
before lights were switched on (Fig. 3(a–e)). Young mice
given HR exercise had a similar pattern of activity to that
of young LR mice (Supporting Information Fig. S1).
When running activities at equal resistance were compared (Supporting Information Fig. S1(a–e)), HR mice
outperformed LR mice at both a 1 and 2 g load (W2 and
W3, respectively; Supporting Information Fig. S1(b,c)),
but were similar for the 3 and 4 g load (Supporting
Information Fig. S1(d,e)).
Phenotypic characterization of ageing SED mice
Body weights of old mice aged 105 and 117 weeks were
11% lower than for young adults aged 25 weeks
(P < 0.05) and this was due in part to a marked decrease
in epididymal fat pad weight (62% and 58%, respectively, between 25 weeks and 105/117 weeks, P < 0.05;
Table 3). Because tibial length was smaller (2.2% and
3.3%) in old (105 and 117 weeks) compared with young
(25 weeks) mice, indicating that the older cohort had
smaller body size, all muscle weights were standardized
to tibia length to account for the difference in body size
(Table 3).
The relative muscle mass of quadriceps and gastrocnemius was reduced from 25 to 105 weeks of age by
15.5% (P < 0.05) and 8% (P < 0.05), respectively
(Table 3). There was no further significant mass reduction for these muscles between 105 and 117 weeks of
age. In addition, the relative mass of TA was reduced by
10.3% between 25 and 117 weeks (P < 0.05). Muscle
mass for EDL, soleus, and triceps did not change with
ageing (from 25 to 117 weeks; Table 3).
Impact of LR exercise on muscle and fat mass in young
and old mice
Voluntary LR exercise for 10 weeks increased the mass
of selected limb muscles in both young and old mice,
compared with age-matched SED controls. For quadriceps, gastrocnemius, and soleus muscles, a two-way
ANOVA showed an effect of age (P < 0.001), activity
state (P < 0.005), and an interaction between these
factors (P < 0.001; Fig. 4(a–c)).
The standardized weights of quadriceps, gastrocnemius, and soleus muscles from young LR (25 weeks)
mice were heavier by 16.5%, 23.5%, and 52%, respectively, compared with SED young mice (P < 0.001,
independent sample t-test; Fig. 4(a–c)). Exercise did
not affect the mass of young TA, EDL, or triceps
muscles (Fig. 4(d–f)). Unlike young mice, only soleus
increased in mass (18%) in old mice subjected to LR
exercise compared with SED age-matched controls
(P < 0.04, independent sample t-test; Fig. 4c). Such LR
exercise in old mice did not affect the muscle mass of
quadriceps, gastrocnemius, TA, EDL nor triceps
(Fig. 4(a,b,d–f)).
Epididymal fat pad mass was influenced by age
(P < 0.001) and activity state (P < 0.001) with an interaction between these factors (P = 0.05; Fig. 4(g)). Young
mice subjected to LR exercise had ∼ 50% less epididymal fat compared with age-matched SED mice
(P < 0.002; independent samples t-test). In contrast, the
weights of epididymal fat pads were similar for old LR
exercised and SED mice, but were less compared with
young SED mice.
Impact of HR exercise on young mice
HR exercise increased the mass of quadriceps and soleus
muscles, to the same extent as did LR exercise
(Fig. 4(a,c)). Quadriceps and soleus muscles from young
HR exercised mice were heavier by 14% and 37.5%
compared with muscles from young SED mice
(P = 0.004 and P < 0.001, respectively; independent
samples t-test; Fig. 4(a,c)). Unlike LR exercise, HR
wheel running did not increase the gastrocnemius mass
(Fig. 4(b)). HR exercise decreased epididymal fat pad
weight to the same extent as LR exercise, and contributed to a 42% decrease relative to SED controls
(P = 0.005; independent samples t-test; Fig. 4(g)). Given
the similar impact of HR and LR exercise on muscle
mass, only LR exercised muscles were analyzed in
depth.
Soleus muscles: myofiber number and size in SED and
LR exercised young and old mice
The number and size of individual myofiber profiles
were quantified on transverse sections of soleus muscles
immunostained with laminin antibody, which defines
individual myofiber contours. The soleus was selected
for detailed myofiber analyses because of the significant
increase in size after LR exercise for both young and old
mice.
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Soffe et al.
Fig. 3. Average distances run by young and old LR mice per hour over 24 h. Each hourly value represents an average over 2 weeks.
Values were calculated bi-weekly throughout the exercise protocol corresponding with an increase in wheel resistance: weeks (W) 1
and 2 with resistance set at 0 g (a); W3 and W4 (1 g) (b); W5 and W6 (2 g) (c); W7 and W8 (3 g) (d); W9 and W10 (4 g) (e). n = 7 mice
per group. Black and white bars under the X-axis indicate dark (19:00–07:00 h) and light (07:00–19:00 h) phases. Asterisk (*) indicates
significant differences between young and old LR mice at *P < 0.05; **P < 0.01; ***P < 0.001.
The number of myofiber profiles in soleus was
increased by 28% in young LR mice compared with
SED controls (P = 0.05; independent samples t-test;
Fig. 5(a)). This was associated with a striking (21%)
increase in myofibers with displaced myonuclei
(P = 0.01; independent samples t-test; Fig. 5(b)); where
myonuclei were not in the normal peripheral juxtasarcolemmal position (Fig. 6(a)), but were instead
located centrally or well within the sarcoplasm
(Fig. 6(b); arrows). Myofiber numbers were similar in
the soleus muscles of young and old SED mice
(Fig. 5(a)); however, the number of myofibers that contained displaced myonuclei increased by ∼ 3% with age
alone (P = 0.04; independent sample t-test; Fig. 5(b);
Fig. 6(c); arrows). LR exercise did not further increase
the number of myofibers with displaced nuclei in old
soleus muscles (Fig. 5(b); Fig. 6(d); arrows). Overall, no
change in average myofiber CSA was detected by twoway ANOVA in the soleus (Fig. 5(c)). Analysis by independent samples t-test showed a trend for decreased
6
myofiber CSA between young and old SED mice
(P = 0.065; Fig. 5(c)).
Further analysis of myofiber CSA distribution and of
histology of soleus muscles showed that young SED
mice have a normally distributed myofiber profile
(clustered at 2000 μm2; Fig. 5(d)) and uniform muscle
architecture (Fig. 6(a)). LR exercise in young mice
shifted myofiber CSA toward smaller myofibers, with
13.5% of all myofibers measured approximating
≤500 μm2 (Fig. 5(d)). This is likely due to the branching of myofibers, and contributes to loss of structural
uniformity and clustering of smaller myofibers
throughout sections of the young exercised soleus
(compare Fig. 6(a, b)). Age also influenced the distribution of myofiber CSA, with both SED and LR
cohorts of old mice displaying an overall shift in
myofiber CSA toward smaller myofibers (Fig. 5(d)),
compared with young mice. As for young mice, old LR
mice had a modest shift toward larger myofibers relative to old SED controls.
Exercise and muscle hypertrophy in old mice
Table 3. Phenotypic characterization of muscle weights standardized to tibia length in SED male C57Bl/6J mice aged 25, 105, and 117 weeks
Absolute mass
Relative mass, mg/tibia length, cm
25 weeks (n = 10) 105 weeks (n = 8) 117 weeks (n = 9) 25 weeks (n = 10) 105 weeks (n = 8) 117 weeks (n = 9)
Phenotype
Body weight (g)*
35.9 ± 0.7
Epididymal fat (g)*** 1.2 ± 0.1
Tibia length (mm)*
17.9 ± 0.14
32.0 ± 1.2 #
0.46 ± 0.1 #
17.5 ± 0.15#
32.4 ± 0.8 #
0.53 ± 0.1 #
17.3 ± 0.09#
Absolute mass, mg
20.1 ± 0.4
n/a
n/a
18.3 ± 0.6#
n/a
n/a
18.7 ± 0.5#
n/a
n/a
Relative mass, mg/tibia length, cm
25 weeks (n = 10) 105 weeks (n = 8) 117 weeks (n = 9) 25 weeks (n = 10) 105 weeks (n = 8) 107 weeks (n = 9)
Whole muscle weight(x1)
Quadriceps***
228.2 ± 3.7
Gastrocnemius*
175.7 ± 3.9
Tibialis anterior*
52.1 ± 1.3
EDL
11.8 ± 0.8
Soleus
8.6 ± 0.3
Triceps
113.7 ± 5.3
188.1 ± 6.9#
158.2 ± 7.8#
48.5 ± 1.7
10.7 ± 0.8
8.2 ± 0.9
105.0 ± 5.4
187.2 ± 3.2#
150.5 ± 4.3#
45.3 ± 1.1#
11.2 ± 1.1
8.0 ± 0.6
109.9 ± 2.5
127.5 ± 2.4
98.1 ± 2.3
29.2 ± 0.7
6.6 ± 0.4
4.8 ± 0.1
63.6 ± 3.2
107.8 ± 4.3#
90.5 ± 4.0#
27.8 ± 0.9
6.1 ± 0.4
4.7 ± 0.5
60.1 ± 3.1
108.3 ± 1.9#
87.1 ± 2.6#
26.2 ± 0.7#
6.5 ± 0.6
4.4 ± 0.2
63.6 ± 1.4
Values are means ± SE; asterisk (*) indicates an effect of age. *P < 0.05; ***P < 0.001. Hash (#) indicates significantly different from 25 weeks, P < 0.05.
No significant differences were observed between mice aged 105 and 117 weeks.
n, number of mice; n/a, not applicable; SED, sedentary.
Central myonuclei and branched myofibers in soleus of
young LR mice
To further investigate the presence of small myofibers and
central myonuclei in young LR mice, serial sections from
the soleus stained with H&E or pan-laminin and Hoechst
were analyzed (Fig. 7). Figure 7(a) shows two myofibers
with central myonuclei and subsequent serial sections
stained with laminin show that the sarcolemma starts to
invaginate into these myofibers (Fig. 7(b–c)) and progresses to where two distinct myofiber profiles are present
(Fig. 7(d–f)); each still retaining a central myonucleus.
Additional young mice aged 12 weeks were exercised
for 1 week on a voluntary running wheel (0 g resistance)
and soleus muscles from these exercised and agematched sedentary controls were sectioned and stained
with H&E (Fig. 8(a,b)). Control sedentary soleus had
normal architecture (Fig. 8(a)), whereas the exercised
soleus showed many small and large myotubes/young
myofibers with central myonuclei, confirming that
myonecrosis had occurred rapidly in response to the
unaccustomed exercise with resultant regeneration over
the 7 days (Fig. 8(b)).
Quadriceps muscles: myofiber number and size in
young and old SED and young LR mice
The quadriceps was selected for detailed analyses
because of the decline in relative muscle weight with
age, and increased muscle mass in young LR mice. As
there was no increase in mass of old LR quadriceps,
myofiber number and CSA were not further investigated.
No difference in total myofiber number was detected
between quadriceps of young and old SED mice
(Supporting Information Fig. S2(a)). Thus, the decline in
quadriceps weight with age can be attributed in part to a
significant reduction in the CSA of both fast- (19%) and
slow- (20%) type myofibers (P = 0.009 and P = 0.031
respectively; independent samples t-test; Supporting
Information Fig. S2(b)). Percentage distribution of fasttype myofibers demonstrated a striking shift in the
CSA of all myofibers toward smaller sizes with age
(Supporting Information Fig. S2(c)), while for slow-type
myofibers, there was a selective loss of larger myofibers
(Supporting Information Fig. S2(d)).
LR exercise in young mice contributed to an 18.5%
increase in the number of myofibers in quadriceps compared with young SED controls (P = 0.02; independent
samples t-test; Supporting Information Fig. S3(a)). Only
a trending increase in fast-type myofiber CSA was
observed between young LR and SED quadriceps
(P = 0.085; independent samples t-test; Supporting
Information Fig. S3(b)). The young LR quadriceps
showed a striking shift in the CSA of all fast-type
myofibers, and a modest shift in slow-type myofibers
toward larger sizes relative to young SED controls (Supporting Information Fig. S3(c,d)).
There was no evidence of myofiber splitting in the
quadriceps of young SED or old SED/LR mice (Supporting Information Fig. S4(a,c,d)), although this was seen
for young LR mice (Supporting Information Fig. S4(b)).
Central/displaced myonuclei were conspicuous on H&E
sections of the quadriceps (in rectus femoris and vastus
lateralis, medialis, and intermedialis) of young LR mice
(but not young SED; compare Supporting Information
Fig. S4(a,b)). While myofibers contained displaced
myonuclei in old (SED and LR) mice (Supporting Information Fig. S4(c,d)), there appeared to be little myofiber
necrosis, since myofibers with fragmented sarcoplasm
7
Soffe et al.
Fig. 4. Average relative muscle weights (a–f) and epididymal fat pad weight (g) from young and old, SED, and exercised (LR and HR)
mice. One-way ANOVA was used to determine the impact of LR and HR protocols on young mice and significant differences are
indicated by differing capital letters. The impact of age and LR exercise (activity) was determined by two-way ANOVA and differing
lower case letters indicate significant differences. P-values for the impact of age and activity and the interaction between these factors
(age × activity) are indicated within each graph. Data are mean ± SEM. n = 7–10 mice per group.
and inflammatory cells were not seen (and areas with
myoblasts and small myotubes resulting from early
regeneration as a result of myonecrosis were not evident)
in old muscles.
Discussion
When young (15–25 weeks) and old (107–117 weeks)
mice were subjected to increasing resistance exercise
8
using voluntary running wheels for 10 weeks, the old
mice ran less distance, were slower, and there was a
striking age-related difference in their diurnal running
patterns. While some skeletal muscles responded to the
resistance exercise by muscle hypertrophy, others did
not, and the increase in muscle mass was more pronounced in young, compared with old, mice.
Because all exercise was voluntarily and old mice ran
less, the intensity and duration of exercise were not
Exercise and muscle hypertrophy in old mice
Fig. 5. Morphometric characterization of soleus muscles from young and old, SED and LR mice. Entire transverse sections of soleus
muscles immunostained with antibodies to laminin or with H&E, were used to quantify the number of individual myofiber profiles (a),
proportion of myofibers with displaced nuclei (b), average myofiber size as cross-sectional area (CSA) (c) and myofiber size
distribution (d). P-values for the impact of age and activity and the interaction between these factors (age × activity) are indicated
within each graph. Data are mean ± SEM. n = 5–7 mice per group.
equivalent between young and old groups. Clearly, this
aspect can influence the extent of hypertrophy in
response to resistance exercise and we are aware that this
complicates comparisons between young and old mice
in our study.
Exercise performance and running patterns in young
and old mice
Young mice were subjected to two resistance protocols:
either low (LR) or high (HR) resistance. Only LR
9
Soffe et al.
Fig. 6. Transverse sections of soleus muscles stained with H&E from young SED (a), young LR (b), old SED (c), and old LR (d) mice.
Small myofibers with displaced (e.g., central) myonuclei (arrows) are visible in young LR mice (b). Myofibers with displaced
myonuclei were also seen in old soleus muscles from SED (c) and LR (d) mice (arrows). Scale is 50 μm.
exercise was tested in old mice because of limited
numbers of aged mice. Exercise performance and
running patterns were clearly affected by ageing with
decreased distance and speed for old mice. In addition,
while two distinct peaks of activity were seen in young
mice over 24 h (one 2 h after lights out (19:00 h) and the
other at 2 h prior to lights on (07:00 h)), old mice ran
almost exclusively during the dark phase (night) with
activity peaking around 19:00 h. An age-related decline
in wheel running activity is well documented in rodents
(Ingram, 2000; Cheng et al., 2013; McMahon et al.,
2014). In our study, shorter distances run by old mice
were attributed to a decrease in speed and, although not
measured in this study, a decline in the amount of time
spent running is also common for ageing mice
(McMahon et al., 2014). The different diurnal patterns of
running between young and old mice are similar to those
reported by others in C57Bl/6J males (Houtkooper et al.,
2011). The precise reason why old mice did not display
the second peak of activity (around 07:00 h) seen in
young mice is not clear, although contributing factors
may be age-related diminished endurance (decreased
oxygen consumption, V02max and maximal exercise
capacity; Schefer & Talan, 1996) and altered circadian
rhythms (Valentinuzzi et al., 1997), since there are
strong circadian patterns of gene regulation that control
metabolism (Shavlakadze et al., 2013). Testosterone is
also a strong driver for exercise in mice, as removal of
endogenous testosterone by orchiectomy results in
almost complete cessation of voluntary wheel running
(Ibebunjo et al., 2011). Whether testosterone concentrations decline in old male mice is unclear. One study
10
showed no difference in serum testosterone of 6- and
29-month-old C57Bl/6J mice (Hamrick et al., 2006),
whereas another reported that serum testosterone concentrations are halved in 24-month-old C57Bl/6J mice
compared with 4 months (Kovacheva et al., 2010). It is
possible that testosterone is high in young 4-month-old
mice and declines by 6 months, with no further decline
with ageing. While administration of testosterone to
28-month-old male C57Bl/6J mice in combination with
low-intensity exercise (treadmill three times/week)
showed many benefits and it was stated that testosterone
concentrations declined at this late age, no measurements of testosterone levels were provided (Guo et al.,
2012).
While C57Bl/6J mice are less avid runners than some
other mouse strains (Irintchev & Wernig, 1987;
Lightfoot et al., 2004; Nehrenberg et al., 2009), male
C57Bl/6J mice aged 8–26 weeks run approximately
7.5 km per night on wheels without resistance (Allen
et al., 2001; Lerman et al., 2002). This distance is similar
to our study where young male mice ran approximately
6–7 km per night on voluntary running wheels (without
resistance), with decreased distance when a 2–4 g load
was applied. The inverse relationship between applied
resistance and exercise performance (distance run and
speed) has been demonstrated previously (Konhilas
et al., 2005); although young adult (∼3 months)
C57Bl/6J male mice tolerated a 7 g increase in wheel
load (∼ 25% of total body weight) before the distance
run decreased significantly (Konhilas et al., 2005). The
difference between these two studies (using similar
mice) regarding the tolerance to increasing resistance
Exercise and muscle hypertrophy in old mice
Fig. 7. Transverse serial sections of soleus muscles stained with H&E (a) and pan-laminin with Hoechst (b–f) from young LR mice.
Arrows indicate split or branched myofibers with central myonuclei. Sections are cut at 8 μm thickness. Serial sections are sequential,
apart from b and c (8 μm apart) and e and f (16 μm apart). Scale bar is 50 μm.
may be due to different exercise protocols (the timing of
resistance application) and the wheel setups used.
The initial distance run by old male mice on wheels
without resistance in our study (∼3 km over 24 h) was
about half that for young mice, although old mice
maintained stable levels of daily running distance over
many weeks (W1 to W8). Decreased voluntary exercise
performance with age has been previously reported by
our group for male FVB mice (McMahon et al., 2014)
and by others in female (Cheng et al., 2013) and male
(Valentinuzzi et al., 1997) C57Bl/6J mice. In the
present study, while old mice ran less than young mice,
they had the capacity to run voluntarily even when a
load of ∼ 13% of total body weight (4 g) was applied to
the wheels. Notably, the most enthusiastic old male
mouse maintained initial levels of running activity
(W1) well into W6 when 2 g of wheel resistance was
applied, and remained the highest achiever until
W8 (3 g).
Impact of resistance exercise on adult and old mice
Food consumption in adult and old sedentary and
exercised mice
The apparent food consumption of all mice was measured (by removal of food from the feeder box)
throughout the 10 weeks study. It should be noted that
because our mice were housed on pine shavings,
instead of wire bottom cages, we were unable to quantify the amount of dropped food: yet, old mice are
messy eaters and drop more food and this confounds
many measurements of food consumption in ageing
rodents (reviewed in Starr & Saito, 2012). A detailed
comparison of food intake in sedentary C57Bl/6J male
mice aged about 2, 6, 12, 20 and 29 months showed
that the amount of food removed from the feeder box
was constant throughout life, apart from a significant
(transient) increase at 20 months (Starr & Saito, 2012).
This age corresponded to the time when food dropping
11
Soffe et al.
Impact of resistance exercise on muscle phenotype
Fig. 8. Transverse sections of soleus muscles stained with H&E
from young sedentary (a) and exercised mice (aged 12 weeks)
for 1 week (no resistance) (b). The exercised soleus contains
many myotubes/small myofibers, that are evidence of recent
regeneration (b). Scale bar is 50 μm.
dramatically increased (doubled) for all older mice and
thus there is tendency to overestimate the amount of
food consumed by mice aged more than 20 months.
This study concluded that actual food consumption
only decreased in the very old mice aged 29 months,
although the amount of food intake per body weight
was constant for all mice aged from 6 to 29 months.
Despite this caveat, during our 10-week study, young
SED mice (aged 4–6 months) consumed more food and
their body weight increased significantly (probably
because of increased adiposity), compared with less
apparent food intake by old SED mice (aged 25–27
months).
The voluntary resistance exercise protocols (LR and
HR) in our study did not increase food intake in young
mice, but did slow the rate of weight gain and reduce
epididymal fat. In contrast, for old LR mice there was a
trend for increased food intake, although no impact on
body weight or epididymal fat weight was detected.
Long-term access to a voluntary running wheel (greater
than 4 weeks) increased food intake and decreased adiposity among adult male and female mice (Swallow
et al., 2001) and rats (Tokuyama et al., 1982). However,
studies in adult male C57Bl/6J and DBA/J mice (strains
considered to have lower running activity) found no difference in food consumption after 3 to 13 weeks of
voluntary wheel exposure (Harri et al., 1999; Jung et al.,
2010).
12
In young mice, LR exercise increased the mass of quadriceps, gastrocnemius, and soleus muscles, whereas HR
increased the mass of only quadriceps and soleus
muscles (with similar efficacy to that of LR exercise).
The lack of impact of HR exercise on gastrocnemius
muscle mass may be explained by the decrease in distance run with HR compared with LR. Similar to our
study, no additional benefit of HR (capped at 12 g) compared with LR exercise (capped at 5 g) was reported for
increasing soleus muscle mass in adult male C57Bl/6J
mice (Konhilas et al., 2005; Table 4).
In our study, the soleus muscle showed the most
hypertrophic response to resistance exercise in all mice
and this accords with studies in rats and mice subjected
to resistance wheel running, where the soleus hypertrophied more than other limb muscles (Konhilas et al.,
2005; Legerlotz et al., 2008; Table 4). In contrast, voluntary wheel running protocols (no resistance) ranging
between 1 and 8 weeks were insufficient to increase the
mass of soleus, gastrocnemius or TA in adult C57Bl/6J
mice (Allen et al., 2001; Konhilas et al., 2005;
Pellegrino et al., 2005; Table 4). The lack of a hypertrophic response by many hindlimb muscles to resistance
wheel running in our study (TA, EDL) also agrees with
other reports (Konhilas et al., 2005; Landisch et al.,
2008; Legerlotz et al., 2008). Although not analyzed in
this study, the plantaris is widely studied in rats and
shows a strong hypertrophic response to exercise
(Table 4).
While the impact of resistance wheel running has been
studied in juvenile and adult mice and rats (Table 4), we
could not find any reports of studies using resistance
wheels for old rodents. Whether old muscle has a diminished capacity to undergo a compensatory increase in
mass and strength in response to increased loading compared with young muscle is not clear. In humans, progressive resistance training matches exercise intensity
between young and old participants by measuring each
individual’s one-repetition maximum, with every
increase in resistance/intensity then calculated as a percentage of that maximum (as in Kosek et al., 2006; Mero
et al., 2012). Thus, young and old participants perform
the same number of repetitions and sets, at the same
relative intensity, allowing for a fair comparison between
both ages. The difficulty in matching exercise intensities
between young and old mice under voluntary conditions
complicates the comparison of the outcome of exercise
in animals of different ages.
The benefits of exercise on ageing muscles have been
described for many animal models and human subjects.
Long-term exercise, even without resistance, initiated
in young adult rats (Brown et al., 1992) and mice
(McMahon et al., 2014) benefits selected skeletal
muscles with ageing. Short-term voluntary wheel
running of old mice for 1 month (from 22 to 23 months)
Exercise and muscle hypertrophy in old mice
Table 4. Summary of selected voluntary wheel running protocols (with and without resistance loading) in mice and rats and the effects on muscle mass
Rodent model
Species/strain
Resistance protocol
Sex
Age
Duration
Resistance wheel running (mice and rats)
Mouse ICR
M
19 weeks
4 weeks
Mouse C57BL/6J M
12 weeks
7 weeks
Rat SD
M
4 weeks
6.5 weeks
Rat SD
M
5 weeks
8 weeks
Rat SD
M
5 weeks
10 weeks
30 weeks
19 weeks
16 weeks
Muscle(s) affected
Applied load
Relative increase in muscle
mass
Up to 10 g
2 g (stop inertia)
NC: TA (absolute mass)
NC: SOL, GASTROC, PL,
or TA
+20% (approx.) SOL
+20% (approx.) SOL
+27% in SOL
+29% in SOL; +23% in PL
+17% in VL; +21% in
ECRL-B
NC: GASTROC, or TA
+6% in PL
+31% in PL
NC: PL
+13% in PL
NC: PL
Up to 5 g
Up to 12 g
4.5 g (stop inertia)
Up to 527 g (173% BW)
4.5 g (stop inertia)
Up to 220 g; (74% BW)
Capped 5% BW
Unloaded wheel running (mice and rats)
Mouse C57BL/6J M
8–10 weeks
1,2, and 4 weeks
Mouse C57BL/6J M
Adult
8 weeks
Mouse C57BL/10 M/F 4 weeks
∼ 9 weeks
N/A
N/A
N/A
Mouse FVB
N/A
M
16 weeks
56 weeks
96 weeks
Rat SD
F
Juvenile
4 weeks
N/A
Rat SD
M
Not specified
6 weeks
N/A
Rat LE
F
16 weeks
20 weeks
N/A
92 weeks
NC: GASTROC, PL, or TA
NC: SOL, GASTROC, or TA
+28% in soleus; NC: EDL,
or TA
+76% EDL; NC: QUAD, TA,
or T
+22% QUAD; NC: EDL, TA,
or T
+30–33% in SOL
(absolute mass)
+17% in PL
(absolute mass)
+13–20% in SOL
(absolute mass)
NC: PL
+24.5% in SOL
+10.5% in EDL
+56.5% in SOL; NC: EDL
References
Ishihara et al., 2002
Konhilas et al., 2005
Legerlotz et al., 2008
Ishihara et al., 1998
Kariya et al., 2004
Allen et al., 2001
Pellegrino et al., 2005
Landisch et al., 2008
McMahon et al., 2014
Munoz et al., 1994
Rodnick et al., 1989
Brown et al., 1992
BW, body weight; ECRL-B, extensor carpi radialis longus and brevis; EDL, extensor digitorum longus; F, female; GASTROC, gastrocnemius; g, grams;
LE, Long–Evans; M, male; N/A, not applicable; NC, no change; PL, plantaris; QUAD, quadriceps; SD, Sprague-Dawley; SOL, soleus; TA, tibialis anterior;
T, triceps brachii; VL, vastus lateralis.
prevented the loss of neuromuscular junctions (Valdez
et al., 2010), as did long-term exercise for 4 or 10
months (from 21 to 25 months or 18 to 28 months,
respectively; Cheng et al., 2013), plus life-long exercise
(4 to 28 months) prevented sarcopenia in quadriceps,
with variable benefits on other muscles (McMahon et al.,
2014). An important question that we attempted to
address in the present study was whether exercise introduced later in life will reduce the age-related loss of
muscle mass (sarcopenia). Even though exercise duration and intensity was not matched between young and
old mice, and the old mice ran shorter distances, hypertrophy of soleus muscles did occur in both ages,
although to a lesser extent in the old mice.
We carried out detailed histological analyses on soleus
and quadriceps muscles to more fully understand the
cellular impact of exercise on the young and old mice.
The age-related loss of muscle mass in old mice is
accounted for by myofiber atrophy and reduced numbers
of myofibers that varies in extent with gender and
between different muscles (Sheard & Anderson, 2012).
Our detailed histological analyses showed similar
numbers of individual myofiber profiles in transverse
sections of young and old soleus and quadriceps
muscles, with reduced myofiber size (CSA) only evident
in quadriceps. It should be noted that in transverse sections, numbers of myofiber profiles do not necessarily
reflect the total number of myofibers because myofiber
branching or splitting can be a confounding factor
(Ontell, 1986; Shavlakadze et al., 2010b), and such split
myofibers can appear with age (Pichavant & Pavlath,
2014).
Myofibers with displaced (nonperipheral and often
central) myonuclei were also increased in old SED and
LR soleus muscles. The precise reasons for this are hard
to determine since such central myonuclei can result
from myonecrosis and subsequent regeneration of adult
myofibers (McGeachie et al., 1993) as well as from
13
Soffe et al.
denervation of myofibers (Lu et al., 1997). The incidence of myofiber necrosis (that results in inflammation,
myogenesis, and regeneration) would appear to be low in
mature and old healthy muscles (in the absence of
trauma; Grounds, 2014), whereas there are increasing
reports of denervation of old myofibers (Aagaard et al.,
2010; Deschenes et al., 2010; Valdez et al., 2010; Chai
et al., 2011; Cheng et al., 2013).
A striking observation for young soleus muscles was
that LR exercise resulted in a large number of small
myofibers with central myonuclei and smaller CSA
(≤500 μm2). These small myofibers with displaced
myonuclei most likely result from an initial acute
response of myonecrosis, myogenesis, and regeneration
following the unaccustomed exercise. In a detailed study
of six mouse strains subjected to voluntary wheel exercise, Irintchev and Wernig (1987) reported that the
soleus muscle was the most susceptible to such initial
exercise-induced myonecrosis, although this was
affected by strain and was variable (Irintchev & Wernig,
1987). It is important to note that subsequent
myonecrosis did not seem to result from the same exercise over many months, presumably because of adaptation after the first acute bout of myonecrosis: some
branched/split myofibers remained even at 11 months
(characterized by central myonuclei and small muscle
profiles; Irintchev & Wernig, 1987). It was not clear
whether the abundance of split myofibers in the young
soleus after 10 weeks of LR exercise is just a result of the
more intense exercise regime (compared with no resistance) or might also reflect subsequent cycles of
myofiber necrosis and regeneration associated with
increasing wheel resistance. We showed that many
myotubes had formed by one week after starting wheel
running (without resistance) in young mice (aged 12
weeks) and these can only have resulted from rapid
myofiber necrosis and regeneration. The similarity of
myotube size, and absence of areas of myonecrosis,
myoblasts, or many small myotubes at the time of sampling, strongly suggests that a subsequent bout of
myonecrosis did not occur throughout this first week of
wheel exercise.
As in the soleus, myofiber splitting was evident in the
quadriceps muscle of young LR mice: however, the distribution varied and may be due to different mechanical
loadings on quadriceps muscle groups or variable susceptibility to myonecrosis under these physiological
conditions. While central myonuclei were not quantified
for the quadriceps, they were not observed in quadriceps
muscles of young SED mice, were occasionally
observed in young LR mice and, as for old soleus
muscles, increased in all old quadriceps muscles irrespective of exercise. Increased numbers of centrally
nucleated myofibers with ageing have been previously
reported in quadriceps muscles of C57Bl/6J mice (comparisons between 3 months and 24 months of age;
gender not noted), and appear to be correlated with
14
atrophic myofibers (Sakuma et al., 2008). Notably
however, central myonuclei are rarely observed in old
female C57Bl/6J mice (aged up to 29 months; Barns
et al., 2014), thus indicating that this might be a feature
of male, but not female, mice. An increase in branched
myofibers with central myonuclei is well documented
after myonecrosis and regeneration (either because of
experimental injury or endogenous damage in dystrophic muscle; Ontell, 1986; Faber et al., 2014; Pichavant
& Pavlath, 2014). In ageing muscle (20–21 months),
branched/split myofibers are also evident and a recent
study in isolated myofibers demonstrates that they occur
without evidence of regeneration, indicating a different
and unknown mechanism for this phenomenon
(Pichavant & Pavlath, 2014). Another pathology that is
widely reported only in ageing male mouse muscles is
the accumulation of tubular aggregates: while these
increase in male muscles from 6 months of age they were
not seen in any muscles of female mice examined up to
19 (Agbulut et al., 2000; Nishikawa et al., 2000;
Chevessier et al., 2004) or even at 24 months of age
(Kuncl et al., 1989). Thus, issues of gender need to be
carefully considered when discussing such pathological
changes in old rodent muscles.
Perspectives
Resistance exercise is widely proposed as a strategy to
combat age-related loss of muscle mass and function
(sarcopenia) in humans. This is the first study to assess
the impact of increasing resistance exercise using voluntary wheel running on muscles of old mice. The low
resistance exercise regime (with weight increasing from
1–4 g) over 12 weeks showed that old male C57Bl/6J
mice ran less than young mice, and had only a single
peak of nocturnal running activity. In response to exercise, the most striking increase in mass (hypertrophy)
was seen for the soleus of young mice and was associated with histological evidence of exercise-induced
myonecrosis and muscle regeneration. Exercise-induced
hypertrophy also occurred for the quadriceps and gastrocnemius muscles in young, but not old, mice: other
muscles did not hypertrophy at either age. Hypertrophy
was less pronounced and there was no evidence of
myofiber damage in old mice: this might reflect reduced
exercise capacity with age. While resistance exercise
resulted in some hypertrophy of old muscles, exercise
may also have benefits on innervation which would help
maintain other aspects of muscle function and coordination, although this was not evaluated in the present study.
It is emphasized that gender needs to be considered in
ageing rodents and that benefits cannot be generalized
across all muscles.
Key words: Exercise, ageing, hypertophy, old skeletal
muscle, myofiber splitting.
Exercise and muscle hypertrophy in old mice
Acknowledgements
This study was supported by funding from the Australian
Research Council (Grant LP120100520 to M. G.) and postgraduate research scholarships from the University of Western Australia and the Centre for Cell Therapy and Regenerative Medicine
Top-Up Scholarship, School of Medicine and Pharmacology,
University of Western Australia and Harry Perkins Institute of
Medical Research, Perth, Western Australia (Z. S.).
Author contributions
Z. S., H. G. R, M. D. G., and T. S. conception and design
of research; Z. S. performed experiments; Z. S.,
C. D. M., T. S. analyzed data; Z. S. , H. G. R., C. D. M.,
M. D. G., and T. S. interpreted results of experiments;
Z. S. and T. S. prepared figures; Z. S., C. D. M., M. D. G.
wrote the manuscript; Z. S., H. G. R., C. D. M., M. D. G.,
and T. S. approved final version of the manuscript.
Supporting information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
Figure S1. Average distance run by young HR mice per
hour over 24 h. Each hourly value represents an average
over 1–2 weeks. Values are calculated for week (W) 1
with resistance set at 0 g (A); W2 (1 g) (B); W3 (2 g)
(C); W4 (3 g) (D); W5 and W6 (4 g) (E); W7 and W8
(5 g) (F); W9 and W10 (6 g). Where young LR mice ran
at the same resistance (G), distances run per hour over
24 h were added for comparison (A–E). Asterisk (*)
indicates significant differences between young LR and
HR mice at P < 0.05; (*) P < 0.01; (**); P < 0.001 (***).
n = 7 mice per group. Black and white bars under the
X-axis indicate dark (1900-0700 h) and light (0700–
1900 h) phases.
Figure S2. Morphometric characterization of quadriceps muscles from young and old, SED mice. Entire
transverse sections of soleus muscles stained with
NADH-TR were used to quantify number of individual
myofiber profiles (A), average myofiber size as crosssectional area (CSA) (B) and myofiber size distribution
of both fast (C) and slow (D) type myofibers. Asterisks
(*) indicates significant differences between young and
old SED mice at P < 0.05. Data are mean ± SEM.
n = 4–7 mice per group.
Figure S3. Morphometric characterization of quadriceps muscles from young SED and LR mice. Entire
transverse sections of soleus muscles stained with
NADH-TR were used to quantify number of individual
myofiber profiles (A), average myofiber size as crosssectional area (CSA) (B) and myofiber size distribution
of both fast (C) and slow (D) type myofibers. Asterisk (*)
indicates significant differences between young SED
and LR mice at P < 0.05. Data are mean ± SEM. n = 4–7
mice per group.
Figure S4. Morphometric characterization of quadriceps muscles from young and old, SED and LR mice.
Transverse sections of quadriceps muscles (rectus
femoris) stained with H&E from young SED (A), young
LR (B), old SED (C) and old LR (D) mice (40× magnification). Myofibers with displaced (e.g., central)
myonuclei (arrows) are visible in young LR mice (B).
Myofibers with displaced myonuclei were also seen in
old quadriceps muscles from SED (C) and LR (D) mice
(arrows). Scale bar is 50 μm.
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