Experimental Gerontology 154 (2021) 111519

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Experimental Gerontology
journal homepage: www.elsevier.com/locate/expgero

Prolonged caloric restriction ameliorates age-related atrophy in slow and

fast muscle fibers of rat soleus muscle
Yuhei Mizunoe a, 1, Masaki Kobayashi b, 1, Hiroki Saito b, Akifumi Goto b, Ryota Migitaka b,
Kumi Miura b, Naoyuki Okita c, Yuka Sudo b, Ryoma Tagawa b, Miki Yoshida b, Ai Umemori b,
Yoshimi Nakagawa d, e, Hitoshi Shimano a, e, f, g, Yoshikazu Higami b, *
a
Department of Internal Medicine (Endocrinology and Metabolism), Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
b
Laboratory of Molecular Pathology & Metabolic Disease, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan
c
Division of Pathological Biochemistry, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Sanyo-onoda, Yamaguchi, Japan
d
Division of Complex Biosystem Research, Department of Research and Development, Institute of Natural Medicine, University of Toyama, Toyama, Japan
e
International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Ibaraki, Japan
f
Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Ibaraki, Japan
g
Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Tokyo, Japan

A R T I C L E I N F O A B S T R A C T

Section Editor: Christiaan Leeuwenburgh Aging causes loss of skeletal muscle mass and function, which is called sarcopenia. While sarcopenia impairs the
quality of life of older adults and is a major factor in long-term hospitalization, its detailed pathogenic mech­
Keywords: anism and preventive measures remain to be identified. Caloric restriction (CR) suppresses age-related physio­
Aging logical and pathological changes in many species and prolongs the average and healthy life expectancy. It has
Skeletal muscle
recently been reported that CR suppresses the onset of sarcopenia; however, few studies have analyzed the effects
Muscle fiber type
of long-term CR on age-related skeletal muscle atrophy. Thus, we investigated the aging and CR effects on soleus
Caloric restriction
(SOL) muscles of 9-, 24-, and 29-month-old ad libitum-fed rats (9AL, 24AL, and 29AL, respectively) and of 29-
month-old CR (29CR) rats. The total muscle cross sectional area (mCSA) of the entire SOL muscle significantly
decreased in the 29AL rats, but not in the 24AL rats, compared with the 9AL rats. SOL muscle of the 29AL rats
exhibited marked muscle fiber atrophy and increases in the number of muscle fibers with a central nucleus, in
fibrosis, and in adipocyte infiltration. Additionally, although the decrease in the single muscle fiber cross-
sectional area (fCSA) and the muscle fibers' number occurred in both slow-type and fast-type muscle fibers,
the degree of atrophy was more remarkable in the fast-type fibers. However, CR suppressed the muscle fiber
atrophy observed in the 29AL rats' SOL muscle by preserving the mCSA and the number of muscle fibers that
declined with aging, and by decreasing the number of muscle fibers with a central nucleus, fibrosis and
denervated muscle fibers. Overall, these results revealed that advanced aging separately reduces the number and
fCSA of each muscle fiber type, but long-term CR can ameliorate this age-related sarcopenic muscle atrophy.

1. Introduction
Sarcopenia is a condition characterized by age-related loss of skeletal
muscle mass and function (Cruz-Jentoft and Sayer, 2019). In humans,
generally, the skeletal muscle mass starts to decrease from approxi­
mately 45 years of age; compared with the peak age (20 years), it de­
clines by 30% to 40% at 80 years of age, which is especially prominent in
the lower limbs (Lexell et al., 1988; McCormick and Vasilaki, 2018). In
male Wistar rats, hindlimb muscle mass also progressively declined with
age, reaching approximately 50% atrophy by 24 months of age
compared with controls at 8 months of age (Pannérec et al., 2016).
While many mechanisms, such as mitochondrial dysfunction (Marzetti
et al., 2013), increased apoptosis (Dupont-Versteegden, 2005), sup­
pression of satellite cell function (Fukada, 2018), increased reactive
oxygen species (Gomes et al., 2017), and abnormal regulation of auto­
phagy (Jiao and Demontis, 2017), have been reported, the onset

* Corresponding author at: Laboratory of Molecular Pathology & Metabolic Disease, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641
Yamazaki, Noda, Chiba 278-8510, Japan.
E-mail address: higami@rs.tus.ac.jp (Y. Higami).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.exger.2021.111519
Received 16 March 2021; Received in revised form 5 August 2021; Accepted 10 August 2021
Available online 17 August 2021
0531-5565/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Mizunoe et al.
mechanism of sarcopenia has not been identified in detail yet (Wagat­
suma and Sakuma, 2012; McCormick and Vasilaki, 2018).
Skeletal muscle mainly comprises multinucleated muscle fibers. Each
muscle fiber mostly expresses only one myosin heavy chain (MyHC)
isoform, and the MyHC isoforms are MyHC I (type I), MyHC IIA (type
IIA), MyHC IIDX (type IID/X), and MyHC IIB (type IIB). Generally, in
rodents, muscle fibers include one type of slow-twitch fiber (MyHC I)
and three types of fast-twitch fibers (MyHC IIA, MyHC IID/X, and MyHC
IIB). Notably, human skeletal muscles do not contain MyHC IIB
(Schiaffino and Reggiani, 2011). MyHC I and MyHC IIA fibers are
oxidative, whereas MyHC IIB fibers are primarily glycolytic, although
the fiber type specification varies between species (Schiaffino and
Reggiani, 2011; Wang and Pessin, 2013; Talbot and Maves, 2016).
Additionally, MyHC IIDX fibers are intermediate to MyHC IIA and MyHC
IIB fibers in regard to fiber size and oxidative potential (Delp and Duan,
1996). It has been reported that aging reduces the muscle mass of the
extensor digitorum longus muscle (mainly composed of fast-type fibers)
by 16% and of the soleus (SOL, mainly composed of slow-type fibers)
muscle by 18% between 6 and 36 months of age (Brown and Hasser,
1996). To date, to distinguish fiber types, immunohistochemistry or
immunoblotting analysis using monoclonal antibodies specific to
various isoforms of MyHCs have been used (Schiaffino et al., 1989). It
has been reported that MyHC I fibers are more sensitive to inactivity and
denervation-induced atrophy, whereas MyHC II fibers are more
vulnerable to cancer cachexia, diabetes, and aging sarcopenia (Larsson
et al., 1993; Larsson et al., 1995; Picard et al., 2011; Schiaffino and
Reggiani, 2011; Wang and Pessin, 2013).
Caloric restriction (CR) is the most robust, reproducible, and simple
experimental manipulation known to extend lifespan and delay onset of
many age-associated pathophysiological changes in various laboratory
rodents (Sinclair, 2005; Ingram and de Cabo, 2017). While the mecha­
nisms by which CR extends lifespan are not completely understood, the
action of CR is mediated by various signaling pathways, such as growth
hormone/insulin-like growth factor/insulin signaling, adenosine
monophosphate-activated protein kinase signaling, and sirtuin activity
(Hoshino et al., 2018). Notably, it has been reported that CR is an effi­
cient nonpharmacological intervention for preventing sarcopenia in
rodents (Hepple et al., 2008; Marzetti et al., 2008; Wohlgemuth et al.,
2010). Recently, Faitg et al. have reported that 13 months of CR exerted
anti-aging effects specifically on SOL muscle oxidative fibers via
improved mitochondrial morphology in Sprague-Dawley rats (Faitg
et al., 2019). They also showed that CR exerted no effect on skeletal
muscle mass, but it prevented the aging effect on the muscle fiber type
composition (Faitg et al., 2019). Additionally, McKiernan et al. have
reported that the loss of muscle mass between 21 and 36 months was
significantly greater in the AL rats compared with the CR rats: 40% loss
in AL soleus verses 10% in CR soleus in Fischer 344/BN F1 hybrid rats
(McKiernan et al., 2004). However, little is known about the long-term
effects of CR (> 2 yr) on each muscle fiber type. Additionally, few
studies have thoroughly analyzed CR-related changes with aging in
skeletal muscle, e.g. separately for each fiber type using multicolor
imaging. Moreover, it is important to consider a strong interaction be­
tween genotype and the degree of CR effects (Sohal and Forster, 2014;
Ingram and de Cabo, 2017).
Therefore, in this study, focusing on the SOL muscle, which com­
prises slow-type and fast-type muscle fibers, we investigated the age-
related muscle atrophy of each fiber type and the long-term effect of
CR on muscle sarcopenic atrophy in Wistar rats.
2. Materials and methods
2.1. Animals and diets
All animal experiments and protocols were conducted in accordance
with the Fundamental Guidelines for Proper Conduct of Animal Exper­
iment and Related Activities in Academic Research Institutions under
2
Experimental Gerontology 154 (2021) 111519
the jurisdiction of the Ministry of Education, Culture, Sports, Science
and Technology of Japan, and were approved by the Ethics Review
Committee for Animal Experimentation at the Tokyo University of Sci­
ence (approval numbers: Y17051, Y18060, Y19056, Y20045). Male 5–7-
week-old Wistar rats were purchased from Clea Inc. (Tokyo, Japan) and
maintained under specific pathogen-free conditions in the Laboratory
Animal Center in the Faculty of Pharmaceutical Sciences, Tokyo Uni­
versity of Science. The animals, their husbandry, and diet have previ­
ously been described in detail (Okita et al., 2015; Narita et al., 2018). All
rats were provided with water and fed ad libitum with a Charles River
Formula-1 diet (Oriental Yeast, Japan). At 12 weeks of age, the rats were
divided into two groups: one was fed ad libitum (AL group) and the other
was calorie restricted (CR group; 70% of AL energy intake). At 9, 24, and
29 months of age, rats were euthanized under isoflurane anesthesia
(Mylan, Canonsburg, PA, USA) 3–5 h after the lights came on. CR rats
were fed every other day. We studied a 9-month-old AL group (9AL, n =
3), a 24-month-old AL group (24AL, n = 6), a 29-month-old AL group
(29AL, n = 4), and a group of 29-month-old rats subjected to CR for 26
months (29CR, n = 9). After euthanasia, the SOL muscle from the right
leg of every rat was dissected and its mass and the total body mass were
measured. Each SOL muscle was immediately frozen in cold 2-methylbu­
tane with liquid nitrogen for subsequent histological examination. The
histological experiments were performed using the randomly selected
rats from each group (9AL, n = 3; 24AL, n = 3; 29AL, n = 4; 29CR, n =
4). The frozen muscles were sliced into 10-μm cross sections by cutting
at the mid-belly of the muscle using a cryostat for histological and
immunofluorescence analysis. The SOL muscle from the left leg of each
rat was removed and immediately frozen in liquid nitrogen and stored at
− 80 ◦ C for immunoblotting analysis.
2.2. Histological analysis
The muscle sections were stained with hematoxylin and eosin (H&E)
and Masson's trichrome staining. The stained sections were scanned
under a microscope, using a charge-coupled device camera (Nikon,
Tokyo, Japan) at a magnification of 100×, 200×, and 400×. Total
muscle cross-sectional area (mCSA) and single muscle fiber cross-
sectional area (fCSA) were defined under H&E staining using the BZ-
H3A application software (Keyence, Osaka, Japan) and ImageJ 1.43u/
Java1.6.0_22 software (http://rsbweb.nih.gov/ij/), and the fiber num­
ber was counted. Muscle fiber with central nuclei and denervated
muscle fiber were counted using image J.
2.3. Immunohistochemical analysis
For immunohistochemistry, frozen muscle sections were fixed with
4% paraformaldehyde for 15 min on ice. After permeabilization with
cold methanol for 10 min and blocking with Blocking One Histo (Nacalai
Tesque, Inc., Kyoto, Japan) for 90 min at room temperature, the speci­
mens were incubated with primary antibodies at 4 ◦ C overnight, fol­
lowed by secondary antibodies for 60 min at room temperature. Images
were acquired by a BZ-X800 microscope (Keyence, Osaka, Japan) at a
magnification of 200×. The antibodies used in this study are shown in
Supplementary Tables 1 and 2.
2.4. Immunoblotting analysis
Immunoblotting was performed with Immunostar® LD chemilumi­
nescent substrates (Wako). Signals were detected with an LAS-3000
image analyzer (Fujifilm, Tokyo, Japan) as previously described (Miz­
unoe et al., 2017). The antibodies used in this study are shown in Sup­
plementary Tables 1 and 2.
2.5. Statistical analysis
Statistical analyses of the four groups were performed using one-way
Y. Mizunoe et al.
analysis of variance followed by a Tukey–Kramer test (with R software)
or a Student's t-test. Analyses were primarily performed using GraphPad
Prism 8 (GraphPad Software, Inc., San Diego, CA, USA), which was also
used to draw Gaussian plots (Hansson et al., 2020) and box plots, and to
perform statistical analysis of the box plots. Data are presented as mean
± standard deviation (SD). A p value <0.05 was considered statistically
significant.
3. Results
3.1. Muscle atrophy rapidly accelerates at a late stage of aging in rats
In AL rats, the body mass increased with age up to 24 months (24AL),
and was significantly decreased at 29 months (29AL) compared with
that in the 24AL rats (Fig. 1A, B). Similarly, the SOL muscle mass and its
value normalized to body mass, a sarcopenia index (Edström et al.,
2006), were significantly reduced in the 29AL rats, suggesting that
prolonged aging causes muscle atrophy in the SOL muscle (Fig. 1C, D).
Subsequently, we analyzed various morphometrical parameters of the
SOL muscle, such as mCSA, total fiber number, the proportion of the
muscle fibers to the mCSA, and the proportion of the fibrosis area, by
H&E and Masson's trichrome staining. mCSA and the proportion of
muscle fiber area to mCSA were markedly reduced in the 29AL rats
compared with that in the 9AL or 24AL rats (Fig. 2A–C). Furthermore,
the total fiber number was significantly decreased in the 29AL rats
(Fig. 3B). These morphological observations indicate that the muscle
atrophy was attributable to the decline in both muscle fiber area and
muscle fiber number. In contrast, the proportion of fibrosis area mark­
edly increased in the 29AL rats (Fig. 2A, D). Additionally, the presence of
ectopic fat infiltration and many small angular fibers, which are simply
indirect signs of muscle fiber denervation (Scelsi et al., 1980; Rowan
et al., 2011; Si et al., 2020), were detected only in the 29AL rats (Fig. 2A
and Supplementary Fig. 1). Furthermore, the proportion of central
nuclei-containing muscle fibers, an indicator of muscle regeneration
(Forcina et al., 2020), also remarkably increased in the 29AL rats
(Fig. 2A, E). Neither of these morphological phenotypes was observed in
the 9AL or 24AL rats (Fig. 2A, C, E). These results indicate that age-
related muscle atrophy and fibrosis in the rat SOL muscle dramatically
progressed after 24 months of age.
Next, to investigate muscle atrophy in detail, we distinguished each
muscle fiber type in the SOL muscle using immunostaining with anti­
bodies against MyHC IIA and MyHC I. In the MyHC IIA fibers, the fiber
number did not change in the 24AL rats compared with that in the 9AL
rats, but it was significantly decreased at 29 months compared with that
at 24 months (Fig. 3A–C). The fCSA of the MyHC IIA fibers significantly
A B
9 24 29
700
600
Body mass (g)
Body mass (g)
500
400
300
200
AL
100
CR
0 0
10 25 40 55 70 85 100 115 130
Age (week)
Fig. 1. Changes in SOL muscle mass with aging and CR. We compared AL rats (9AL, 24AL, 29AL) with CR rats (29CR) until 29 months of age. (A) Body mass with
time of 29AL and 29CR rats. (B) Body mass and (C) SOL muscle mass of 9AL, 24AL, 29AL, and 29CR rats. (D) SOL muscle mass normalized to body mass. 9AL, n = 3;
24AL, n = 6; 29AL, n = 4; 29CR, n = 9. Values are mean ± SD. Differences between values were statistically analyzed by Tukey's test after one-way ANOVA. Statistical
significance is shown as *p < 0.05, **p < 0.01.
Experimental Gerontology 154 (2021) 111519
decreased already at 24 months in the AL rats, and more remarkably at
29 months compared with that at 9 months (Fig. 3E). Additionally, when
the fCSA was plotted to analyze the MyHC IIA fibers in more detail, in
the 9AL rats, most of the muscle fibers were 3000 to 5000 μm2, while in
the 29AL rats, the majority were less than 1000 μm2 (Fig. 3F–G). Even in
the 24AL rats, the fCSA distribution was smaller and showed a broader
distribution compared with that in the 9AL rats (Fig. 3G). Namely, with
aging, the peak of the fCSA distribution of the MyHC IIA fibers shifted to
the smaller size (< 2000 μm2) (Fig. 3F–G). In the MyHC I fibers, the fiber
number and the fCSA were significantly decreased in the 29AL rats
compared with those in the 9AL rats (Fig. 3A–B, D, E). Moreover, the
fCSA distribution of the MyHC I fibers showed that the peak in the 24AL
rats was similar to that in the 9AL rats (3000 to 5000 μm2), whereas the
peak in the 29AL rats shifted to the smaller myofiber size (1000 to 2000
μm2; Fig. 3H, I). Unlike the MyHC IIA fibers, MyHC I fibers in the 24AL
and 29AL rats included muscle fibers larger than 6000 μm2 (Fig. 3H).
Additionally, the median of the muscle MyHC IIA fCSA was significantly
decreased in both 24AL (2682 ± 586 μm) and 29AL rats (636.5 ± 96 μm)
compared with that in the 9AL rats (3795 ± 34 μm), whereas the MyHC I
fCSA was decreased only in the 29AL rats (2122 ± 608 μm) compared
with that in the 9AL rats (3864 ± 365 μm; Fig. 3J). These results suggest
that the decrease in the fCSA of the MyHC IIA fibers preceded the
decrease in the number of myofibers in the aging process, whereas in the
MyHC I fibers, the change in both parameters occurred at a late stage of
aging. The proportion of each fiber type with central nuclei was larger in
the MyHC I fibers compared with that in the MyHC IIA fibers (Fig. 3K).
These results suggest that skeletal muscle atrophy developed in different
time courses in the MyHC IIA and MyHC I muscle fibers. Moreover, the
ratio of each muscle fiber type to the total fiber number (the fiber oc­
cupancy ratio) in the 24AL rats decreased in the MyHC I muscle fibers
and increased in the MyHC IIA muscle fibers compared with that in the
9AL rats, while that in the 29AL rats increased in the MyHC I muscle
fibers and significantly decreased in the MyHC IIA muscle fibers,
compared with that in the other two groups (Fig. 3L). These data show
that the number of fast-type muscle fibers is more likely to decrease with
age than slow-type fibers, and these changes are induced between 24
and 29 months of age.
3.2. Long-term CR mitigates the phenotype of sarcopenic muscle atrophy
The body mass of the CR group remained almost unchanged until 29
months of age and reached approximately 66% and 75% of the 24AL and
29AL rats' body mass, respectively (Fig. 1A, B). The SOL muscle mass of
the 29CR group was similar to that of the 29AL group, but when
normalized to body mass it was significantly greater, suggesting
ANOVA
C ANOVA
D ANOVA
p<0.0001 p<0.0015 p<0.0027
** ** * **
* * ** **
SOL/Body mass (mg•g-1)
800 600 1.5
500
600
SOL ( mg)
400 1.0
400 300
200 0.5
200
100
0 0.0
9 24 29 29 9 24 29 29 9 24 29 29
AL CR AL CR AL CR
3
Y. Mizunoe et al. Experimental Gerontology 154 (2021) 111519

A
9AL 24AL 29AL 29AL

H&E
Masson trichrome

B ANOVA p<0.0097 C ANOVA p<0.0037 D ANOVA p<0.0037 E ANOVA p<0.0001

Muscle fiber area/mCSA(%)

** ** ** **
**

Fibrosis area / mCSA(%)

18 120 24 ** 35

central nuclei ratio (%)

**
16 100 20 30
14
mCSA (mm2)

25
12 80

Average of
16
10 20
8 60 12
15
6 40 8 10
4
2 20 4 5
0 0 0 0
9AL 24AL 29AL 9AL 24AL 29AL 9AL 24AL 29AL 9AL 24AL 29AL

Fig. 2. Muscle atrophy progresses in rat SOL muscle because of aging. The patterns of fiber atrophy in the SOL muscle in 9AL, 24AL, and 29AL rats were analyzed by
H&E staining and Masson's trichrome staining (A). Total muscle cross-section area (mCSA) (B), muscle fiber area per mCSA (C), fibrosis area per mCSA (D), and
average of muscle fiber with central nuclei (E) were quantified from images. Representative images are shown (9AL, n = 3; 24AL, n = 3; 29AL, n = 4). Muscle fibers
with central nuclei (white arrows), denervated muscle fibers (black arrows), adipocyte infiltration (black arrowheads) are shown. Scale bars, 200 μm. Values are
mean ± SD. Differences between values were statistically analyzed by Tukey's test after one-way ANOVA. Statistical significance is shown as *p < 0.05, **p < 0.01.

prolonged CR prevented muscle atrophy (Fig. 1C, D). While the mCSA of fibers, that were observed in the 29AL rats. Furthermore, the proportion
the SOL muscle did not significantly change, the proportion of the of fibers with central nuclei in each muscle fiber type was similar be­
fibrosis area was markedly lower in the 29CR rats compared with that in tween the MyHC IIA fibers and the MyHC I fibers (Fig. 5K), indicating no
the 29AL rats (Fig. 4A–C). Consistently, the ratio of the muscle fibers to significant difference in the degree of CR-induced preservation between
the total muscle area was significantly increased by CR (Fig. 4D). the two fiber types. Moreover, the ratio of each muscle fiber type to the
Furthermore, the total number of muscle fibers was greater in the 29CR total fiber number in 29CR rats tended to decrease in the MyHC I muscle
rats compared with that in the 29AL rats (Fig. 5B), indicating that CR can fibers and increase in the MyHC IIA muscle fibers compared with that in
protect both the area and number of muscle fibers against aging. the 29AL rats (Fig. 5L). These data indicate that the occupancy of fast
Moreover, CR significantly reduced both the proportion of central muscle fibers in the SOL muscle was moderately preserved by CR at 29
nuclei-containing muscle fibers and the denervated muscle fibers months.
(Fig. 4E, F). These results indicate that long-term CR ameliorated aging-
related muscle atrophy.
We next investigated each muscle fiber type of the SOL muscle in the 3.3. Involvement of ubiquitinated-protein accumulation in MyHC I fibers
29AL and 29CR rats. The number of MyHC IIA fibers tended to be higher
in the 29CR rats than in the 29AL rats, while the number of MyHC I Aging has been reported to be accompanied by numerous functional
fibers did not change (Fig. 5A, C, D). Moreover, the fCSA of both the alterations, including the accumulation of ubiquitinated proteins, a
MyHC IIA and MyHC I fibers that was decreased in the 29AL rats was process mainly driven by increased levels of oxidative stress (Fernando
significantly preserved by CR (Fig. 5E and Supplementary Fig. 2). The et al., 2019). We investigated the levels of ubiquitin accumulation as a
fCSA distribution showed that the peak of the MyHC IIA fibers shifted to marker of aggregated proteins in the SOL muscle of 29AL and 29CR rats.
the larger size (> 2000 μm2), and the proportion of fibers smaller than We found that the abundance of ubiquitinated-proteins was increased in
2000 μm2 was drastically reduced in the 29CR rats compared with that the 29 AL rats (Supplementary Fig. 3A, B). In contrast, CR decreased the
in the 29AL rats (Fig. 5F, G). Similarly, the proportion of MyHC I fibers accumulation of ubiquitinated proteins (Supplementary Fig. 3C, D).
smaller than 1000 μm2 remarkably decreased, while fibers larger than Additionally, immunostaining of ubiquitinated proteins in continuous
4000 μm2 significantly increased in the 29CR rats compared with that in sections showed that the accumulation of ubiquitinated proteins
the 29AL rats (Fig. 5H, I). Additionally, the median of the muscle fCSA occurred specifically in MyHC I muscle fibers in 29AL, but this accu­
was significantly increased in both fiber types in the 29CR rats compared mulation was decreased in 29CR rats (Fig. 6A).
with the 29AL rats (2314 ± 386 μm vs. 636.5 ± 96 μm in MyHC IIA;
3437 ± 631 μm vs. 2122 ± 608 μm in MyHC I; Fig. 5J). These data show 4. Discussion
that CR prevented the age-related decreases in both the number and
fCSA of the MyHC IIA fibers, and the decrease in fCSA of the MyHC I In the present study, we examined the chronological changes in age-
related muscle atrophy in slow- and fast-muscle fibers by comparing

4
Y. Mizunoe et al. Experimental Gerontology 154 (2021) 111519

A
9AL 24AL 29AL 29AL

*
SOL
*

MyHC Ⅰ MyHC ⅡA
B C D
ANOVA p<0.0073 ANOVA p<0.0371 ANOVA p<0.0347

Number of MyHC ⅡA fibers

*
Number of muscle fibers

600

Number of MyHC I fibers

MyHC ⅡA MyHC Ⅰ * 2500
** 500
3000 2000
2500 * 400
1500
2000 300
1500 1000
200
1000
500 100 500
0 0 0
9AL 24AL 29AL 9AL 24AL 29AL 9AL 24AL 29AL

E 9AL 24AL 29AL F G 9AL

ANOVA ANOVA 24AL

MyHC IIA distribution (%)

MyHC ⅡA fiber CSA (%)

p<0.0001 p<0.0275 9AL 24AL 29AL 100 29AL

** ** 100 **
6000 ** 80
Mean fCSAs (μm2)

** p=0.08 80
5000 **
60
4000 60 **
**
3000 40 ** 40
2000 20 * 20
1000
0 0
0

10 0
2000
3000
4000
5000
6000
7000
8000
00
MyHCⅡA MyHCⅠ
fCSA (μm2)
fCSA (μm2)
H 9AL
J
9AL 24AL 29AL I 60 24AL 8000 **
MyHC I fCSAs (%)

100
MyHC I distribution (%)

29AL
80 6000
fCSA (μm2)

**
40
60 **
** * * 4000
40 ** 20 **
20 2000
0 0
0
10 0
2000
3000
4000
5000
6000
7000
8000
00

24 L
29 L
9 AL
24 L
29 L
L
A
A

A
A
9A

fCSA (μm2)
fCSA (μm2) MyHC ⅡA MyHC Ⅰ
K L
/ Number of MyHC fiber
Number of central nuclei

40 * 86.2% 80.6% 91.6% MyHC Ⅰ

/total fiber number (%)

150
13.8% 19.4% 8.4% MyHC ⅡA
Fiber occupancy

30
100
20
(%)

10 50

0 0 *
MyHC
TypeⅡA
ⅡAMyHC
TypeⅠ Ⅰ 9AL 24AL 29AL
29AL

Fig. 3. MyHC IIA and MyHC I muscle fibers in SOL muscle atrophy with age. (A) Immunohistochemical images of SOL muscle sections analyzed for each muscle fiber
type [MyHC IIA fiber (red) and MyHC I fiber (green)]. (B–D) Quantification of the number of muscle fibers (B), the number of MyHC IIA fibers (C), and the number
of MyHC I fibers (D). (E) The average of single muscle fiber cross-sectional area (fCSA) in each fiber type was quantified from images. (F–I) The proportion of fCSA to
total fiber number of MyHC IIA (F) and MyHC I (H) muscle fibers; the non-linear lines in (G) and (I) were plotted by the Gaussian plot function. The vertical axis
shows each fiber distribution (%) and the horizontal axis shows fCSA (μm2). (J) The boxplot median of each muscle fiber type. (K) The number of muscle fibers with
central nuclei per total fiber number in each muscle fiber type. (L) Quantification of fiber occupancy to total fiber number. Representative images and the quan­
titative data (9AL, n = 3; 24AL, n = 3; 29AL n = 4) are shown. Asterisks show muscle fibers containing central nuclei. Scale bars, 100 μm. Values are mean ± SD.
Differences between values were statistically analyzed by Tukey's test after one-way ANOVA. Statistical significance is shown as *p < 0.05, **p < 0.01. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

5
Y. Mizunoe et al. Experimental Gerontology 154 (2021) 111519

A
29AL 29CR

H&E
H&E
Masson trichrome

B C D E F

Average of central nuclei ratio (%)

Muscle fiber area / mCSA (%)

denervated muscle fiber (%)

* **
12 25
Fibrosis area / mCSA(%)

** 100 30 25
10 20 25
80 20
mCSA (mm2)

8 20
15 60 15
6 15
10 40 10
4 10
2 5 20 5
5
0 0 0 0 0
29AL 29CR 29AL 29CR 29AL 29CR 29AL 29CR 29AL 29CR

Fig. 4. Caloric restriction (CR) ameliorates aging-related muscle atrophy. (A) The patterns of fiber atrophy in the SOL muscle of 29AL and 29CR rats were analyzed
by H&E staining and Masson's trichrome staining. Total muscle cross section area (mCSA) (B), fibrosis area per mCSA (C), muscle fiber area per mCSA (D), average of
muscle fibers with central nuclei (E), and the percentage of denervated muscle fiber (F) were quantified from images. Representative images are shown (29AL, n = 4;
29CR, n = 4). Muscle fibers with central nuclei (white arrows), denervated muscle fibers (black arrows), adipocyte infiltration (black arrowheads) are shown. Scale
bars, 200 μm. Values are mean ± SD. Differences between values were statistically analyzed by Student's t-test. Statistical significance is shown as *p < 0.05, **p
< 0.01.

9AL, 24AL, and 29AL Wistar rats, and evaluated whether prolonged CR MyHC IIA fibers was significantly larger than that in the MyHC I fibers,
prevents muscle atrophy. We found that: 1) at least in rat SOL muscles, implying more susceptibility of the MyHC IIA fibers to age-related fiber
the age-related changes up to 24 months of age were mild, and the number loss in the rat SOL muscle (Fig. 3L). These results imply that the
sarcopenic pathological condition rapidly progressed after 24 months of cell death of muscle fibers in late aging occurs predominantly in MyHC
age in Wistar rats, and 2) CR significantly suppressed the progression of IIA fibers. However, the accumulation of ubiquitinated proteins, a
sarcopenic muscle atrophy at 29 months of age. marker of protein aggregation that occurs before cell death, was
significantly observed in MyHC I muscle fibers, rather than in MyHC IIA
muscle fibers in the 29AL rats (Fig. 6). The proportion of fibers with
4.1. Age-related muscle atrophy by muscle fiber type
central nuclei was also larger in MyHC I fibers (Fig. 3K). In general,
muscle fibers with central nuclei are characterized as the marker of
We observed a significant decrease in fiber number in both MyHC I
regenerative muscle fibers (Ciciliot and Schiaffino, 2010). Additionally,
and MyHC II fibers in the 29AL rats. Generally, the decrease in the
collateral innervation occurs in MyHC I muscle fibers, leading to age-
number of muscle fibers is considered to be attributed to the induction of
related remodeling of motor units (Saini et al., 2013; Miljkovic et al.,
cell death (apoptosis), functional decline of satellite cells, and degen­
2015). In contrast, MyHC II fibers reportedly have less satellite cells than
eration of muscle fibers due to denervation (Marzetti et al., 2012; Sheard
MyHC I fibers (Verdijk et al., 2007; Hunter et al., 2016). Furthermore, a
and Anderson, 2012; Lee et al., 2017). Given that the fibrosis area
preferential denervation of MyHC II muscle fibers occurs during the
significantly increased only in the 29AL rats, it is conceivable that the
normal aging process (Saini et al., 2013). Based on these findings, it is
cell death of muscle fibers was dramatically induced between 24 and 29
possible that MyHC I muscle fibers are more susceptible to cell death and
months of age (Fig. 1). This is supported by the report that significant
simultaneously have a higher regeneration capacity. Considering the
muscle atrophy does not occur until after 28 months of age in Fischer
finding that muscle atrophy also occurred in MyHC I fibers (Fig. 3D), the
344/BNF1 rat hybrids (Brown and Hasser, 1996). As shown in Fig. 3,
balance between degeneration and regeneration in aging may be slightly
while both fiber number and fiber fCSA significantly decreased in MyHC
inclined toward degeneration in MyHC I fibers, resulting in the slight
I and MyHC IIA fibers in the 29AL rats, the degree of decrease in the

6
Y. Mizunoe et al. Experimental Gerontology 154 (2021) 111519

Fig. 5. MyHC IIA and MyHC I muscle

A 29AL 29CR 29CR fibers in SOL muscle atrophy in 29CR
rats. (A) Immunohistochemistry im­
ages of SOL muscle analyzed for each
muscle fiber type; MyHC IIA fiber (red)
and MyHC I fiber (green). (B–D)
Quantification of the number of muscle
ː fibers (B), the number of MyHC IIA fi­
ː
bers (C), and the number of MyHC I
fibers (D). (E) Average fCSA of each
MyHC ⅡA MyHC Ⅰ fiber type was quantified from images.
B C D (F–I) The proportion of fCSA to total
fiber number of MyHC IIA (F) and
Number of MyHC ⅡA fibers

Number of MyHC I fibers

p=0.09
* MyHC I (H) muscle fibers; the non-
Number of muscle fibers

400 2000
2000 linear lines in (G) and (I) were plotted
1500 300 1500 by the Gaussian plot function. The
vertical axis shows each fiber distribu­
1000 200 1000 tion (%) and the horizontal axis shows
fCSA (μm2). (J) The boxplot median of
500 100 500
each muscle fiber type. (K) Number of
0 0 0 muscle fibers with central nuclei per
29AL 29CR 29AL 29CR 29AL 29CR total fiber number in each muscle fiber
type. (L) Quantification of fiber occu­
E 29AL 29CR F G 29AL
pancy to total fiber number. Repre­
sentative images and the quantitative

MyHC IIA distribution (%)

29AL 29CR 100
data (29AL, n = 4; 29CR, n = 4) are
MyHC ⅡA fCSAs (%)

P=0.06 100 29CR

4000 **
shown. Asterisks show muscle fibers
Mean fCSAs (μm2)

80 80
3000 containing central nuclei. Scale bars,
60 60
100 μm. Values are mean ± SD. Dif­
2000 40 ** 40 ferences between values were statisti­
** cally analyzed by Student's t-test.
1000 20 ** 20
Statistical significance is shown as *p
* P=0.06
0 0 0 < 0.05, **p < 0.01. (For interpretation
MyHC ⅡA MyHC Ⅰ 0 00 00 00 00 00 00 00 00 of the references to colour in this figure
10 20 30 40 50 60 70 80 legend, the reader is referred to the
fCSA (μm2) web version of this article.)
fCSA (μm2)
H I 29AL
J 8000
MyHC I distribution (%)

29AL 29CR 50
29CR **
100 **
MyHC I fCSA (%)

40 6000
fCSA (μm2)

80
30
60 4000
P=0.06 20
40 *
* 2000
20 10
**
0 0 0
0 00 00 00 00 00 00 00 00 L L
10 20 30 40 50 60 70 80 A 9CR 9A 9CR
fCSA (μm2) 29 2 2 2
fCSA (μm2) MyHC ⅡA MyHC Ⅰ
K L
/ Number of Type fiber (%)
Number of central nuclei

25 91.6% 87.8% MyHC Ⅰ

/total fiber number (%)

150
Fiber occupancy

20 8.4% 12.2% MyHC ⅡA

15 100
10
50
5
0 0
MyHC ⅡA MyHC
TypeⅡA TypeⅠ Ⅰ 29AL 29CR
29CR

decline in the muscle fiber number in MyHC I fibers compared with isoform ratio due to fiber-type transformation. It has been reported that
MyHC IIA fibers. In other words, cell death due to aging may be blunted myofibers temporarily become hybrid types during this process (Talbot
in MyHC I fibers, resulting in a lower degree of decrease in the fCSA of and Maves, 2016). In fact, hybrid fibers, characterized by MyHC I/
MyHC I fibers, despite the increased accumulation of ubiquitinated MyHC IIA double-positive staining, were occasionally observed in our
proteins and increased number of muscle fibers with central nuclei. images (Fig. 3A and 5A). Therefore, a transformation of MyHC IIA into
Alternatively, the age-related preferential decrease in MyHC IIA fibers MyHC I might occur with aging, thereby contributing to a preferential
could be explained by a proportional shift in the MyHC IIA to MyHC I decrease in MyHC IIA fibers. Additionally, the length of the SOL tendon

7
Y. Mizunoe et al. Experimental Gerontology 154 (2021) 111519

A 29AL 29CR

fiber type fiber type

Multi Ubiquitin Laminin α MyHC ⅡA MyHC Ⅰ Multi Ubiquitin Laminin α MyHC ⅡA MyHC Ⅰ

Serial section Serial section

Fig. 6. Ubiquitinated proteins accumulate in MyHC I fibers. Immunohistochemistry images of a serial section of 29AL (left) and 29CR (right) SOL muscle using anti-
Multi-ubiquitin (red)/Laminin α (green) and MyHC I (green)/MyHC IIA (red) antibodies. Representative images are shown. Arrows show muscle fibers with multi-
ubiquitin. Scale bars, 500 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and myotendinous junction reportedly increases with age (Nielsen et al., aged rats.
2018), which could affect the fibrosis area or fCSA in the muscle section
near the myotendinous junction. Such a change could be a possible
4.2. Long-term CR ameliorates the sarcopenic muscle atrophy in the rat
mechanism for the age-related myofiber loss observed in this study,
model
although we have no direct evidence. The elucidation of these possi­
bilities will require further research.
A previous study has reported that between 21 and 36 months of age,
The number of both MyHC IIA and MyHC I fibers significantly
the SOL muscle weight was reduced by 10% in Fischer 344/BN F1
decreased in the 29AL rats, although the fCSA of the MyHC IIA fibers
hybrid rats with 40% CR, whereas it reduced by 40% in AL rats
already decreased at 24 months (Fig. 3). These data suggest that the
(McKiernan et al., 2004). In the present study, we showed that the SOL
decrease in the fCSA of the MyHC IIA fibers may precede the decrease in
muscle weight loss in the CR group was approximately 25% between 9
the number of myofibers in the process of age-related muscle atrophy.
and 29 months of age, while that in the AL group was approximately
Previous studies have demonstrated that skeletal muscles of older adults
35%. When compared per unit of body weight, the SOL muscle weight in
include MyHC II muscle fibers that are 10%–40% smaller in size,
the 29CR rats was recovered to almost the 9AL level (Fig. 1). Moreover,
compared with young adults (Kosek et al., 2006; Verdijk et al., 2007;
CR significantly counteracted the age-related changes in the SOL mus­
Nilwik et al., 2013). Notably, consistent with our data in 24AL rats,
cle, such as the increase in fibrosis, fat infiltration, and muscle fibers
Nilwik et al. have reported that the reduced muscle mass with aging in
with central nuclei, the decrease in muscle fiber number and fCSA, and
older men is mainly attributed to decreased MyHC II muscle fiber size,
the accumulation of ubiquitinated proteins. Consistently, it has been
which is unlikely to be accompanied by muscle fiber loss (Nilwik et al.,
reported that CR ameliorated muscle atrophy in the upper leg and
2013). In contrast, it has been reported that the MyHC I muscle fiber size
especially the fCSA of MyHC II fibers in Rhesus monkeys between the
is hardly affected by aging, which is consistent with our results that the
ages of 16 and 22 years (McKiernan et al., 2011). Moreover, CR
MyHC I muscle fibers did not change in the 24AL rats' SOL muscle (Glass
reportedly attenuated the effect of aging on MyHC IIA fiber size in the
and David, 2003; Verdijk et al., 2007; Sandri et al., 2013). Given that
SOL muscle (Marzetti et al., 2008), which is consistent with our data that
muscle fibers with central nuclei and fibrosis areas did not increase at
both the number and fCSA of MyHC IIA fibers were preserved by long-
24 months of age, the MyHC IIA muscle fiber atrophy in the 24AL rats
term CR. These finding suggest that CR suppresses muscular atrophy
was probably independent of cell death or fiber transformation.
by protecting fast-type muscle fibers, which are significantly impaired
Generally, the balance between myofiber protein synthesis and degra­
with aging. We also confirmed that long-term CR significantly improved
dation is important for maintaining myofiber size (Glass and David,
muscle fibrosis and cell death (Fig. 4). It has been suggested that loss of
2003; Sandri et al., 2013). Additionally, MyHC II fibers reportedly have
satellite cells causes age-related muscle fibrosis (Fry et al., 2015), which
lower protein synthesis activity, which is necessary for skeletal muscle
raises the possibility that CR-induced prevention of muscle fibrosis may
growth and repair, compared with MyHC I fibers (Wall et al., 2015;
be attributed to maintenance of satellite cells.
Hunter et al., 2016). Therefore, the reduced synthesis is highly likely to
At present, it remains unclear to what extent long-term CR inter­
contribute to MyHC IIA muscle fiber atrophy in SOL muscle of advanced
vention can attenuate muscle loss during aging in humans. However,

8
Y. Mizunoe et al.
several studies have demonstrated the effects of CR on age-related
physiological changes in human skeletal muscles. For example, CR is
associated with reduced inflammation in human muscle (Yang et al.,
2016). Additionally, Mercken et al. have shown that long-term CR
changed the transcription profile of skeletal muscle of older individuals
to that of younger individuals, such as decreased inflammation
(Mercken et al., 2013). These data suggest that CR can beneficially affect
human muscles, although more work is needed to evaluate this idea
(McCormick and Vasilaki, 2018).
This study provided the following insights: 1) muscle atrophy pro­
gressed rapidly in a late stage of aging, and led to pathological changes
in skeletal muscle; 2) the degree of muscle fiber atrophy with aging was
more remarkable in MyHC IIA fibers than in MyHC I fibers; and 3) long-
term CR delayed sarcopenic muscle atrophy. Potential gains from these
findings may be limited because muscle fibers that can be categorized as
slow or fast types include MyHC IIX and MyHC IIB fibers, as well as
MyHC I and MyHC IIA fibers. Nevertheless, we anticipate that further
analysis using late-stage aging models will enable more precise in­
vestigations of the cause(s) of sarcopenia.
CRediT authorship contribution statement
Y. Higami; Conceptualization. Funding acquisition. Supervision.
Project administration.
Y. Mizunoe. and M. Kobayashi; Investigation. Project administration.
Writing- Reviewing and Editing.
H. Saito., A. Goto., R. Migitaka., K. Miura., A. Umemori., and M.
Yoshida. Support investigation.
R. Tagawa., Y. Sudo., N. Okita., Y. Nakagawa., and H. Shimano;
Support investigation.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
We are deeply grateful to all members of the Laboratory of Molecular
Pathology and Metabolic Disease of the Faculty of Pharmaceutical Sci­
ences, Tokyo University of Science (TUS) for their cooperation. We also
thank Edanz Group (https://jp.edanz.com/ac) for editing a draft of this
manuscript.
Funding
This work was supported by Grants-in-Aid for Young Scientists (No.
18K17953) from the Japan Society for the Promotion of Science (JSPS)
and the JSPS Fellowship for Young Scientists (No. 19J00407) to Y.M.;
Grants-in-Aid for Young Scientists (B) (No. 17K13231) from JSPS to M.
K.; and Grants-in-Aid for Scientific Research (B) (No. 17H02179) from
JSPS and the Ministry of Education, Culture, Sports, Science and Tech­
nology (MEXT) Supported Program for the Strategic Research Founda­
tion at Private Universities, 2014–2018, to Y.H.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.exger.2021.111519.
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