Journals of Gerontology: Biological Sciences

cite as: J Gerontol A Biol Sci Med Sci, 2017, Vol. 72, No. 12, 1595–1606
doi:10.1093/gerona/glx089
Advance Access publication May 13, 2017

Original Article

Resveratrol Enhances Exercise-Induced Cellular and

Functional Adaptations of Skeletal Muscle in Older Men

Editor’s Choice
and Women
Stephen E.  Alway,1–3 Jean L.  McCrory,1,2 Kalen  Kearcher,1,2 Austen  Vickers,1,2
Benjamin  Frear,1,2 Diana L.  Gilleland,1 Daniel E.  Bonner,1 James M.  Thomas,1 David
A. Donley,1 Mathew W. Lively,1,4 and Junaith S. Mohamed1,3
Division of Exercise Physiology, Department of Human Performance and Applied Exercise Science, West Virginia University School of
1

Medicine, Morgantown. 2West Virginia Clinical and Translational Science Institute, Morgantown. 3Center for Neuroscience, Morgantown,
West Virginia. 4Section of Rheumatology, Department of Medicine, West Virginia University School of Medicine, Morgantown.

Address correspondence to: Stephen E. Alway, PhD, Division of Exercise Physiology, Department of Human Performance and Applied Exercise
Science, West Virginia University School of Medicine, 1 Medical Center Drive, PO Box 9227, Morgantown, WV. E-mail: salway@hsc.wvu.edu

Received: September 6, 2016; Editorial Decision Date: April 8, 2017

Decision Editor: Rafael de Cabo, PhD

Abstract
Older men (n = 12) and women (n = 18) 65–80 years of age completed 12 weeks of exercise and took either a placebo or resveratrol (RSV) (500
mg/d) to test the hypothesis that RSV treatment combined with exercise would increase mitochondrial density, muscle fatigue resistance, and
cardiovascular function more than exercise alone. Contrary to our hypothesis, aerobic and resistance exercise coupled with RSV treatment did
not reduce cardiovascular risk further than exercise alone. However, exercise added to RSV treatment improved the indices of mitochondrial
density, and muscle fatigue resistance more than placebo and exercise treatments. In addition, subjects that were treated with RSV had an
increase in knee extensor muscle peak torque (8%), average peak torque (14%), and power (14%) after training, whereas exercise did not
increase these parameters in the placebo-treated older subjects. Furthermore, exercise combined with RSV significantly improved mean fiber
area and total myonuclei by 45.3% and 20%, respectively, in muscle fibers from the vastus lateralis of older subjects. Together, these data
indicate a novel anabolic role of RSV in exercise-induced adaptations of older persons and this suggests that RSV combined with exercise might
provide a better approach for reversing sarcopenia than exercise alone.
Key words: Mitochondria—Sarcopenia—Fiber type—Exercise—Muscle—Strength—Fatigue

Sarcopenia is predictive of falls and is associated with periods of
hospital-associated immobilization (1). This is noteworthy, as aging
muscles display an impaired or failed recovery following immobili-
zation-induced muscle atrophy (2,3). Several potential countermeas-
ures to unloading-induced atrophy have been tested, but most have
only been partially successful. For example, taurine was shown to
prevent the slow to fast myosin shift with unloading (4), but it did
not prevent the loss of muscle mass. Insulin growth factor-I overex-
pression during hindlimb unloading in rodents induced some poten-
tial molecular signaling improvements, especially the atrogen Muscle
RING-finger protein-1 (MuRF1), but there was no overall change
in muscle protein loss or fiber type transition with this treatment
(5). In addition, β-hydroxy-β-methylbutyrate, a leucine metabolite,
© The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com.
provided modest protection against skeletal loss of muscle mass
during disuse in sarcopenic rats (6). We have found that green tea
extract increased satellite cell proliferation and differentiation in rat
plantaris and soleus muscles during recovery from hindlimb suspen-
sion as compared to vehicle-treated muscles and decreased oxidative
stress and the abundance of the Bcl-2–associated X protein (Bax), a
proapoptotic protein, yet this did not further improve muscle recov-
ery in reloaded muscles over placebo (PL) treatments (7).
In vitro studies have shown that resveratrol (3,5,4′-trihydrox-
ystilbene; RSV) increases protein synthesis (8), inhibits protein deg-
radation, and attenuates atrophy of skeletal muscle fibers (9–12). A
high dose of RSV in vivo (400 mg/kg/d) was reported to attenuate
muscle fiber atrophy following hindlimb suspension (13) in rodents.
1595

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 1596 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12
We have found that a low dose (12.5 mg/kg/d) of RSV (14) had a
trend (p = .06) to blunt the loss of muscle mass during hindlimb
suspension in old rats but it did not improve recovery or increase
the number of satellite cells or muscle mass after disuse in old ani-
mals (15). Nevertheless, other data failed to identify RSV-mediated
improvements in metabolic function in rodents (16). Furthermore,
while exercise adaptations in the elderly are typically attenuated
as compared to responses in young adults (17), it is not known if
RSV coupled to exercise provides a stronger stimulus for reducing or
reversing sarcopenia in humans (18) than exercise alone.
Thus, the purpose of this study was to determine if 12 weeks of
exercise would improve muscle fatigue resistance more than exer-
cise alone in older men and women. We tested the hypothesis that
RSV treatment would increase mitochondrial density, which would
decrease muscle fatigue resistance, and improve indices for cardio-
vascular risk as compared to exercise alone in older men and women.
Methods
Detailed methods are provided in Supplementary Methods.
Subjects
Healthy older men (n = 12) and women (n = 18) completed this
study. The subjects were randomly assigned in a balanced design to
include the same number of males (n = 6) and females (n = 9) in each
experimental group. A resting 12-lead ECG and a symptom-limited
maximal graded exercise test (GXT) were conducted to rule out
heart disease and to establish proper cardiovascular function. The
study was approved by the West Virginia University Institutional
Review Board for Human Research, and all participants signed
informed consent prior to participating in the study.
Body Mass Index and Body Fat
Trained personnel measured height, weight, and waist and hip cir-
cumference. Body composition was estimated via air displacement
plethysmography (BOD POD, COSMED, Inc.). The body mass
index was calculated as weight (kg) divided by height (m) squared.
Blood Indices
Venous blood from the antecubital vein was obtained after a
12-hour overnight fast. Fasted post-training samples were collected a
minimum of 48 hours after the last exercise session. Plasma obtained
from blood sampling was analyzed at West Virginia University
Hospital’s analytical laboratory.
Assessment of Maximal Oxygen Uptake
Oxygen consumed (VO2), carbon dioxide produced (VCO2), respira-
tory exchange ratio = VCO2/VO2, ventilation-expired (VE), ventila-
tion-inhaled (VI), ventilatory threshold (VT), ventilatory equivalent
for CO2 (VE/VCO2), and O2 (VE/VO2) were measured using a vali-
dated (19), twenty-second averaging breath-by-breath analysis sys-
tem (TrueOne 2400; ParvoMedics, Sandy, UT).
Muscle Function and Fatigue
Maximal torque
Measures of muscle strength and fatigue were obtained using a veloc-
ity-dependent Biodex System 3 dynamometer (Biodex, Inc., Shirley,
NY) because function and fatigue are compromised in aging (20).
Maximal knee extension torque was evaluated at 60, and 240°/s.
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Data were calculated for peak torque (Newton-meters), total work
in one repetition (Joules), total work over seven repetitions (Joules),
work fatigue (%), average power (Watts), and average peak torque
over seven repetitions (Newton-meters). Subjects were not provided
with encouragement but the investigator counted each repetition
loudly during the test.
Isokinetic fatigue
Fatigue data were calculated from the decline in torque over 32
isokinetic contractions at 240°/s on the Biodex unit. This repre-
sents a moderate load for older subjects (21). Muscular fatigue was
expressed as a fatigue index (fatigue index = [1 − (initial torque −
final torque)/initial torque]). These are similar to methods used pre-
viously in our labs (22,23).
Muscle Samples
Needle biopsies of the vastus lateralis muscle were collected under
local anesthesia with 2% lidocaine using a Bergstrom needle as
described previously (17,24,25). The subjects refrained from strenu-
ous activity for 36 hours prior to the biopsy and for 48 hours after
the biopsy. Visible blood and connective tissue were removed from
the biopsy sample and the tissue was frozen in isopentane cooled to
the temperature of liquid nitrogen, and stored at −80°C until analysis.
Muscle Myosin Composition and Morphology
Frozen tissue sections were cut at −20°C and placed on glass slides,
air-dried, and incubated overnight at 4°C with antibodies from the
Hybridoma Bank against: myosin heavy chain (MHC) IIA (SC-71),
MHCI (BAF8S), and MHC IIX (6H1). Fiber area and the minimum
Feret diameter was determined from the digital images and quanti-
fied by Image J software (NIH) from a minimum of 500 fibers. All
analyses were performed with the investigator blinded to treatment
(RSV or PL) using established methods (26,27).
Muscle Ultrastructure
Fifty fibers were randomly selected from five specimen blocks for
ultrastructural analyses. The electron micrographs were analyzed
by a stereological 168-point short-line test grid (28). Morphometric
measures were made of the proportion of fibers occupied by the
mitochondria. The stereological measures were made without the
investigator’s knowledge of the group that was analyzed.
Mitochondria Biogenesis
The relative number of copies of human mitochondrial DNA
(mtDNA) was quantified by real-time PCR using a Human mtDNA
Monitoring Primer Set (Takara/Clontech). Nuclear DNA (nDNA)
content was used as a standard. Two mtDNA targets (ND1, ND5)
and two nDNA targets (SLCO2B1, SERPINA1) were used for the
detection assay. The manufacturer has designed the primers to avoid
amplification of pseudogenes. DNA was purified from skeletal mus-
cle biopsies (Qiagen) and amplified with the primers according to the
manufacture’s recommendation. The CT value for each mitochon-
dria and genomic DNA primer was determined and 2(−ΔΔCT) for the
primer pairs and the mtDNA copy number was determined.
PCR Arrays for Mitochondrial Metabolism
RNA was purified from muscle biopsies and mRNA for mito-
chondrial genes was assessed using the Human Mitochondria RT2
Profiler PCR Array (Qiagen). ΔCT was calculated for each gene
 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12 1597

and the housekeeping genes and the fold change between pre-
exercised and post-exercised groups for each gene was calculated
using 2(−ΔΔCT). The fold differences of 2(−ΔΔCT) between exercise and
PL groups were reported as log base 2 fold differences between
genes in pre- to post-training conditions, or between pre-exercise
and between post-exercise conditions for PL and RSV treatments.
Satellite Cell and Myonuclei
Antibodies against paired box transcription factor 7 (Pax7;
Developmental Studies Hybridoma Bank, Iowa city, IA) was used
to identify satellite cells and laminin (Developmental Studies
Hybridoma Bank) was used to identify the boundaries of muscle
fibers. The sections were counter stained with 4′,6-diamidino-2-phe-
nylindole (DAPI) to visualize all of the nuclei in each tissue section
(Vector Laboratories, Burlingame, CA). The number of nuclei that
were positive for Pax7 and the total number of DAPI-positive nuclei
were counted from digital confocal images (Zeiss) by an investigator
who was blinded to the experimental groups.
The subjects were randomly assigned in a double-blind manner
into either a 12-week exercise group that took 500 mg RSV/d in cap-
sule form (RSV; 80% female) or a 12-week exercise control group
that took 500 mg/d of a corn starch PL in capsule form (PL; 80%
female). The code identifying the PL and RSV groups was not broken
until the data analysis was completed, and then the subjects were
placed in the appropriate groups for data analyses.
Statistical Analysis
Pre-exercise variables were compared between groups with a one-
way analysis of variance (ANOVA) and post-training data were
compared with a two-way repeated measures ANOVA as group (PL
vs RSV) and condition (pre- vs post-exercise). A Tukey’s post hoc test
was conducted if a main effect or an interaction effect was identified
by the ANOVA. A Condition (ie, training) × Group interaction indi-
cates that the response to training differed between the PL and RSV
groups. The data are reported as means ± SEM. P ≤.05 was defined
as statistically significant.

DNA Oxidative Damage Results

Fixed tissue sections were incubated with 8-hydroxyguanosine
(8-OHdG; Santa Cruz) an indication of oxidative damage to skel-
etal muscle nuclear DNA. To confirm that the nuclei were inside the
boundary of the muscle fibers, the tissue sections were also incubated
with antibodies against dystrophin and laminin (Developmental
Studies Hybridoma Bank). The sections were counter stained with
DAPI and then examined by an investigator who was blinded to the
experimental groups.
Exercise Training and RSV Treatment
As exercise provides health benefits for older subjects, all subjects
were exercised for 12 weeks in this study. Resistance training was
given to provide an anabolic effect for muscle growth. Aerobic train-
ing was added to improve mitochondrial biogenesis and function, as
mitochondria are central to aging-associated signaling for cell death
including apoptosis (7,29,30) and autophagy (31,32).
Effects of Exercise Training on Body Composition
and Metabolic Biomarkers
One subject in the PL group and four subjects in the RSV group
reported a lack of time to commit to training and withdrew from the
study and additional subjects were recruited to the subject groups
so that 15 subjects completed the study in both PL and RSV groups.
The characteristics of the subjects that completed the study are
shown by treatment group in Table 1. Data are separated by sex
for each group and are given in Supplementary Table 1. Both PL
and RSV groups had similar characteristics at the beginning of the
study. Three-way ANOVA analysis showed that there were subject
group differences based on the sex of the subjects with men having
greater body weight (p < .001), body fat (p = .026), and lean body
mass (p < .001) than women, but there were no differences in these
variables between women in the two groups or men in the PL or RSV
groups (Supplementary Table 1); however, there were no differences

Table 1.  Effects of Exercise Training and Resveratrol on Body Composition, and Metabolic Biomarkers in Older Subjects

Grouped (male + female) p Values

Condition Group Condition × Group

Pre-PL Post-PL Pre-RSV Post-RSV (pre, post) (PL, RSV) Interaction

Age (y) 67.9 ± 1.1 67.9 ± 1.1 68.1 ± 1.1 68.1 ± 1.1 1 .513 1

Height (cm) 66.9 ± 1.0 66.9 ± 1.0 65.9 ± 1.0 65.9 ± 1.1 .645 <.001 <.001
Weight (kg) 74.1 ± 3.7 73.2 ± 4.3 73.0 ± 3.8 73.6 ± 4.1 .935 .860 .454
Lean mass (kg) 50.1 ± 2.4 49.5 ± 4.3 48.5 ± 3.1 46.5 ± 3.2 .565 .464 .218
Body fat (%) 32.6 ± 2.1 32.1 ± 2.1 35.2 ± 2.0 33.4 ± 2.0 .572 .117 .111
BMI (kg/m2) 26.2 ± 1.3 25.8 ± 1.4 25.3 ± 1.3 26.4 ± 1.2 .835 .802 .397
Hemoglobin A1C (% of 6.0 ± 0.3 5.3 ± 0.2 5.4 ± 0.3 5.3 ± 0.2 .182 .124 .18
Hb)
Triglycerides (mg/dL) 132.8 ± 10.7 124.7 ± 4.5* 117.1 ± 23.7 109.7 ± 11.9* .002 .112 .942
Total cholesterol (mg/dL) 228.9 ± 10.3 219.6 ± 10.1 214.9 ± 9.2 207.8 ± 9.3 .401 .196 .907
HDL (mg/dL) 44.1 ± 3.7 53.8 ± 4.7 51.1 ± 3.3 53.4 ± 3.4 .094 .363 .281
LDL (mg/dL) 147.1 ± 7.8 122.5 ± 7.1* 131.6 ± 7.2 114.4 ± 7.9* .007 .120 .624
Insulin (µU/mL) 10.4 ± 1.1 7.9 ± 0.9* 10.8 ± 1.2 8.0 ± 0.8* .002 .827 .795

Note: BMI = body mass index; HDL = high-density lipoprotein; LDL = low-density lipoprotein; PL = placebo; RSV = resveratrol. Older male and female subjects
were given a PL or 500 mg of RSV and exercised for 12 wk. The data are reported for blood triglycerides, total cholesterol, HDL, LDL, as well as body weight, body
fat, lean body mass, and oxygen uptake (VO2 max) (n = 15 PL; n = 15 RSV; 60% of each group was comprised of women: 9 women and 6 men were randomly
assigned to each group in a balanced design). *p < .05, PL vs RSV.

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 1598 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12
between groups when males and females were averaged within each
group.
Exercise resulted in significant reductions in total triglycerides,
high-density lipoprotein cholesterol, low-density lipoprotein choles-
terol, and serum insulin, but RSV did not change these metabolic
variables further (Table 1). This exercise effect and lack of RSV
effect was similar in both men and women (Supplementary Table
1). Neither the exercise nor the RSV treatment significantly altered
bodyweight, body fat, body mass index, or lean body mass in older
subjects (Table 1).
Effects of RSV on Improvements in Oxygen Uptake
to 12 Weeks of Exercise
Cardiovascular adaptations to exercise training are best reflected by
changes in absolute maximal oxygen uptake as relative measures of
oxygen uptake can be affected by changes in bodyweight. Before
exercise, absolute VO2 max was similar in PL- and RSV-treated
groups. The ANOVA analysis revealed a significant main effect (p <
.05) in absolute VO2 max on condition (pre- to post-exercise). While
the post hoc analyses to identify the source of the main effect showed
that the absolute VO2 max was significantly improved by exercise
in only the RSV-treated subjects (p = .028), there appeared to be no
central improvement in absolute VO2 max in the exercise-trained PL
group (p = .286). There was no statistically significant interaction
(p = .09) between Group (PL vs RSV) × Condition (pre- vs post-
exercise). The absolute VO2 max data are shown in Figure 1A.
Similarly, the ANOVA analysis showed that there was a signifi-
cant main effect for condition (pre- vs post- exercise differences; p <
.05) in the relative maximal oxygen uptake when the subjects were
considered as a single group. There was not a statistically significant
interaction between Group (PL vs RSV) × Condition (pre- vs post-
exercise) (p = .71). Post hoc analyses identified a significant improve-
ment in relative VO2 max in RSV-treated subjects (24.0 ± 1.3 mL/
kg/min vs 28.7 ± 1.7 mL/kg/min, p < .05) but relative VO2 max was
unchanged in PL-treated subjects who exercised (23.7 ± 1.3 mL/kg/
min vs 26.1 ± 1.4 mL/kg/min, p = .13). This improvement in relative
maximal oxygen uptake likely reflects a central adaptation in RSV-
treated subjects because bodyweight did not differ before or after
exercise in RSV-treated subjects, and bodyweight in PL and RSV
groups were not changed by treatment or exercise (Table 1).
Mitochondrial Density
There was a significant main effect of condition (pre- vs post-exer-
cise) for mitochondrial volume density (p < .05). Without consider-
ing the apparent health of the mitochondria, quantification of the
volume density of mitochondria from longitudinal sections was
9.3% ± 1.3% before exercise, and 13.5% ± 2.3% post-training in
muscle samples from the PL group but post hoc analyses following
the identification of the significant main effect of condition showed
that this did not represent a statistically significant improvement in
mitochondrial volume density (p = .093). On the other hand, post
hoc analysis of the significant main effect of condition revealed that
mitochondria volume density in the vastus lateralis significantly
increased from 9.8% ± 1.7% pre-training to 16.8% ± 2.5% (p <
.05) in the RSV group post-training. The fiber type of the samples
could not be determined from the electron micrographs. Of inter-
est, apparently morphologically normal (red arrows) and abnormal
mitochondria (blue arrows) could be identified before and after
exercise in PL- and RSV-treated samples (Figure 1B), although these
were not quantified. Nevertheless, it was apparent that mitochondria
with normal morphological characteristics (eg, normal cristae and
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mitochondria boundaries etc.) were abundant in the RSV-treated
longitudinal muscle samples, but abnormal mitochondria (broken
cristae etc.) were readily obvious in the post-trained PL-treated sam-
ples. However, the two-way ANOVA did not identify a significant
effect for group (PL vs RSV) and no significant interaction effect
between group and condition were found.
Signaling for Mitochondrial Biogenesis and
Mitochondrial Function
Total RNA was isolated from muscle biopsies before and after
exercise after PL and RSV treatments. The RNA was used for mito-
chondrial microarray analyses. The fold difference values of altered
mRNAs are presented as heat maps of log2 transformed fold change
from pre- to post-exercise (Figure 1C). Scatter plots showing nor-
malized log 10-transformed signal intensities for each comparison
show global gene fold differences between PL or RSV groups before
and after exercise training (Supplementary Figures 1–4).
RSV promoted proapoptosis signaling
The expression of apoptotic genes BCL2 antagonist/killer 1 (BAK1),
BH3-interacting domain death agonist (BID), cyclin-dependent
kinase inhibitor 2A (CDKN2A), phorbol-12-myristate-13-acetate-
induced protein 1 (PMAIP1), strafin (SFN), dynamin 1-like protein
(DNM1L), and prefoldin-like chaperone (UXT) had a 3- to 11-fold
greater pre- to post-exercise change in RSV as compared to PL mus-
cle samples (Table 2; Supplementary Results).
RSV promoted antiapoptotic changes
Two antiapoptotic proteins, B-cell lymphoma 2 (BCL2) and BCL2-
like 1 (Bcl-xL), had approximately twofold greater gene expres-
sion differences pre- to post-exercise in RSV as compared to PL
treatments.
RSV regulation of mitochondrial metabolic genes
Several mitochondrial metabolic genes were expressed to higher lev-
els from pre- to post-exercise in RSV versus PL subjects. This included
carnitine/choline acetyltransferase (CPT1B, 4-fold), COX18 (3-fold),
uncoupling protein 2 (UCP2, 6-fold), uncoupling protein 3 (UCP3,
5-fold), and hypoxanthine phosphoribosyltransferase 1 (HPRT1,
16-fold) (Table 2).
RSV regulation of mitochondrial morphology
Mitofusin 1 (MFN1), mitofusin 2 (MFN2), and morphology regula-
tor (MSTO1) had approximately 7-, 2.5-, and 3-fold greater gene
level pre- to post-exercise in muscles from RSV versus PL subjects,
respectively (Table 2; Supplementary Results).
Carrier/shuttle/import proteins
Compared to PL, RSV had two- to fourfold greater pre- to post-
exercise increases in gene expression for solute carrier family 25
(SLC25) members, including SLC25A10, SLC25A22, SLC25A27,
SLC25A31, and SLC25A37, and StAR-related lipid transfer domain
containing 3 (STARD3) (Table 2). Similarly, genes for members of
the translocase of inner mitochondrial membrane (TIMM) fam-
ily transport protein, including TIMM10, TIMM10B, TIMM44,
TIMM50, and TIMM8A, and translocase of outer mitochondrial
membrane (TOMM) family members, TOMM34 and TOMM40L,
inner mitochondrial membrane peptidase subunit 2 (IMMP2L), and
leucine-rich pentatricopeptide repeat containing (LRPPRC) protein
had 3- to 13-fold greater gene differences in muscles of RSV-treated
 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12 1599

Figure 1. (A) Maximal oxygen uptake (VO2 max) was determined before (Pre) and after (Post) 12 wk of exercise in subjects who consumed a placebo (PL) or resveratrol
(RSV) throughout the training period. The data are expressed as absolute oxygen consumed. Maximal oxygen consumption increased in RSV-treated subjects but
not in PL-treated subjects after 12 wk of exercise. (B) Representative electron micrographs of vastus lateral muscle sections prepared for electron microscopy before
training (i: scale bar = 500 µm) and after 12 wk of exercise training in PL-treated (ii: scale bar = 500 µm) and RSV-treated (iii: scale bar = 500 µm) samples. A lower
magnification of post-exercise RSV-treated muscle (iv: scale bar = 800 µm). Examples of apparently morphologically normal mitochondria are shown with the red
arrows, and apparently, abnormal mitochondria are shown with blue arrows. (C) mRNA was extracted from biopsies of the vastus lateralis muscles before and after
exercise. Subjects took either a PL or RSV for 12 wk and exercised throughout the study. Gene expression was analyzed by PCR array and the global data set were
evaluated from 84 genes. Quantitative analysis of genes that were differentially increased in muscles of RSV- vs PL-treated older subjects. The Heat Map provides a
visualization of the fold changes in mitochondrial log2 fold gene expression between the selected groups for every gene with the layout for the Heat Map as follows:

Layout 01 02 03 04 05 06 07 08 09 10 11 12

A ATP5A ATP5B ATP51 ATP5F ATP51 ATP5 ATP53 ATP5 ATP5I ATP5 ATP5J ATP5
B ATP5O COX4I1 COX5A COX5B COX6A1 COX6A2 COX6B1 COX6C COX7A2 COX7A2 COX7B COX8A
C CYC1 NDUFA1 NDUFA1 NDUFA11 NDUFA3 NDUFA3 NDUFA4 NDUFA5 NDUFA6 NDUFA8 NDUFAB1 NDUFB1
D NDUFB2 NDUFB3 NDUFB4 NDUFB5 NDUFB6 NDUFB7 NDUFB8 NDUFB9 NDUFC1 NDUFC2 NDUFS1 NDUFS2
E NDUFS3 NDUFS4 NDUFS5 NDUFS6 NDUFS7 NDUFS8 NDUFV1 NDUFV2 NDUFV3 PPA1 SDHA SDHB
F SDHC SDHD UQCR11 UQCRC1 UQCRC2 UQCRFS1 UQCRH UQCRQ ARRDC3 ASB1 CYB561D DNAJB1
G EDN1 GADD45B HSPA1A HSPA1B LRP5L MitoH1 MitoH2_ MitoH2_ MitoH2_4 MitoH2_ RNU11S SLC25A25

The legend for the color intensity is shown below each heat map.
(D) Isokinetic measures of knee extensor muscle torque (Newton-meters), average peak torque, and average power (watts) were obtained on a Biodex at 60°/s.
The data represent mean data from the subjects in each group. *p < .05, pre-training vs post-training. **p < .05, RSV vs PL treatment. Pre-PL, before training in
the placebo group; Post-PL, after 12 wk of training in the placebo group. Pre-RSV, before training in the resveratrol group; Post-RSV, resveratrol-treated muscles
after 12 wk of exercise. *p < .05, pre-training vs post-training.

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Table  2. Normalized Mitochondrial mRNA Log2 Fold Differences With 2 Fold or Greater Increases in Resveratrol vs Placebo Treatment
Pre–Post Exercise

Gene Symbol Pre-PL vs Pre-RSV Post-PL vs Post-RSV Pre- vs Post-Exercise PL Pre- vs Post-Exercise RSV

Apoptosis
BAK1 3.14122 3.92495 5.70290 12.38249
BCL2 3.29122 −0.81512 3.69288 5.78463
BCL2L1 (Bcl-xl) 0.74122 1.06497 6.82299 8.24248
BID 2.59122 1.06497 9.78299 13.05249
CDKN2A 3.91122 2.08488 −1.01711 4.59464
PMAIP1 2.07122 2.82488 4.51289 9.02448
SFN 2.61122 3.09494 7.76291 13.08249
DNM1L 6.75122 0.71488 1.61289 8.69461
UXT 11.55122 −0.18512 0.21289 11.19463
Metabolism
AIP 4.12122 5.84495 2.16289 11.74249
COX18 3.47122 3.81488 −4.13711 2.76463
CPT1B 3.93122 1.06497 5.24299 9.85249
UCP2 5.25122 1.06497 6.70299 12.63249
UCP3 4.46122 1.06497 8.67299 13.81249
IMMP2L 1.21122 3.73494 6.88285 11.44249
LRPPRC 0.53122 7.08495 3.52289 10.75249
HPRT1 7.21122 8.89495 2.94289 18.66249
Morphology
MFN1 6.70122 0.59488 0.62289 7.53461
MFN2 1.80122 1.06497 6.09299 8.57249
MSTO1 2.54122 1.06497 9.10298 12.32249
Carrier/import
SLC25A10 4.18122 1.47485 7.32289 12.59430
SLC25A22 5.75121 1.06497 4.48300 10.91249
SLC25A27 5.91122 3.06488 0.99289 9.58459
SLC25A31 3.33122 1.21488 5.03291 9.19458
SLC25A37 9.65122 −0.90512 −1.67711 6.68463
SLC25A5 4.92122 1.06497 10.73299 16.33249
STARD3 1.43122 2.92487 5.41288 9.38450
TIMM10 2.23122 4.58495 6.05287 12.48249
TIMM10B 5.02122 6.80495 1.23289 12.67249
TIMM44 5.09122 0.64488 4.82291 10.17451
TIMM50 1.59122 1.06497 9.03299 11.30249
TIMM8A 4.77122 8.02495 2.83289 15.24249
TOMM34 2.05122 3.44488 3.57289 8.68457
TOMM40L 5.08122 1.06497 9.45298 15.21249

Note: PL = placebo; RSV = resveratrol.

subjects as compared to PL-treated subjects (Table 2; Supplementary (TIMM23) were two- to eightfold lower in RSV- versus PL-treated
Results). muscles (Table 3; Supplementary Results).

Genes That Had a Lower Increase With RSV versus
PL Treatments
The genes in which there was a twofold or more lower in RSV-
treated versus PL-treated genes are shown in Table 3 and Figure 1C.
Apoptosis/autophagy
SH3 domain containing GRB2-like endophilin B1 (SH3GLB1)
and Ras homolog family (RHOT) members T1 (RHOT1) and T2
(RHOT2) had a approximately 5-, 2.5-, and 7-fold lower gene differ-
ences in RSV versus PL pre- to post-exercise, respectively (Table 3).
Carrier/shuttle/import proteins
Gene expression differences from pre- to post-exercise in SLC25A4,
SLC25A13, SLC25A14, SLC25A15, SLC25A19, SLC25A20,
SLC25A21, and SLC25A30, mitochondrial import proteins, metaxin
2 (MTX2), and translocase of inner mitochondrial membrane 23
Decreased RSV regulation of mitochondrial metabolic genes
The gene expressions levels of COX10, and carnitine palmitoyltrans-
ferase 2 (CPT2), GrpE-like 1, and mitochondrial (GRPEL1) and
mitochondrial intermediate peptidase (MIPEP) were two- to fourfold
lower in RSV-treated muscles as compared to PL-treated muscles.
Muscle Strength and Fatigue
Subjects who exercised and consumed RSV had an 8.5% (p < .05)
increase in knee extensor peak torque that was measured at 60°/s as
compared to the pre-training data, whereas there was no change in
maximal torque production in the subjects who consumed the PL
and exercised for 12 weeks. In a similar fashion, average knee exten-
sion torque that was measured at 60°/s, increased by 14.1% (p < .05)
in subjects that had taken RSV from pre- to post-training, whereas,
there was no significant change in the average peak torque at 60°/s in
subjects who consumed the PL and trained for 12 weeks.

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Table  3. Normalized Mitochondrial mRNA Log2 Fold Differences That Were 2 Fold or More Lower in Resveratrol vs Placebo Treatment
Pre–Post Exercise

Gene Symbol Pre-PL vs Pre-RSV Post-PL vs Post-RSV Pre- vs Post-Exercise PL Pre- vs Post-Exercise RSV

Apoptosis/autophagy SH3GLB1 1.66122 −5.75510 8.21299 3.73460

RHOT1 3.78122 −6.12510 7.06298 4.33465
RHOT2 −1.98879 −4.79518 8.84298 1.67450
Metabolism COX10 −1.65878 −1.94511 8.17299 4.18469
CPT2 1.64122 −3.62510 7.15299 4.78477
GRPEL1 3.05122 −5.59510 10.51299 7.58466
MIPEP 2.07122 −4.56512 2.62289 −0.25537
Carrier/import SLC25A13 −4.45878 1.19494 9.86300 6.21252
SLC25A14 −4.88878 −0.43512 8.10289 2.39473
SLC25A15 −1.26878 −2.49510 9.75299 5.60457
SLC25A19 −2.61878 0.62488 5.39290 3.01469
SLC25A20 −7.17857 1.06497 8.15298 −0.34365
SLC25A21 −3.56879 −1.07510 7.55298 2.52434
SLC25A30 −4.12879 1.06497 9.83299 6.38248
SLC25A4 −0.52878 −0.25512 5.05289 3.88463
MTX2 −8.16857 1.06497 9.14299 −0.34365
TIMM23 2.86122 −5.54518 8.00298 4.93468

Note: PL = placebo; RSV = resveratrol.

Muscle power was unchanged in PL-fed subjects from before to
after 12 weeks of exercise training. However, RSV-treated subjects
had a 14.0% (p < .05) increase in knee extensor muscle power after
training as compared to before training (Figure 1E). The interaction
between Session (pre- or post-training) × Group (PL vs RSV) was
significant for average power in knee extension (Watts) at 60°/s (p
= .004) and average peak torque over seven repetitions in extension
(Newton-meters) at 60°/s (p = .038) (Figure 1D). Other indices of
muscle function were unchanged by RSV treatment as compared to
PL treatment (Supplementary Tables 2–7).
Muscle Fiber Characteristics
Fiber type
An example of tissue cross-sections that were used for fiber type
and fiber size distribution is shown in Figure 2. Before training,
type I fiber distribution was not statistically different in the vastus
lateralis of PL-treated (68.3% ± 6.3%) and RSV-treated (51.9% ±
5.5%) groups. Type I fiber composition was not significantly altered
after 12 weeks of exercise training in PL-treated (50.4% ± 6.9%) or
RSV-treated (43.0% ± 5.5%) subjects. In the PL group, the distribu-
tion of type IIA fibers was unchanged before (31.2% ± 6.0%) as
compared to after training (48.3% ± 5.9%). Similarly, in the RSV-
treated group, the distribution of type IIA fibers was unchanged
from pre-training (47.8% ± 5.5%) to post-training (55.6% ± 5.1%).
A small number of fibers co-expressed type I and type IIA myosin
heavy chain. There was no difference before exercise in the percent
of type IIA/I co-expressing fibers between the PL group (0.45% ±
0.32%) and the RSV group (0.33% ± 0.35%). There was no sta-
tistically significant change in the percent distribution of type IIA/I
co-expressing fibers after exercise in either the PL-treated (1.37% ±
0.96%) or RSV-treated (1.39% ± 0.56%) subjects. Type IIX fibers
were rare and made up less than 0.1% of the total population of
fibers that were examined in muscle biopsies of the subjects in this
study. Consequently, few fibers co-expressed IIA/IIX in this sample.
The IIA/IIX fibers made up 0.014% ± 0.01% and 0.05% ± 0.02%
of the fiber population in the PL group before and after exercise,
respectively. Similarly, the percentage of type IIA/IIX fibers was and
0.017% ± 0.02% and 0.09% ± 0.03% in the RSV group.
Type I fiber size
To test if fiber size were improved with RSV treatment, we obtained
fiber cross-sectional area and Feret diameters from tissue sections
that had been incubated in antibodies against type I, type IIA, and
type IIX myosin heavy chain (Figure 2A). The data suggest that fiber
area adaptations to exercise and treatment were fiber type specific.
Twelve weeks of exercise significantly improved mean fiber type I
area (Figure 2B) and Feret diameter (There was a shift to the right in
the fiber area-frequency histogram of PL-treated subjects especially
in the 5,000–6,000 µm2 range (Figure 2D). RSV-treated fibers also
had a shift to the right of the fiber area-frequency distribution and a
significantly greater percentage of fibers larger than 7,000 µm2 after
training as compared to before training (Figure 2E).
These differences can be seen readily in the cumulative frequency
distribution for type I fibers, where there is a small shift towards
larger fibers in the PL trained group but a clearer shift to larger fib-
ers was found in the RSV-treated group (Figure 2F; Supplementary
Figure 5).
Type IIA fiber size
Unlike the adaptations in type I fibers, 12 weeks of exercise failed to
improve mean type IIA fiber area or Feret diameter in the PL group
(Figure 3A). In contrast to the PL group, in the subjects treated with
RSV, type IIA fiber area and Feret diameter were 32.2% and 11.9%
greater, respectively, after 12 weeks of training as compared to pre-
training (Figure 3B). Exercise training in the PL group had a right-
ward shift in the type IIA fiber area-frequency distribution with a
greater frequency of fibers with sizes exceeding 6,000 µm2 (Figure
3C). Type II fibers from the RSV-treated group had a shift to the right
of the fiber area-frequency distribution and a significantly greater
percentage of fibers larger than 5,000 µm2 after training as com-
pared to before training (Figure 3D). The similarity in the pre- and
post-training type IIA cumulative frequency distribution can been
readily seen for the PL trained group (Figure 3C), whereas a shift to
larger fibers can be seen in the trained RSV treated type IIA fibers as
compared to before training (Figure 3E). Although the cumulative
frequency curves for the type IIA fibers shifted to the right after exer-
cise as compared to before exercise, the shift to larger fibers (shift to

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 1602 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12
Figure  2.  Fibers from placebo (PL)-treated and resveratrol (RSV)-treated
muscles. (A) Mean type I  fiber area. *p  <  .05, pre-training vs post-training.
**p < .05, RSV vs PL treatment. (B) Mean Feret diameter from type I fibers.
*p  <  .05, pre-training vs post-training. **p  <  .05, RSV vs PL treatment. (C)
Fiber area-fiber frequency histograms of type I  fibers from PL-treated
muscles. (D) Fiber area-fiber frequency histograms of type I fibers from RSV-
treated muscles. (E) Cumulative frequency area histograms of type I fibers
from PL treated and RSV treated. (F) Representative fiber sections stained
with DAPI to show muscle nuclei type IIA MHC (green), type IIX MHC (red),
and type I MHC (purple) fibers. The overlay shows co-expression of type IA
and IIX MHC (yellow/green).
the right) was more pronounced in type IIA fibers of the RSV group
(Figure 3D–F; Supplementary Figure 6).
Type IIA/type IIX co-expressing fiber size
There did not appear to be any fibers that were solely expressing type
IIX myosin heavy chain in the biopsy samples. There were very few
fibers that co-expressed type IIA and type IIX myosin heavy chain
before exercise in most subjects. Although there was a tendency for
more fibers to co-express both type IIA and type IIX myosin heavy
chain after exercise, there was considerable variability among the
subjects in both groups. Type IIA/IIX co-expressing fibers were rela-
tively small and averaged 1677.8 ± 246 µm2 after exercise in the
PL group and 1834.4 ± 537.1 µm2 after exercise in the RSV-treated
group but the low number of fibers and the large variation in size
prevented any statistically important observations in this fiber type.
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Figure 3.  Mean fiber area (A) and mean Feret diameter (B) in type IIA fibers
from placebo (PL)-treated and resveratrol (RSV)-treated muscles fibers.
*p < .05, pre-training vs post-training. **p < .05, RSV vs PL treatment. Fiber
area-fiber frequency histograms of type IIA fibers from PL-treated (C) and
RSV-treated (D) muscles fibers. Cumulative frequency area histograms of
type IIA fibers from PL-treated and RSV-treated muscles fibers (E).
Nuclei and Satellite Cell Content
Although there was no statistical change in the number of nuclei/
fiber with the PL treatment after training as compared to the pre-
training data, the total number of nuclei (satellite cell + myonuclei)
was increased by 20% (p < .05) in the RSV-treated tissue samples
of RSV-treated muscles as compared to the pre-training samples.
The number of Pax7 positive nuclei (satellite cells) was significantly
increased from 0.45 to 1.1 Pax7 satellite cells/fiber (p < .05) in the
post-trained sample of the RSV-treated subjects, but total satellite
cell content/fiber did not change in the PL-treated samples (Figure
4A–D).
Oxidative Damage
8-OHdG-positive nuclei were used as an indication of oxidative
DNA damage in nuclei. No nuclei that resided between the basal
lamina and sarcolemma were 8-OHdG positive. However, nuclei
that were either inside the basal lamina or outside the basal lamina
were identified to have oxidative damage. 8-OHdG-positive nuclei
 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12 1603

that were located outside of the basal lamina were not quantified.
Oxidative damage of DNA as measured by 8-OHdG-positive nuclei
inside the basal lamina was 20.9% and 18.8% in muscles from the
pre-training biopsies of PL and RSV treatment groups. Exercise
decreased the number of nuclei that were located inside the basal
lamina with oxidative damage in both PL (5.6% of the total nuclei)
and RSV (3.7% of the total nuclei) groups, but there was some vari-
ability between subjects in each group and there were no statistically
significant differences between RSV and PL groups (Figure 4E–G).
These data suggest that there is less oxidative damage after 12 weeks
of exercise training but that RSV treatment did not reduce the per-
cent of total muscle nuclei (inside the basal lamina) with DNA dam-
age further.

Discussion
VO2 max and fatigue resistance have been reported to improve
when RSV was combined with endurance exercise training (33,34).
However, other preclinical long-term studies have also reported that
RSV provided modest or no increases in reducing aging-associated
decrements in muscle mass or oxidative stress (16,35). In addition,
several clinical RSV studies in humans have shown no, or only minor
improvements in function (36–38). In contrast, our data suggests
that RSV acts synergistically with exercise-induced signaling path-
ways to enhance cellular and molecular adaptations that lead to
improved mitochondria density, oxygen uptake, fiber area, and mus-
cle function in aged persons.

Mitochondria and Oxygen Consumption

Consistent with our hypothesis, we found that RSV improved
maximal oxygen consumption and mitochondrial volume density
more so than exercise alone (Figure 1). We noticed that in the elec-
tron micrographs, some of the mitochondria appeared to be mor-
phologically appropriate, while others appeared to have increased
spacing of the cristae and less dense packing of the mitochondrial
membrane. The RSV-treated samples appeared to have fewer mito-
chondria with abnormal morphology as compared to PL-treated
muscles. The implications are of course that more mitochondria
that are not “healthy” would not be of any advantage to the
muscle; whereas if the mitochondria are morphologically sound
and presumably fully functional then we might assume that, the
increase in mitochondrial density could be of benefit for improved
energy production in the muscle cells. Based on the subjective
evaluation of the micrographs, we speculate that the better mito-
chondrial morphology along with greater mitochondrial volume
density in RSV-treated muscles after exercise training as compared
to PL conditions may have occurred in part to removal or repair
of the mitochondria with abnormal morphology. Nevertheless, we
have no way of knowing if the appearance of abnormal mitochon-
dria had any negative effect on the mitochondria’s ability to gener-
ate ATP or to oxidize substrates.
Consistent with the increase in mitochondria volume density,
there was some enhancement of gene expression with RSV treat-
ment in genes associated mitochondrial morphology, and mito-
chondrial import (Table 2). It was also particularly interesting to
note that RSV also increased genes associated with mitochondrial-
regulated apoptotic signaling. However, apoptotic signaling might
be an important mechanism to remove dysfunctional organelles
and this could be part of a process to “clean” up and remove
Figure  4.  An example of tissue sections cut from human biopsies. (A)
DAPI (blue)-stained nuclei. (B) Pax7-positive satellite cell nuclei are green.
(C) The basal lamina of each fiber was identified by a laminin antibody
(fuchsia). (D) The overlay of panels A–C. White arrows point to Pax7 nuclei
that are located inside the basal lamina. The yellow arrow shows a Pax7
nucleus that is outside of the basal lamina. (E) Total nuclei and satellite
cell (Pax7+) content were quantified and expressed per fiber. Nuclei that
were outside of the basal lamina were assumed to be of non-myogenic
origins. Although Pax7 nuclei that were outside of the basal lamina would
be expected to be capable of myogenic contributions, they were not
counted as it was not possible to tell if they were migrating from another
muscle fiber, or if they would contribute to new de novo muscle fibers.
*p  <  .05, pre-training vs post-training. **p  <  .05, RSV vs PL treatment.
(F) 8-Hydroxyguanosine (8-OHdG)-positive nuclei were identified and
quantified if they resided inside the basal lamina as an indication of
oxidative damage to skeletal muscle nuclear DNA. *p < .05, pre-training vs
post-training. (G) A representative cross-section from a 78-year-old female
after 12 wk of exercise training using a PL. (i) DAPI-stained nuclei (blue).
(ii) 8-OHdG-positive nuclei are green. (iii) The muscle sarcolemma (red)
was indicated by an anti-dystrophin antibody. (iv) The basal lamina was
identified by an antibody to laminin and this is shown in white. (v) The
overlay of the preceding images is shown. White arrows indicate the same
8-OHdG-positive nuclei in each section. Nuclei that were located inside the
basal lamina and were 8-OHdG positive were quantified. Most nuclei that
sat between the basal lamina and sarcolemma would be expected to be
satellite cells, but these cells were only rarely 8-OHdG positive, whereas
more frequently, non-muscle nuclei that resided outside of the basal
lamina and sarcolemma were identified as 8-OHdG positive (yellow arrow)
but these nuclei were not quantified.

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 1604 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12
dysfunctional proteins in aging. However, this is not a simple rela-
tionship because several apoptotic genes were also decreased as
were genes associated with autophagy in RSV- versus PL-treated
muscle cells (Table 3). Furthermore, a number of import genes
were downregulated in RSV-treated samples pre- to post-exercise
as compared to PL-treated samples (Table 3), perhaps as a result
of a greater magnitude of proapoptotic signaling and removal of
dysfunctional proteins (Table 3). This varied gene regulation of
mitochondrial-associated apoptotic and import signaling war-
rants further investigation, to determine if these changes provide
an improved muscle environment for adaptation to exercise with
RSV in elderly subjects.
Our results in older subjects in the current study differ some-
what with data from Scribbans and colleagues (39) who reported
no additive effects of RSV supplementation on aerobic exercise per-
formance. Gliemann and colleagues (40) have reported that RSV
might blunt the effects of aerobic exercise training on low-density
lipoprotein, total cholesterol/high-density lipoprotein ratio, and tri-
glycerides. However, we did not observe any blunting of these meas-
ures in our study, but rather we found a small synergistic effect of
combining RSV with exercise training (aerobic + resistance exercise)
on maximal oxygen uptake but no synergistic improvement in the
lipid profiles in older subjects (Table 1).
It is unlikely that RSV either blunted or prevented some of the
improvements in cardiovascular indices or mitochondrial function
that might have been expected as part of exercise-induced adapta-
tions. Otherwise, we would have expected to have poorer functional
outcomes (VO2 max, fatigue resistance, mitochondrial volume etc.),
in RSV-treated muscles, but this was not the case.
Our data do not support a RSV-mediated improvement in lipid pro-
files, or a reduction in oxidative damage that is additive to exercise in
older subjects. However, it is possible that the dose that we gave the sub-
jects (500 mg/d) was not sufficient to result in some of the metabolic and/
or mitochondrial enhancements that have been reported in preclinical
animal models. Metabolic rates differ in humans and rodents, and after
a first pass through the liver and perhaps the active RSV per se or active
metabolites of RSV may have differed or been proportionally lower in
humans who took RSV and exercised.
Muscle Function
Exercise improvements were observed in knee extension strength
(peak torque, average peak torque, and average power) in RSV-
treated subjects but not in PL-treated subjects who exercised for
12 weeks. As the randomly assigned RSV group tended to have a
lower starting torque as compared to the PL group, we examined if
the greater difference in the strength of the RSV group post-training
could be due at least in part to the lower starting point in this group
as compared to the PL group, and the RSV group simply caught
up to the PL group by the post-training measurement. However, we
observed only small differences from comparing the group means
from the pre- and post-training data for each subject in each group
(see Supplementary Results). Thus, the data strongly suggest that
RSV provided a greater benefit for developing maximal torque as
compared to the PL treatment in response to exercise in elderly
subjects.
However, the longer time to peak torque along with no differ-
ences in power or average absolute torque suggests that while RSV
increased torque production, it may have slowed torque develop-
ment. This conclusion is consistent with the shift towards slower
myosin heavy chain characteristics of type I fibers (41,42) and the
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approximately 30% greater type I fiber area in vastus lateralis mus-
cles of RSV-treated as compared to PL-treated subjects in the current
study. Thus, the greater relative area that is comprised of type I fibers
may account for the lower rate of torque production after exercise
in the muscles of RSV-treated subjects.
Fiber Area and Satellite Cells
The improvement in muscle function (Figure 1E) suggests a new role
for RSV-mediated contractile protein accumulation (Figures 2 and 3),
to regulate at least in part, muscle force and power. Furthermore,
RSV induced an improvement in fiber cross-sectional area of exer-
cised muscles (Figures 2 and 3). Additional evidence for this adap-
tion is observed by a shift to the right in fiber area-fiber frequency
histograms (Figures 2 and 3). Of particular interest, type IIA fiber
area did not improve with exercise in the PL group, but it increased
in the subjects who consumed RSV and exercised. This contrasts to
data that showed a RSV-associated blunting of both hypertrophy and
satellite cell proliferation in muscles of old mice (43). Nevertheless,
our current results are consistent with data from reloading after atro-
phy in rodents, where improvement in especially type II fiber size was
reported in animals given RSV (15).
Along with the larger fibers (Figure 4), RSV induced an increase
in the exercise-associated nuclear and satellite cell numbers (Figure 4).
While it is clear that muscle satellite cells are critical for successful mus-
cle regeneration/repair (44), the idea that satellite cells are essential for
increasing fiber size during muscle hypertrophy (45–49) has been chal-
lenged from preclinical rodent models (50). However, it is not necessary
for RSV to enhance satellite cell proliferation, as RSV could potentially
contribute to a greater total nuclear pool by reducing apoptotic signal-
ing such as proapoptotic proteins Bax, cleaved caspase 3, and cleaved
caspase 9 in exercised muscles (15), and therefore reducing the potential
for elimination of muscle nuclei. As we did not conduct a time course
evaluation, we do not know if the total satellite cell and nuclear abun-
dance was increased early as a result of hypertrophic adaptations to
exercise training, or if it were accomplished more slowly and in part by
eliminating fewer nuclei through apoptotic pathways. The association
of RSV-mediated increase in fiber size and total myonuclei (satellite cells
and muscle nuclei) is consistent with the nuclear domain hypothesis,
but additional time course studies should be conducted to provide a
better documentation of satellite cell and myonuclei accretion with the
combined effects of exercise and RSV treatments.
Study Limitations
A potential limitation of this study is that we used a somewhat
moderate training protocol, where only small or in some cases, no
changes were noted in the variables that where measured. Thus, we
cannot rule out the possibility that if we had used a training protocol
that had been more intensive and had resulted in greater exercise-
induced improvements, that the effects of RSV might have been dif-
ferent than what we report in this study. However, the intensity of
the exercise protocol that we used in this study is in our opinion,
more “translationally relevant” than if we had used a more aggres-
sive training protocol similar to that used in other studies. This is
because our findings will be relevant for average older persons who
are interested in increasing their physical activity but who are not
interested in engaging in training at high intensity. However, it is
exciting to note that even with this modest training approach, we
saw some very important structural adaptations in muscle to exer-
cise with RSV treatment.
 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12
Conclusion
We propose that exercise-initiated an overall improvement in mito-
chondrial function that was linked to improved anabolic signaling
pathway leading to hypertrophy of type I  and IIA fiber sizes, and
an increase in total muscle nuclei (satellite cells and myonuclei) that
was enhanced in muscles from RSV-exercised versus PL-exercised
subjects. The greater fiber sizes account for the RSV-induced eleva-
tion in muscle torque and power. These data support a role for RSV
treatment combined with exercise as a potential means for reducing
or reversing sarcopenia in elderly persons.
Supplementary Material
Supplementary  data  is available at  The Journals of Gerontology,
Series A: Biological Sciences and Medical Sciences online.
Funding
This project was supported by the National Institute of General Medical
Sciences (Grant/Award Number: 5P20RR016440, P20RR016477,
P30RR032138/GM103488, and U54 GM104942).
Acknowledgment
We would like to acknowledge the West Virginia University Microscope
Imaging Facility, which is supported by the Mary Babb Randolph Cancer
Center and NIH grant P20RR016440, P30RR032138/GM103488, and
P20RR016477.
Conflict of Interest
The authors declare no conflicts of interest.
References
1. Castillo EM, Goodman-Gruen D, Kritz-Silverstein D, Morton DJ,
Wingard DL, Barrett-Connor E. Sarcopenia in elderly men and women:
the Rancho Bernardo study. Am J Prev Med. 2003;25:226–231.
doi:S0749379703001971.
2. Suetta C, Hvid LG, Justesen L, et al. Effects of aging on human skel-
etal muscle after immobilization and retraining. J Appl Physiol (1985).
2009;107:1172–1180. doi:10.1152/japplphysiol.00290.2009.
3. Magne H, Savary-Auzeloux I, Vazeille E, et al. Lack of muscle recov-
ery after immobilization in old rats does not result from a defect in
normalization of the ubiquitin-proteasome and the caspase-dependent
apoptotic pathways. J Physiol. 2011;589(Pt 3):511–524. doi:10.1113/
jphysiol.2010.201707.
4. Pierno S, Liantonio A, Camerino GM, et al. Potential benefits of taurine
in the prevention of skeletal muscle impairment induced by disuse in the
hindlimb-unloaded rat. Amino Acids. 2012;43:431–445. doi:10.1007/
s00726-011-1099-4.
5. Pierno S, Camerino GM, Cannone M, et al. Paracrine effects of IGF-1
overexpression on the functional decline due to skeletal muscle disuse:
molecular and functional evaluation in hindlimb unloaded MLC/mIgf-1
transgenic mice. PLoS ONE. 2014;8:e65167. doi:e65167.10.1371/jour-
nal.pone.0065167.
6. Hao Y, Jackson JR, Wang Y, Edens N, Pereira SL, Alway SE. β-Hydroxy-
β-methylbutyrate reduces myonuclear apoptosis during recovery from
hind limb suspension-induced muscle fiber atrophy in aged rats. Am J
Physiol Regul Integr Comp Physiol. 2011;301:R701–R715. doi:10.1152/
ajpregu.00840.2010.
7. Alway SE, Bennett BT, Wilson JC, et al. Green tea extract attenuates mus-
cle loss and improves muscle function during disuse, but fails to improve
Downloaded from https://academic.oup.com/biomedgerontology/article-abstract/72/12/1595/3824856
by guest
on 10 December 2017
1605
muscle recovery following unloading in aged rats. J Appl Physiol (1985).
2015;118:319–330. doi:10.1152/japplphysiol.00674.2014.
8. Montesano A, Luzi L, Senesi P, Mazzocchi N, Terruzzi I. Resveratrol pro-
motes myogenesis and hypertrophy in murine myoblasts. J Transl Med.
2013;11:310. doi:10.1186/1479-5876-11-310.
9. Wyke SM, Russell ST, Tisdale MJ. Induction of proteasome expression in
skeletal muscle is attenuated by inhibitors of NF-kappaB activation. Br J
Cancer. 2004;91:1742–1750. doi:10.1038/sj.bjc.6602165.
10. Russell ST, Wyke SM, Tisdale MJ. Mechanism of induction of muscle
protein degradation by angiotensin II. Cell Signal. 2006;18:1087–1096.
doi:10.1016/j.cellsig.2005.09.009.
11. Wyke SM, Tisdale MJ. Induction of protein degradation in skeletal mus-
cle by a phorbol ester involves upregulation of the ubiquitin-proteas-
ome proteolytic pathway. Life Sci. 2006;78:2898–2910. doi:10.1016/j.
lfs.2005.11.014.
12. Alamdari N, Aversa Z, Castillero E, et al. Resveratrol prevents dexameth-
asone-induced expression of the muscle atrophy-related ubiquitin ligases
atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent
mechanism. Biochem Biophys Res Commun. 2012;417:528–533.
doi:10.1016/j.bbrc.2011.11.154.
13. Momken I, Stevens L, Bergouignan A, et al. Resveratrol prevents the
wasting disorders of mechanical unloading by acting as a physical exer-
cise mimetic in the rat. FASEB J. 2011;25:3646–3660. doi:10.1096/
fj.10-177295.
14. Jackson JR, Ryan MJ, Hao Y, Alway SE. Mediation of endogenous antiox-
idant enzymes and apoptotic signaling by resveratrol following muscle dis-
use in the gastrocnemius muscles of young and old rats. Am J Physiol Regul
Integr Comp Physiol. 2010;299:R1572–R1581. pii: ajpregu.00489.2010.
doi:10.1152/ajpregu.00489.2010.
15. Bennett BT, Mohamed JS, Alway SE. Effects of resveratrol on the recovery
of muscle mass following disuse in the plantaris muscle of aged rats. PLoS
ONE. 2013;8:e83518. doi:10.1371/journal.pone.0083518.
16. Ringholm S, Olesen J, Pedersen JT, et al. Effect of lifelong resveratrol sup-
plementation and exercise training on skeletal muscle oxidative capacity
in aging mice: impact of PGC-1α. Exp Gerontol. 2013;48:1311–1318.
doi:10.1016/j.exger.2013.08.012.
17. Roman WJ, Fleckenstein J, Stray-Gundersen J, Alway SE, Peshock R,
Gonyea WJ. Adaptations in the elbow flexors of elderly males after heavy-
resistance training. J Appl Physiol (1985). 1993;74:750–754.
18. Mankowski RT, Anton SD, Buford TW, Leeuwenburgh C. Dietary antioxi-
dants as modifiers of physiologic adaptations to exercise. Med Sci Sports
Exerc. 2015;47:1857–1868. doi:10.1249/MSS.0000000000000620.
19. Bassett DR Jr, Howley ET, Thompson DL, et  al. Validity of inspiratory
and expiratory methods of measuring gas exchange with a computerized
system. J Appl Physiol (1985). 2001;91:218–224.
20. McNeil CJ, Rice CL. Fatigability is increased with age during velocity-
dependent contractions of the dorsiflexors. J Gerontol A Biol Sci Med Sci.
2007;62:624–629. doi:https://doi.org/10.1093/gerona/62.6.624.
21. Power GA, Dalton BH, Rice CL, Vandervoort AA. Power loss is greater
following lengthening contractions in old versus young women. Age
(Dordr). 2012;34:737–750. doi:10.1007/s11357-011-9263-z.
22.
Ferketich AK, Kirby TE, Alway SE. Cardiovascular and mus-
cular adaptations to combined endurance and strength train-
ing in elderly women. Acta Physiol Scand. 1998;164:259–267.
doi:10.1046/j.1365-201X.1998.00428.x.
23. Alway SE, Hughson RL, Green HJ, Patla AE. Human tibialis anterior
contractile responses following fatiguing exercise with and without beta-
adrenoceptor blockade. Clin Physiol. 1988;8:215–225.
24. Alway SE, Coggan AR, Sproul MS, Abduljalil AM, Robitaille PM. Muscle
torque in young and older untrained and endurance-trained men. J
Gerontol A Biol Sci Med Sci. 1996;51:B195–B201.
25. Sharif S, Thomas JM, Donley DA, et al. Resistance exercise reduces skele-
tal muscle cachexia and improves muscle function in rheumatoid arthritis.
Case Rep Med. 2011;2011:205691. doi:10.1155/2011/205691.
26. Alway SE, Stray-Gundersen J, Grumbt WH, Gonyea WJ. Muscle cross-
sectional area and torque in resistance-trained subjects. Eur J Appl Physiol
Occup Physiol. 1990;60:86–90.
 1606 Journals of Gerontology: Biological Sciences, 2017, Vol. 72, No. 12
27. Alway SE, Starkweather LA. Effect of anabolic steroids on new fiber
formation and fiber area during stretch-overload. J Appl Physiol (1985).
1993;74:832–837.
28. Alway SE, MacDougall JD, Sale DG, Sutton JR, McComas AJ. Functional
and structural adaptations in skeletal muscle of trained athletes. J Appl
Physiol (1985). 1988;64:1114–1120.
29. Siu PM, Alway SE. Response and adaptation of skeletal muscle to denerva-
tion stress: the role of apoptosis in muscle loss. Front Biosci (Landmark
Ed). 2009;14:432–452.
30. Siu PM, Pistilli EE, Butler DC, Alway SE. Aging influences cellular and
molecular responses of apoptosis to skeletal muscle unloading. Am J Physiol
Cell Physiol. 2005;288:C338–C349. doi:10.1152/ajpcell.00239.2004.
31. Chabi B,LjubicicV,Menzies KJ,Huang JH,SaleemA,Hood DA.Mitochondrial
function and apoptotic susceptibility in aging skeletal muscle. Aging Cell.
2008;7:2–12. doi:10.1111/j.1474-9726.2007.00347.x.18028258.
32. O’Leary MF, Vainshtein A, Iqbal S, Ostojic O, Hood DA. Adaptive plasticity
of autophagic proteins to denervation in aging skeletal muscle. Am J Physiol
Cell Physiol. 2013;304:C422–C430. doi:10.1152/ajpcell.00240.2012.
33. Hart N, Sarga L, Csende Z, et al. Resveratrol enhances exercise train-
ing responses in rats selectively bred for high running performance. Food
Chem Toxicol. 2013;61:53–59. doi:10.1016/j.fct.2013.01.051.
34. Murase T, Haramizu S, Ota N, Hase T. Suppression of the aging-associated
decline in physical performance by a combination of resveratrol intake
and habitual exercise in senescence-accelerated mice. Biogerontology.
2009;10:423–434. doi:10.1007/s10522-008-9177-z.
35. Jackson JR, Ryan MJ, Alway SE. Long-term supplementation with res-
veratrol alleviates oxidative stress but does not attenuate sarcopenia in
aged mice. J Gerontol A Biol Sci Med Sci. 2011;66:751–764. doi:10.1093/
gerona/glr047.
36. Gliemann L, Olesen J, Biensø RS, et al. Resveratrol modulates the angio-
genic response to exercise training in skeletal muscles of aged men. Am
J Physiol Heart Circ Physiol. 2014;307:H1111–H1119. doi:10.1152/
ajpheart.00168.2014.
37. Polley KR, Jenkins N, O’Connor P, McCully K. Influence of exercise
training with resveratrol supplementation on skeletal muscle mitochon-
drial capacity. Appl Physiol Nutr Metab. 2016;41:26–32. doi:10.1139/
apnm-2015-0370.
38. Macedo RC, Vieira A, Marin DP, Otton R. Effects of chronic resveratrol
supplementation in military firefighters undergo a physical fitness test—a
Downloaded from https://academic.oup.com/biomedgerontology/article-abstract/72/12/1595/3824856
by guest
on 10 December 2017
placebo-controlled, double blind study. Chem Biol Interact. 2015;227:89–
95. doi:10.1016/j.cbi.2014.12.033.
39. Scribbans TD, Ma JK, Edgett BA, et al. Resveratrol supplementation does not
augment performance adaptations or fibre-type-specific responses to high-
intensity interval training in humans. Appl Physiol Nutr Metab. 2014;39:1305–
1313. doi:10.1139/apnm-2014-0070.
40. Gliemann L, Schmidt JF, Olesen J, et al. Resveratrol blunts the positive
effects of exercise training on cardiovascular health in aged men. J Physiol.
2013;591:5047–5059. doi:10.1113/jphysiol.2013.258061.
41. Degens H, Yu F, Li X, Larsson L. Effects of age and gender on shorten-
ing velocity and myosin isoforms in single rat muscle fibres. Acta Physiol
Scand. 1998;163:33–40. doi:10.1046/j.1365-201x.1998.00329.x.
42. Plas RL, Degens H, Meijer JP, et al. Muscle contractile properties as an
explanation of the higher mean power output in marmosets than humans
during jumping. J Exp Biol. 2015;218(Pt 14):2166–2173. doi:10.1242/
jeb.117655.
43. Ballak SB, Jaspers RT, Deldicque L, et al. Blunted hypertrophic response in
old mouse muscle is associated with a lower satellite cell density and is not
alleviated by resveratrol. Exp Gerontol. 2015;62:23–31. doi:10.1016/j.
exger.2014.12.020.
44. Chang NC, Rudnicki MA. Satellite cells: the architects of skel-
etal muscle. Curr Top Dev Biol. 2014;107:161–181. doi:10.1016/
B978-0-12-416022-4.00006-8.
45. Adams GR, Caiozzo VJ, Haddad F, Baldwin KM. Cellular and molecu-
lar responses to increased skeletal muscle loading after irradiation. Am J
Physiol Cell Physiol. 2002;283:C1182–C1195.
46. Rosenblatt JD, Yong D, Parry DJ. Satellite cell activity is required for hyper-
trophy of overloaded adult rat muscle. Muscle Nerve. 1994;17:608–613.
47. Rosenblatt JD, Parry DJ. Adaptation of rat extensor digitorum longus mus-
cle to gamma irradiation and overload. Pflugers Arch. 1993;423:255–264.
48. Rosenblatt JD, Parry DJ. Gamma irradiation prevents compensatory
hypertrophy of overloaded mouse extensor digitorum longus muscle. J
Appl Physiol (1985). 1992;73:2538–2543.
49. Mitchell PO, Pavlath GK. A muscle precursor cell-dependent pathway
contributes to muscle growth after atrophy. Am J Physiol Cell Physiol.
2001;281:C1706–C1715.
50. McCarthy JJ, Mula J, Miyazaki M, et al. Effective fiber hypertrophy in sat-
ellite cell-depleted skeletal muscle. Development. 2011;138:3657–3666.
doi:10.1242/dev.068858.