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Resistance Exercise-induced Changes in Muscle Metabolism are Load-

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DOI: 10.1249/MSS.0000000000002088
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Resistance Exercise-induced Changes in Muscle Metabolism
are Load-dependent
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Changhyun Lim1,2, Hyo Jeong Kim3, Robert W. Morton1, Roger Harris4, Stuart M. Philips1,
Tae Seok Jeong5, Chang Keun Kim2,6
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Department of Kinesiology, McMaster University, Ontario, Canada, 2Human Physiology, Korea
National Sport University, Seoul South Korea, 3Aging Physiology, Korea National Sport
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University, Seoul, Korea, 4Sport, Education and Sciences, University of Chichester, Chichester,
United Kingdom, 5SPIK Sport Medicine Clinic and Performance Centre, Seoul, South Korea,
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Exercise and Metabolism Research Centre, Zhejiang Normal University, Jinhua, China
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Accepted for Publication: 17 June 2019

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Copyright © 2019 American College of Sports Medicine

Medicine & Science in Sports & Exercise, Publish Ahead of Print
DOI: 10.1249/MSS.0000000000002088
Resistance Exercise-induced Changes in Muscle Metabolism

are Load-dependent
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Changhyun Lim1,2, Hyo Jeong Kim3, Robert W. Morton1, Roger Harris4, Stuart M. Philips1, Tae
Seok Jeong5, Chang Keun Kim2,6
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Department of Kinesiology, McMaster University, Ontario, Canada, 2Human Physiology, Korea
National Sport University, Seoul South Korea, 3Aging Physiology, Korea National Sport
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University, Seoul, Korea, 4Sport, Education and Sciences, University of Chichester, Chichester,
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United Kingdom, SPIK Sport Medicine Clinic and Performance Centre, Seoul, South Korea,
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Exercise and Metabolism Research Centre, Zhejiang Normal University, Jinhua, China
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Author for correspondence
Prof. Chang Keun Kim, Ph.D.

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Human physiology, Korea National Sport University,
88-15 Oryun-dong, Songpa-gu, Seoul South Korea
T: +82-2-410-6815
E: ckkim@knsu.ac.kr
Copyright © 2019 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
No research grant was received for this study. CONFLICT OF INTEREST. The authors
declare that the results of the study are presented clearly, honestly and without fabrication,
falsification or inappropriate data manipulation. The results of the present investigation do not
constitute endorsement by ACSM. No conflict of interest or financial are declared by the authors.
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ABSTRACT
Introduction: Lower-load (LL), higher-repetition resistance exercise training (RET) can
increase muscle mass similar degree as higher-load (HL), lower-repetition RET. However, little
is known about how LL and HL RET modulate other aspects of the RET phenotype such as
satellite cells, myonuclei, and mitochondrial proteins. We aimed to investigate changes in muscle
mass, muscle strength, satellite cell activity, myonuclear addition, and mitochondrial protein
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content following prolonged RET with LL and HL RET. Methods: We recruited 21 young men
and randomly assigned them to perform 10 weeks RET (leg press, leg extension and leg curl)
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three times per week with the following conditions: 80FAIL (80% one repetition maximum
performed [1RM] to volitional fatigue), 30WM (30%1RM with volume matched to 80FAIL),
and 30FAIL (30%1RM to volitional fatigue). Skeletal muscle biopsies were taken from the
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vastus lateralis pre- and post-RET intervention. Results: After 10 weeks of RET, only 30FAIL
and 80FAIL showed an increase in peak torque and type I fiber cross-sectional area (CSA)
(p<0.05). Moreover, only 30FAIL resulted in a significant decrease in the myonuclear domain of
type II muscle fibers and an increase in mitochondrial proteins related to autophagy, fission and
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fusion (all p<0.05). Conclusion: We discovered that LL RET was effective at increasing the
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content of a number of mitochondrial proteins. Similar to previous research, we found that
changes in muscle mass and strength were independent of load when repetitions were performed
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to volitional fatigue.
Key words: resistance training, muscle function, mitochondria, skeletal muscle, low-load high-
repetition
INTRODUCTION
Maintaining strength and muscle mass is often part of athletic performance and requisite for
healthy aging. Importantly, resistance exercise training (RET) is a more potent stimulus for
increasing muscle mass than aerobic exercise training (1), but aerobic exercise is a more potent
stimulus for increasing mitochondrial content, function (2) and protein turnover (3, 4). Indeed,
the apparently divergent nature of the phenotype achieved with regular training with either RET
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or aerobic exercise is a core principle of exercise adaptation (5).
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The American College of Sports Medicine (ACSM) recommends RET be performed at a load
of at least 65-70% repetition maximum (RM) for individuals who wish to build muscle mass (6);
however, recent studies have shown that lower-load RET performed to volitional fatigue can
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generate similar changes in muscle mass and strength as traditional, higher-load RET performed
to volitional fatigue (7, 8). In addition, RET activates satellite cells (9) and may (depending on
particular RET variables) improve mitochondrial capacity (10, 11). It is plausible that performing
lower-load, higher-repetition RET yields the benefits of both resistance- and endurance-like
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training by eliciting positive changes in muscle mass, satellite cell activation, and mitochondrial
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metabolism. For example, earlier work from Burd et al. (12) showed that changes in protein
synthesis might support greater expansion of mitochondrial proteins as well as additional

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myofibrillar proteins. However, that study consisted of only one bout 4 sets of unilateral leg
extension, so chronic phenotypic adaptations to that type of protocol remain unknown.
To our knowledge, no training study has compared lower- vs. higher-load RET and also
included an external work-matched group. We also lack basic information on important co-
regulatory processes of satellite cell regulation and mitochondrial protein expression that are
associated with divergent forms of RET. Thus, the aim of the present study was to compare the
effects of performing 10 weeks of RET with lower- vs. higher-loads, include a lower-load
externally work-matched group, on muscle function, muscle size, satellite cell activation,
myonuclear addition, mitochondrial function after performing lower-load, high-repetition vs.
higher-load, low-repetition RET for 10 weeks. Given previous work in this area we hypothesized
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that lower-load contractions, so long as they were performed to voluntary fatigue, would result in
equivalent hypertrophy to that seen with performance of higher-load contractions. In contrast,
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lower load contractions performed to volume-match the external work performed in higher-load
would not stimulate hypertrophy. We also hypothesized that given the greater volume of work
able to be performed with lower loads, when performed to voluntary fatigue, that it may also be a
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more effective stimulus for mitochondrial biogenesis than the other loading schemes.
METHODS
Participants. The study was carried out in compliance with the Declaration of Helsinki.
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Approval from the Bioethics Committee of Korea National Sport University (1263-201706-BR-
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002-01) was obtained. All participants were fully informed of the purpose of the study and of the
experimental procedures to be used. The study was conducted after subjects had given their
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written consent to participate in all procedures. The study subjects were men (n=21) who had no
self-reported musculoskeletal, cardiovascular, and respiratory system disorders and had not
undertaken any regular RET in the past 2 years.
Study Design. All tests were carried out at the same time of day to avoid the effects of circadian
rhythms, and were conducted under similar standard environmental conditions and the
participants were asked to follow their normal diet throughout the experimental period. To assess
muscle function of the lower extremities, an isokinetic muscle function test (60°/sec, 240°/sec)
was conducted to measure peak torque. Finally, a muscle biopsy of vastus lateralis in the
quadriceps femoris was taken pre- and post-training to evaluate the changes in cross-sectional
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area, number of satellite cells and myonuclei, and mitochondrial metabolism.
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Subjects were randomly assigned to one of three experimental groups: a high-load low-
repetition group exercising at 80% of single repetition maximum (1RM) to volitional failure
(80% of 1RM set-to-fatigue: 80FAIL); a group exercising at 30% of 1RM with the same total
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external workas 80FAIL (30% 1RM work-matched to 80FAIL: 30WM); and a low-load high-
repetition group exercising at 30% to volitional fatigue (30% of 1RM set-to-fatigue: 30FAIL).
Subject characteristics are presented in Supplementary Digital Content 1 (See Table, SDC 1,
Subjects’ descriptive characteristics)..

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Resistance Training Protocol. All groups participated in the RET three times per week for 10
weeks. The RET three sets of leg press, leg extension, and leg curl. Subjects’ 1RM was
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determined using the indirect measurement method. After the measurement of 1RM, 80FAIL
performed muscle contractions to fatigue at 80% of 1RM per set, while 30WM exercised at 30%
of 1RM and continued until they reached the same total volume as that of 80FAIL group in every
set. For all groups, 1RM was re-measured in each phase of the RET (phase 1: 1st–4th week,
phase 2: 5th–8th week, phase 3: 9th–10th week) to revise the workload, and all the participants
were subjected to a warm up exercise of 15-minute fast walking (6 km/h) on a treadmill prior to
the main RET program.
Isokinetic Muscle Function Test. Muscle function of the lower extremity (knee joint) was
examined using an isokinetic dynanometer (Humac Norm, CSMI, USA). Before undertaking the
RET, subjects performed a warm-up exercise of fast walking (6 km/h) for 10 minutes. Subjects
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were asked to move their knee joints from 0° to 90° and perform five repetitions at an angular
speed of 60°/sec and 15 repetitions at 240°/sec. Their peak torque per kg body weight were then
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evaluated.
Muscle Biopsies. A muscle biopsy was taken prior to training and 72 hours after the final
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training session from the vastus lateralis. Participants were instructed not to exercise for at least
72 hours or consume caffeine or alcohol for 48 hours prior to each muscle biopsy. In addition,
they did not have food for 9 hours prior to each biopsy and rested for 30 minutes on the biopsy
bed prior to each biopsy. For each muscle biopsy, an area of skin and fascia covering the vastus
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was anesthetized locally with 1% lidocaine and ~80-100 mg of skeletal muscle was taken (13).
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Part of the sample skeletal muscle was placed in optimal cutting temperature compound (Tissue-
Tek, The Netherlands) for histological analysis of muscle fiber phenotype, satellite cells, and
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myonuclei of skeletal muscle, while the rest of the samples were snap-frozen in liquid nitrogen
and stored at –80°C before being analysed for protein expression.
SDS-PAGE. For analysis of mitochondria-related protein expression, 40 mg of muscle tissue
was immersed in ice-cold lysis buffer [25 mM tris-Cl (ph 7.5), 250 mM NaCl, 5mM EDTA, 1%
NP-40, 1 mM phenymethylsulfony1 fluoride (PMSF), and 5 mM dithiothreitol (DTT)], left at
4°C overnight, and then centrifuged at 14,000 RPM for 30 minutes at 4°C. After quantifying the
resulting total cytosolic protein fraction via BCA protein assay (Thermo Scientific, Rockford,
IL), 2X SDS loading buffer (60 mM tris, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM 2-
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mercaptoethanol, and 0.1% bromophenol blue) was added. In addition, separating gel (30%
acrylamide, 1.5 M tris, pH 8.8, 10% ammonium persulfate, TEMED) and 5% stacking gel (30%
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acrylamide, 1 M tris, pH 6.8, 10% ammonium persulfate, TEMED) that fit the molecular weight
of the protein to be analysed were made and added to the samples, and each was subjected to
electrophoresis with a standard marker (1610374, Bio-rad, USA) at 80 V.

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Western Blotting. After completing the preparations for transfer to a nitrocellulose blotting
membrane (Life Science, Germany) with 3M paper (Whatman) in a transfer buffer (0.2M
glycine, 50 mM tris base, 0.05% SDS, 20% methanol), the transfer was carried out by setting the
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voltage and transfer time suitable for the molecular amount. After transfer of proteins to the
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membrane, the membranes were blocked with 5% bovine serum albumin (BSA) solution (10
mM tris base, HCl pH 7.6, 0.5 M NaCl, 0.05% Tween 20) for 1.5 hours at 4°C. The primary
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antibodies, for the number of mitochondria (COX IV, sc-376732, Santa Cruz, USA; cytochrome
c, sc-8385, Santa Cruz, USA), mitochondrial biogenesis (PGC-1α, sc-13067, Santa Cruz, USA;
TFAM, sc-37667, Santa Cruz, USA; NRF1, sc-515360, Santa Cruz, USA), mitochondrial fusion
(Mfn1, sc-50330, Santa Cruz, USA; Mfn2, sc-50331, Santa Cruz, USA; Opa1, sc-367890, Santa
Cruz, USA), mitochondrial fission (Drp1, sc-32898, Santa Cruz, USA; Fis1, sc-98900, Santa
Cruz, USA), mitophagy (PINK1, sc-23707, Santa Cruz, USA; Parkin, ab15954, Abcam, USA),
anti-oxidative enzyme (SOD1, sc-11407, Santa Cruz, USA; SOD2, sc-30080, Santa Cruz, USA),
and β-actin (sc-47778, Santa Cruz, USA) were incubated in the blocking solution at a
concentration of 1:1000 for 12 hours at 4°C. After incubation with the primary antibodies, the
membranes were washed three times for 10 minutes with TBS-T solution. Membranes were
incubated with the secondary antibodies in 5% skim milk at a concentration of 1:5000, for 1.5
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hours. Lastly, the secondary antibodies were removed by washing with TBS-T solution three
times for 10 minutes, and the membrane band images were obtained by incubating in WBLR
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solution (Luminata Crescendo Western HRP Substrate, Millipore, USA) and scanned using a
ChemiDoc XRS system (Bio-Rad, USA).

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Immunohistochemistry. For analysis of phenotype, satellite cells, and myonuclei of muscle fibers,
tissue samples embedded in OCT compound were cut into 7 µm thick sections in a microtome
(CM1850, Leica, Germany) maintained at a temperature of -20°C. The sections were transferred
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to a glass slide and were fixed in a 4% PFA solution for 5 minutes. To remove fat, the tissues
were soaked in methanol, which had been kept at -20°C for 10 minutes and then were put in a
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blocking solution (donkey serum and 0.1% triton/PBS at a 9:1 ratio), for another 20 minutes. To
check changes in satellite cells and muscle cross-sectional area membranes were incubated with a
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primary antibody of anti-Pax7 (MAB 1675, R&D system, USA) and anti-laminin (L9393, Sigma,
USA) in 1% BSA/TPBS at the ratio of 1:100 for over 12 hours at 4°C. The primary antibodies
were removed by washing with 0.1% triton/PBS three times for 10 minutes, and the secondary
antibodies (Alexa Fluor 594 donkey anti-rabbit IgG antibody and Alexa Fluor 488 donkey anti-
mouse IgG antibody) diluted with 1% BSA/TPBS at a ratio of 1:500 were applied incubated for 4
hours in a darkroom at room temperature (20-25°C). Additionally, for analysis of the muscle fiber
phenotype a primary antibody to MyHCI (A4.951, Developmental Studies Hybridoma Bank,
USA) was applied incubated for 2 hours. Then, the second antibody (Alexa Fluor 546 donkey
anti-mouse IgG antibody) was applied and incubated for a further hour. Finally, sections were
washed with 0.1% triton/PBS three times for 10 minutes, sprayed with a mounting medium (H-
1200, VECTOR, USA) containing 4,6-diamidino-2-phenylindole (DAPI) to dye the nuclei, and
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fixed with a cover glass. The dyed tissues were converted to images by using a confocal
microscope (Leica TCS SP8 confocal microscope, Leica microsystem, Germany).
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To examine the proliferation of progenitor satellite cells, the same muscle was sectioned (7μm),
and a primary antibody of Myf5 (ab125078, Abcam, USA) was applied and incubated with 1%
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BSA/TPBS at a ratio of 1:100. After the incubation process, a secondary antibody (horseradish
peroxidase-conjugated goat anti-rabbit ZYMED, 65-6120, USA) was incubated for 2 hours,
followed by stimulation for 30 minutes by using Vector Elite ABC horseradish peroxidase kit (PK
6100K, Vector Laboratories, UK), and sprayed with diaminobenzidine (DAB) substrate (SK-4105,
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Vector Laboratories, UK) to visualize Myf5. Finally, the section was sprayed with a mounting
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medium, placed under a cover glass, and captured as an image using a confocal microscope (Leica
DM2500, Leica microsystem, Germany).

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To ensure reliability of sample analysis, more than 200 muscle fibers per sample were examined to
analyse the cross-sectional area (CSA) by type, the number of satellite cells (Pax7, Myf5), the
number of myonuclei, the myonuclear domain, and the number of central nuclei (14). All image
analyses were done blinded for both group and time.
Statistical Analyses. The data were analysed using SPSS/PASW Statistics 18.0. A two-way
repeated ANOVA was performed to examine main effects and interactions. When a significant
interaction was detected a post hoc test (Tukey HSD) was performed to explore the pairwise
differences. Exercise volume data were analysed with a one-way ANOVA with group as the
independent variable. Statistical significance level of all the tests was set at α=0.05 and values are
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presented as means ± standard deviation (SD).
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RESULTS
Exercise Volume. Volume data for each exercise in each group are presented in Table 1. One-
way ANOVAs revealed significant differences between groups for each exercise. Post-hoc tests
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revealed that there were no differences between 80FAIL and 30WM; however, 30FAIL had
more volume in each exercise than both 80FAIL and 30WM (p<0.05).
Isokinetic Muscle Function Tests. Muscle function tests are presented in Table 2. Briefly, 10
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weeks of RET resulted in an increase in peak torque measured at 60°/s and 240°/s. There were
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group by time interactions with the peak torque measured at 240° /s, such that only the 30FAIL
group increased peak torque compared to pre-values.

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Muscle Fiber Cross Sectional Area. There was a group by time interaction (p=0.023) for type I
muscle fiber area with increases in type I CSA in 30FAIL (p=0.001) and 80FAIL (p=0.04) but
not 30WM (p=0.126; Figure 1A). Conversely, all groups showed an increase in type II CSA
following RET (p<0.001) with no differences between groups (p>0.05).
Satellite cells and myonuclei. All satellite cell and myonuclear analyses conducted on each fiber
type are reported in Figure 2 and Supplementary Digital Content 2 (See Figure, SDC 2,
Representative immunohistochemical image of fiber type-specific analysis). There was an
increase in the number of satellite cells surrounding type I fibers in all groups (p<0.05); however,
there was no group by time interaction (p>0.05; Figure 2A). In contrast there was a significant
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group by time interaction with the number of satellite cells surrounding type II (p<0.05) with
significant changes in each group (30FAIL: 187%, 80FAIL: 137%, 30WM: 137%; p<0.05);
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however, 30FAIL increased to a greater extent compared to the other conditions (Figure 2E). In
addition, there was a significant group by time interaction in the number of satellite cells
expressing Myf5 following RET (p=0.003) with significant increases in each group (See Figure,
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SDC 3, Representative staining image of Myf5 analysis and result). There was an increase in the
number of myonuclei surrounding type II fibers (p<0.05; Figure 2B) but not type I fibers (p>0.05;
Figure 2F) following the RET intervention. Moreover, there were no group by time interactions
for either type I or type II fibers (p>0.05). Interestingly, there was a trend towards an increase in
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myonuclear domain following RET in type I fibers (Figure 2C) and a decrease in myonuclear
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domain in type II fibers (Figure 2G). Lastly, there was a group by time interaction in centrally
located nuclei for both type I (p=0.004; Figure 2D) and type II fibers (p=0.001; Figure 2H) such
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that there was an increase in the centrally-located nuclei in type I fibers in 80FAIL and 30WM
(p<0.05) but not 30FAIL (p=0.483) and an increase in the centrally-located nuclei in type II
fibers in 80FAIL and 30FAIL (p<0.05) but not 30WM (p=0.253).
Mitochondrial Capacity and Mitophagy. Proteins assessed to determine mitochondrial
capacity and turnover are displayed in Figure 3 and representative western blot band in
Supplementary Digital Content 4 (See Figure, SDC 4, Representative Western blot band). In
regards to mitochondrial capacity, COX IV expression increased following RET (p=0.006) with
no group by time interaction (p=0.102; Figure 3A). In addition, there was no change or group by
time interaction in cytochrome c expression (p>0.05; Figure 3B).
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In regards to mitochondrial biogenesis, there was no change or group by time interaction in
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PGC-1 alpha, NRF1 or TFAM expression (See Figure, SDC 5, Expression of mitochondria
biogenesis-related proteins). Lastly, in regards to mitophagy, there was a significant group by
time interaction in Parkin expression (p=0.011), and post hoc test revealed that Parkin expression
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increased in 30FAIL (p=0.004) but not 80FAIL or 30WM (Figure 3D). However there was a
significant change in PINK1 expression following RET (p=0.042) with no group by time
interaction (Figure 3C).

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Mitochondrial Function. Proteins assessed to determine mitochondrial function are displayed in

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Figure 4. Briefly, there was no change in Opa1 or Fis1 expression following RET (p>0.05);
however, there were significant group by time interactions (p<0.05). We observed a significant
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increase in 30FAIL (p<0.01) but not 80FAIL or 30WM (Figure 4A and D). In SOD1, SOD2 and
Mfn2, there were no group by time interactions but SOD1 was increased following RET (p<0.05;
Figure 4E and F). Moreover, there was a group by time interaction in Drp 1 expression
(p=0.016), and post hoc test revealed that Drp1 expression increased in 30WM and 30FAIL
(p<0.05) but not 80FAIL (p>0.05; Figure 4C). In addition, there was no change in Mfn2/Drp1
ratio following RET, but there was a significant group by time interaction (p=0.047) and post
hoc test revealed that Mfn2/Drp1 ratio increased in 80FAIL (p=0.037) but not 30FAIL or 30WM
(See Figure, SDC 6, Ratio of Mfn2 and Drp1 expressions).
DISCUSSION
It is clear that load is not a determinant of the hypertrophic response to RET when loads are
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lifted to the point of volitional fatigue (15). There are, however, few studies examining the
cellular-level changes observed with RET programs of varying loads and some have speculated
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that hypertrophy may be fiber-type specific with lower-load RET (16). Congruent with the
conclusion of equivalent hypertrophy (15), we show here that when RET was performed to
volitional fatigue there were significant and similar changes in muscle fiber hypertrophy (Figure
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1). We also observed equivalent increases in strength, contrary to what some have reported (15)
with higher versus lower loads; nonetheless, our data are in-line with some other work (7, 8)
showing no difference in strength gains, regardless of the load lifted. We also show, again
congruent with previous work (9), that RET increases the number satellite cells and myonuclei;
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however, that varying load and volume did not affect the magnitude of change in these variables
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(Figure 2). Lastly, we found that a number of mitochondrial proteins related to turnover and
metabolism are increased with RET, but add that mitochondrial adaptations were greater with
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performance of higher-volume, lower-load RET (Figure 3 and 4). Our work adds to a growing
body of knowledge (15) that confirms that changes in RET-induced muscle hypertrophy are not
load-dependent when loads are lifted to the point of volitional fatigue. We did not find a muscle
fiber-level specific hypertrophy as some have speculated occurs when lifting lighter loads (16)
and in fact saw robust increases in both type I and type II fiber hypertrophy in 80 FAIL and
30FAIL a finding consistent with fatigue being achieved and full muscle fiber activation.
Both RET volume (17) and load (18, 19) can influence RET-induced muscle hypertrophy and
strength when they are work- or volume-matched (i.e., not performed to volitional fatigue).
However, similar to recent research (7), we observed similar RET-induced increases in both type
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I and type II muscle fiber CSA when RET was performed to volitional fatigue (i.e., in 30FAIL
vs. 80FAIL; Figure 1). Additionally, similar to previous findings (19, 20), we found that neither
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load nor volume influenced RET-induced changes in muscle strength when muscle strength was
assessed using an unpractised test (e.g., a dynamometer test as opposed to a 1RM test of an
exercise that the participants were performing during the RET intervention). Accordingly, we
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found that the improvement in isokinetic knee extension peak torque (240°/sec) was only
significant in 30FAIL, not 80FAIL or 30WM (Table 2). Thus, there is now substantial evidence
that changes in muscle mass and strength are not driven by load (21), and, along with the results
from recent meta-analyses (15), we advocate that RET guidelines (22) require revision to reflect
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this science.
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To our knowledge, no study has compared the RET-induced changes in satellite cell activity
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between RET with varying load and volume. First, similar to what many have shown (14, 23),
we found that RET increased the number of active and proliferating satellite cells (Figure 2A and
D). Consequently, following 10 weeks of RET, there was an increased number of myonuclei
(particularly surrounding type II fibers), which resulted in a decreased myonuclear domain in
type II fibers but an increased myonuclear domain in type I fibers (Figure 2C and G). Moreover,
the greatest increase in satellite cell activation and the largest decrease in type II myonuclear
domain were in the 30FAIL group. Altogether, our results suggest that satellite cell activity may
be related to RET volume, but not load when RET is performed to volitional fatigue.
Previous research has demonstrated that exercise modality (e.g., endurance vs. resistance)
determines the post-exercise mitochondrial and myofibrillar rates of protein turnover (3, 4), and
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that RET can improve mitochondrial protein content (11, 24). Thus, we hypothesized that
performing more endurance-like RET (higher-volume/lower-load) would result in more
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endurance exercise-like increases in mitochondrial proteins. In previous studies that compared
the effects of performing RET to volitional fatigue with different intensities (i.e., higher- vs.
lower-load RET), load did not affect the RET-induced improved peripheral central arterial
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stiffness (25) or capillary density (26). In contrast, data from the present study found that the
RET-induced increases in mitophagy (i.e., PINK1), mitochondrial fusion (i.e., Opa1), and
mitochondrial fission (i.e., Drp1 and Fis1) proteins occurred only in 30FAIL (Figure 3 and 4).
Accordingly, our data suggest that performing higher-volume, lower-load RET is a more potent
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stimulus for mitochondrial metabolism and turnover than lower-volume, higher-load RET. We
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hypothesize that higher-volume RET results in greater substrate utilization and metabolic
demand that may be driving changes in mitochondrial proteins.

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Limitations of the present study include the fact that we did not control for protein intake,
which can influence hypertrophy but the overall magnitude of which is not large (27). Also, we
acknowledge that the 30FAIL group performed a greater amount of work than the 30WM and
80FAIL, which may have accounted for the changes seen in the 30FAIL group compared to the
other groups. And we did not directly measure mitochondrial oxidative metabolism (28), which
would be advisable in future trials. We also acknowledge that our sample size was small;
however, we note that many of the effects we observed (i.e., hypertrophy) were robust and in-
line with previous work in this area suggesting we were not underpowered to detect similar
adaptations, for example in hypertrophic outcomes, as have been previously observed (15).
However, having no prior knowledge of many of the outcomes we measured as secondary
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objectives we may not have been sufficiently powered to see changes in those variables. Finally,
we acknowledge that without evidence the results from the present study may not be the same in
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trained individuals; however, we speculate that given the observation that one is always able to
perform more work with lower versus higher loads would mean that the differences we observed
here would be broadly consistent even with progressive training.

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We investigated the changes in muscle mass, muscle strength, satellite cell activity,
myonuclear addition, and a number of proteins related to mitochondrial metabolism following 10
weeks of RET with varying load and volume. Lower-load RET performed to volitional fatigue
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resulted in similar changes in muscle mass and muscle strength when compared to higher-load
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RET performed to volitional fatigue. In addition, lower-load RET performed to volitional fatigue
for 10 weeks was the only condition that resulted in a decrease in type II myonuclear domain and
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increase in mitochondrial autophagy, fission-, and fusion-related proteins. In accordance with
previous research, performing RET to volitional fatigue, independent of load, results in
significant changes in muscle mass; however, lighter-load, higher-volume RET appears to
provide a greater stimulus for an increase in mitochondrial function-related proteins.
ACKNOWLEDGMENTS
No research grant was received for this study.
CONFLICT OF INTEREST
The authors declare that the results of the study are presented clearly, honestly and without
fabrication, falsification or inappropriate data manipulation. The results of the present
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investigation do not constitute endorsement by ACSM. No conflict of interest or financial are
declared by the authors.
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Figure Legends
Figure 1. Muscle fiber cross-sectional area for (A) type I fiber and (B) type II fiber. 80FAIL,
80%RM set-to-fatigue; 30WM, 30%RM work matched to 80FAIL; 30FAIL, 30%RM set-to-
fatigue. * p<0.05, significant difference from pre within each group, # p<0.05, main effect for
time.
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Figure 2. Satellite cells and myonuclei analysis conducted on each fiber type. (A) pax7/type I
fiber, (B) myonuclei/type I fiber, (C) myonuclear domain of type I fiber, (D) central nuclei of
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type I fiber, (E) pax7/type II fiber, (F) myonuclei/ type II fiber, (G) myonuclear domain of type
II fiber, (H) central nuclei of type II fiber. 80FAIL, 80%RM set-to-fatigue; 30WM, 30%RM
work matched to 80FAIL; 30FAIL, 30%RM set-to-fatigue. * p<0.05, significant difference from
pre within each group, # p<0.05, main effect for time.
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Figure 3. Expression of mitochondria capacity and turnover-related proteins. (A) COX IV, (B)
cytochrome C, (C) PINK1, (D) Parkin. 80FAIL, 80%RM set-to-fatigue; 30WM, 30%RM work
matched to 80FAIL; 30FAIL, 30%RM set-to-fatigue; NRF1, nuclear respiratory factor 1; TFAM,
mitochondrial transcription factor A; PINK1, PTEN-induced putative kinase 1. # p<0.05, Main
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effect for time; * p<0.05, significant difference from pre within that group; † p<0.05, significant
difference from 80FAIL post; ‡ p<0.05, significant difference from 30WM post.
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Figure 4. Expression of mitochondrial function-related proteins. (A) Opa1, (B) Mfn2, (C) Drp1,
A
(D) Fis1, (E) SOD1, and (F) SOD2. 80FAIL, 80%RM set-to-fatigue; 30WM, 30%RM work
matched to 80FAIL; 30FAIL, 30%RM set-to-fatigue; Opa1, optic atrophy type 1; Mfn2,
mitofusin 2, Drp1, dynamin-related protein 1; Fis1, mitochondrial fission 1 protein, SOD1,
superoxide dismutase 1; SOD2, superoxide dismutase 2. # p<0.05, Main effect for time, *
p<0.05, significant difference from pre within that group, † p<0.05, significant difference from
80FAIL post, ‡ p<0.05, significant difference from 30WM post.
Supplementary Digital Content
Supplementary Digital Content contains 1 Table (SDC 1) and 5 figures (SDC 2-6).
Supplementary Digital Content 1. Table.
SDC 1 that illustrate the subjects’ descriptive characteristics. doc.
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Supplementary Digital Content 2. jpg
SDC 2. Representative immunohistochemical image of fiber type-specific analysis. (A) Pax7
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(green), (B) laminin (red) and myosin heavy chain (MyHC) Ⅰ(red), (C) DAPI (blue), (D)
Pax7+laminin+MyHCⅠ+DAPI. Yellow circle, Pax7; white arrow, central nuclear; black fiber,
type II fiber. Scale bar represents 100 ㎛.

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SDC 3. Representative staining image of Myf5 analysis and result. (A) Representative DAB
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staining image of Myf5 analysis and (B) the number of satellite cells expressing Myf5. 80FAIL,
80%RM set-to-fatigue; 30WM, 30%RM work matched to 80FAIL; 30FAIL, 30%RM set-to-
C
fatigue. * p<0.05, significant difference from pre within each group, red circle: Myf5. Scale bar
represents 50 ㎛.
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SDC 4. Representative Western blot band. 80FAIL, 80%RM set-to-failure; 30WM, 30%RM
work matched to 80FAIL; 30FAIL, 30%RM set-to-failure; PGC-1α, peroxisome proliferator-
activated receptor gamma coactivator 1-alpha; NRF1, nuclear respiratory factor 1; TFAM,
mitochondrial transcription factor A, Opa1, optic atrophy type 1; Mfn1, mitofusin 1; Mfn2,
mitofusin 2, Drp1, dynamin-related protein 1; Fis1, mitochondrial fission 1 protein, PINK1,
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PTEN-induced putative kinase 1, SOD1, superoxide dismutase 1; SOD2, superoxide dismutase 2.
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SDC 5. Expression of mitochondria biogenesis-related proteins. (A) PGC-1α, (B) NRF1, (C)
TFAM. 80FAIL, 80%RM set-to-failure; 30WM, 30%RM work matched to 80FAIL; 30FAIL,
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30%RM set-to-failure; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator
1-alpha; NRF1, nuclear respiratory factor 1; TFAM, mitochondrial transcription factor A.

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SDC 6. Ratio of Mfn2 and Drp1 expressions. 80FAIL, 80%RM set-to-failure; 30WM, 30%RM
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work matched to 80FAIL; 30FAIL, 30%RM set-to-failure; Mfn2, mitofusin 2; Drp1, dynamic-
related protein 1. * p<0.05, significant difference from pre within each group.
A
Figure 1
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Figure 2
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Figure 3
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Figure 4
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Table 1. Exercise volume.
80FAIL 30WM 30FAIL
Leg
1117±463 1139±117 1571±476*
extension
Leg press 2774±931 2833±208 3315±800*
Leg curl 517±159 547±56 765±288*
Values are shown as means ± SD, Unit; kg, Volume = load lifted per repetition multiplied by
the number of repetitions per set. Data shown are the average volume per set in each phase.
80FAIL, 80%RM set-to-fatigue; 30WM, 30%RM work matched to 80FAIL; 30FAIL,
30%RM set-to-fatigue, * p<0.05, significantly difference from 80FAIL and 30WM.
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Table 2. Muscle function test.
80FAIL 30WM 30FAIL P value
pre post pre post pre post Group Time GxT
Peak
torque, 60° 262±63 275± 67 277±50 292±37 274±43 316±33 0.73 > 0.001 0.24
(Nm)
Peak
†
torque, 156±30 170±26* 163±29 172±18* 160±28 196±19* 0.51 > 0.001 0.03
240° (Nm)
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1 RM, Leg
extension 128±21 157±29 117±28 152±25 120±22 153±22 0.77 > 0.001 0.88
(kg)
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Values are shown as means ± SD, 80FAIL, 80%RM set-to-fatigue; 30WM, 30%RM work
matched to 80FAIL; 30FAIL, 30%RM set-to-fatigue, * p<0.05 different from pre-intervention.
† p<0.05 different from other groups post-intervention.
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Supplementary digital content
Supplementary Digital Content 1 Table.
SDC 1. Subjects’ descriptive characteristics.

80FAIL (n=7) 30WM (n=7) 30FAIL (n=7) P
Age (y) 24±2 23.1±2 23±1 0.22
Height (cm) 175±6 175±6 173±8 0.84
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Weight (kg) 79.4±12.1 77.1±13.1 73.5±8.4 0.26
BMI (kg/m2) 25.9±3.9 25.0±3.1 24.4±1.3 0.64
Values are shown as means ± SD, 80FAIL, 80%RM set-to-fatigue; 30WM, 30%RM work
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matched to 80FAIL; 30FAIL, 30%RM set-to-fatigue; BMI, body mass index; y, year.
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Resistance Exercise-induced Changes in Muscle Metabolism are Load-

Article in Medicine & Science in Sports & Exercise · July 2019

Changhyun Lim Robert Morton

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The user has requested enhancement of the downloaded file.

Accepted for Publication: 17 June 2019

Copyright © 2019 American College of Sports Medicine

Resistance Exercise-induced Changes in Muscle Metabolism

Seok Jeong5, Chang Keun Kim2,6

Author for correspondence

Prof. Chang Keun Kim, Ph.D.

Human physiology, Korea National Sport University,

88-15 Oryun-dong, Songpa-gu, Seoul South Korea

Introduction: Lower-load (LL), higher-repetition resistance exercise training (RET) can

content of a number of mitochondrial proteins. Similar to previous research, we found that

synthesis might support greater expansion of mitochondrial proteins as well as additional

extension, so chronic phenotypic adaptations to that type of protocol remain unknown.

myonuclear addition, mitochondrial function after performing lower-load, high-repetition vs.

equivalent hypertrophy to that seen with performance of higher-load contractions. In contrast,

undertaken any regular RET in the past 2 years.

Subjects’ descriptive characteristics)..

the main RET program.

and stored at –80°C before being analysed for protein expression.

NP-40, 1 mM phenymethylsulfony1 fluoride (PMSF), and 5 mM dithiothreitol (DTT)], left at

electrophoresis with a standard marker (1610374, Bio-rad, USA) at 80 V.

ChemiDoc XRS system (Bio-Rad, USA).

phenotype a primary antibody to MyHCI (A4.951, Developmental Studies Hybridoma Bank,

microscope (Leica TCS SP8 confocal microscope, Leica microsystem, Germany).

DM2500, Leica microsystem, Germany).

analyses were done blinded for both group and time.

group increased peak torque compared to pre-values.

following RET (p<0.001) with no differences between groups (p>0.05).

Representative immunohistochemical image of fiber type-specific analysis). There was an

fibers in 80FAIL and 30FAIL (p<0.05) but not 30WM (p=0.253).

time interaction in cytochrome c expression (p>0.05; Figure 3B).

biogenesis-related proteins). Lastly, in regards to mitophagy, there was a significant group by

interaction (Figure 3C).

Mitochondrial Function. Proteins assessed to determine mitochondrial function are displayed in

(See Figure, SDC 6, Ratio of Mfn2 and Drp1 expressions).

(particularly surrounding type II fibers), which resulted in a decreased myonuclear domain in

performing more endurance-like RET (higher-volume/lower-load) would result in more

demand that may be driving changes in mitochondrial proteins.

However, having no prior knowledge of many of the outcomes we measured as secondary

here would be broadly consistent even with progressive training.

myonuclear addition, and a number of proteins related to mitochondrial metabolism following 10

increase in mitochondrial autophagy, fission-, and fusion-related proteins. In accordance with

previous research, performing RET to volitional fatigue, independent of load, results in

significant changes in muscle mass; however, lighter-load, higher-volume RET appears to

provide a greater stimulus for an increase in mitochondrial function-related proteins.

No research grant was received for this study.

fabrication, falsification or inappropriate data manipulation. The results of the present

declared by the authors.

Review and Meta-Analysis. Sports medicine (Auckland, NZ). 2019;49(2):233-54. Epub

2018/10/21. doi: 10.1007/s40279-018-1008-z. PubMed PMID: 30341595.

doi: 10.1152/japplphysiol.01081.2012. PubMed PMID: 23221957.

phosphorylation and protein synthesis in human muscle. The Journal of physiology.

2008;586(15):3701-17. Epub 2008/06/17. doi: 10.1113/jphysiol.2008.153916. PubMed PMID:

18556367; PubMed Central PMCID: PMCPMC2538832.

4. Vissing K, McGee S, Farup J, Kjolhede T, Vendelbo M, Jessen N. Differentiated mTOR but

not AMPK signaling after strength vs endurance exercise in training-accustomed individuals.

10.1111/j.1600-0838.2011.01395.x. PubMed PMID: 23802289.

adaptation. Cell Metab. 2013;17(2):162-84.