Accepted Manuscript
The Type of Manuscript: Regular Article
Modulation of mitochondrial dynamics in skeletal muscle during endurance
training: early activation of fission and late induction of fusion protein expression

Hisashi Takakura1, Hitomi Banba2, Tatsuya Yamada3, Rikuhide Koma2,4, Tsubasa
Shibaguchi5, Yudai Nonaka5, and Kazumi Masuda2*

1

Faculty of Health and Sports Science, Doshisha University, Kyotanabe, Kyoto, 6100394, Japan.

2

Faculty of Human Sciences, Kanazawa University, Kanazawa, Ishikawa 920-1192,
Japan.

3

Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588-0664,
USA.

4

Research Fellow of Japan Society for the Promotion of Science, Tokyo, Japan.

5Institute of Liberal Arts and Science, Kanazawa University, Kanazawa, Ishikawa, 9201192, Japan.

Running Title: Mitochondrial dynamics in skeletal muscle
Number of Tables: 0
Number of Figures: 4*Corresponding author: Kazumi Masuda, Ph.D., Professor

Faculty of Human Sciences, Kanazawa University, Kakuma-machi, Kanazawa-city,
Ishikawa, 920-1192, Japan. E-mail: masudak@staff.kanazawa-u.ac.jp, Phone: [+81] 76264-5568, Fax: [+81] 76-264-5574

Abstract
The mitochondria are highly plastic organelles. Exercise training alters
mitochondrial dynamics (mitochondrial fission and fusion) in skeletal muscles andimproves overall mitochondrial function by altering mitochondrial morphology andforming new networks. However, changes in mitochondrial dynamics over time during
exercise have not previously been observed. In the present study, we examined thechanges in mitochondrial fission and fusion protein expression in rats over four weeks ofendurance swimming training. The expression of the GTPase protein dynamin-related
protein 1 (Drp1), a mitochondrial fission protein, increased during the early phase of the
training period. In contrast, the expression of optic atrophy type 1 (OPA1), a
mitochondrial fusion protein, was increased in the late phase of the training period. These
data suggest that mitochondrial fission was increased in the early phase, andmitochondrial fusion was initiated partially in the late phase, of the training period. In
conclusion, mitochondrial dynamics may be modulated depending on the phase of
muscular adaptation to exercise training. This modulation contributes to enhanced
mitochondrial function in skeletal muscle.
Keywords: endurance training; mitochondrial fusion and fission; protein expression;
skeletal muscle

論文タイトル: ミトコンドリアの分裂と融合は持久力的トレーニング期間中の異なるタイミングで促進される
著者: 高倉久志 1, 番場瞳 2, 山田達也 3, 小間陸嗣 2, 4, 芝口翼 5, 野中雄大 5,
増田和実 2

所属
1: 同志社大学スポーツ健康科学部（〒610-0394 京都府京田辺市多々羅都谷 1-3）
2: 金沢大学人間社会研究域人間科学系（〒920-1192 石川県金沢市角間町）
3: ネブラスカ州立大学リンカーン校 生化学部門（アメリカ合衆国ネブラスカ州リンカーン市）
4: 日本学術振興会特別研究員
5: 金沢大学国際基幹教育院 GS 教育系（〒920-1192 石川県金沢市角間町）

ランニングタイトル
筋組織内のミトコンドリアダイナミクスについて

抄録
ミトコンドリアは非常に可塑性の高い細胞小器官である。運動トレーニング
は骨格筋のミトコンドリアダイナミクス（ミトコンドリアの分裂と融合）に影響を及ぼす。その結果として、ミトコンドリア形態は変化し、新しいネットワークが形成されることにより、ミトコンドリア機能は改善する。しかしながら、運動トレーニング期間中のミトコンドリア動態の経時的変化はこれまで検討されていなかった。本研究では、ラットに 4 週間の持久的水泳トレーニングを行わせ、ミトコンドリアの分裂および融合タンパク質の発現量の変化を調べた。ミトコンドリア分裂タンパク質である GTPase protein dynamin-related
protein 1（Drp1）の発現は、トレーニング期間の初期に増加した。一方、ミトコンドリア融合タンパク質である optic atrophy type 1（OPA1）の発現は、トレーニング後期に増加した。これらの結果から、ミトコンドリアの分裂はトレー
ニング期間の初期に、ミトコンドリアの融合は後期に部分的に生じていることが示唆された。結論として、ミトコンドリアダイナミクスは運動トレーニング
に対する適応の段階に応じて調節される可能性が考えられた。また、この変化は骨格筋におけるミトコンドリア機能の増強に寄与していると考えられる。

1

Introduction

2

Mitochondria are intracellular organelles that produce adenosine triphosphate (ATP)3through oxidative phosphorylation and are essential for endurance exercise performance in4skeletal muscles. Mitochondria exhibit high plasticity in response to physiological and5pathological stimuli

6(fusion/fission), and degradation (mitophagy) of mitochondria, ultimately affecting their

7

metabolic functions and oxidative capacity 1). While mitochondrial biogenesis in skeletal

8

muscle has been well studied in response to endurance training 3-4), mitochondrial fusion and9fission responses have not been fully elucidated.

1-2). These stimuli influence the content (biogenesis), dynamics

10

The mitochondrial dynamics of fusion and fission have emerged as important

11

processes for the maintenance of functional mitochondria 5-6). Mitochondrial fission utilizes

12

dynamin-like protein 1 (Drp1) and membrane-bound adaptor proteins, including fission 1 (Fis131) to cleave the outer mitochondrial protein membranes 7-8). In contrast, mitochondrial fusion

14

utilizes the GTPase transmembrane proteins, mitofusion 1 (Mfn1) and mitofusion 2 (Mfn2),

15

located on the outer mitochondrial membrane, to fuse the outer membrane of the mitochondria

16

9)

17

and is responsible for the fusion of the inner mitochondrial membrane 7).

. The optic protein atrophy 1 (OPA1) protein is located in the inner mitochondrial membrane

18

Previous studies on mitochondrial dynamics in response to exercise have typically19

focused on changes in the expression of mitochondrial dynamics-related proteins and mRNAs

20

in three contexts: acutely exercised muscle, fully adapted muscle to endurance training, and21

knockout muscle of mitochondrial dynamics-related proteins 10-12). Indeed, Ding et al. 11)

22

observed that continuous low-intensity exercise rapidly increased (within 30 min) Fis1 mRNA

23

levels, followed by increases in Mfn1 (3 h post) and Mfn2 (12 h post) mRNA levels.

24

Additionally, Arribat et al.10) reported that 16wks of continuous moderate-intensity training in25elderly subjects did not increase Mfn1 or Mfn2 content but increased OPA1 content and26

mitophagy effectors. Moreover, in mice with a muscle-specific heterozygous deficiency of

27

dynamin-related protein 1 (Drp1), a mitochondrial fission protein, a reduction in Drp1 signaling28

was associated with decreased muscle endurance and running performance, suggesting that29

Drp1 signaling plays a role in adaptations to endurance exercise training 12). However, changes

30

in mitochondrial dynamics over time during exercise training were not evident. Therefore, we

31

aimed to examine the changes in mitochondrial dynamics-related proteins over time during four32

weeks of endurance swimming training.

33

34

Materials and Methods

35

Ethical approval

36

All the experimental procedures were conducted in accordance with the Guide for the37

Care and Use of Laboratory Animals of the Physiological Society of Japan. This study was

38

approved by the Ethics Committee on Animal Experimentation at Kanazawa University

39(Protocol # AP-132797).

40
41

Animal care and exercise endurance training program

42

Male Wistar rats were obtained from the Japan SLC Corporation. All rats were43

acclimated to their new environment for 7 days. Experimental animals were housed in an air-44

conditioned room under laboratory environmental conditions (12:12 light/dark cycle, room45

temperature: 23 ± 2 ˚C, humidity: 55 ± 5%). A standard diet (MF oriental yeast) and water were46

provided ad libitum.

47

Eight-week-old Wistar rats (body weight: 194–227 g) were randomly divided into

48

control (Con) and training (Tr) groups. The Tr groups were further divided into four groups

49

according to the training period from 1 to 4 wks (9 wks , 10 wks, 11 wks, and 12 wks). Age-

50

matched Con groups corresponding to the Tr groups were prepared as follows: 9 wks, 10 wks,

51

11 wks, and 12 wks. The rats in the 1 wk training group swam for 2 h in four 30-min bouts

52

separated by 5 min of rest, during which a weight equaling 2% of the rats’ body weight was

53

tied to the rats. The training protocol for the other swimming groups was as follows: on the first54

and second days, the rats swam for 1 h in two 30-min bouts separated by 5 min of rest. On the55

third and fourth days, the rats swam for 1.5 h in three 30-min bouts, separated by 5 min of rest.

56

On and after the fifth day, the rats swam for 2 h in four 30-min bouts separated by 5 min of rest.

57

Except for the first bout of swimming training until the sixth day, a weight equal to 2% of their

58

body weight was tied to the rats. The rats performed the swimming protocol six days/wk. The

59

water temperature was maintained at approximately 35 ˚C. The tank was square-shaped: 48 cm

60

deep, 80 cm long, and 60 cm wide. It had an average surface area of at least 400 cm2/rat. In

61addition, we monitored the rats to prevent climbing, diving, and bobbing during swimming

62

training. Cases in which these behaviors were observed were dealt with immediately.

63
64

Preparation of tissue and sample homogenization

65

All surgical procedures were performed under anesthesia with an intraperitoneal (i.p.)66

injection of medetomidine (0.4 mg/kg), midazolam (2 mg/kg), or butorphanol (2.5 mg/kg).

67

Twenty-four h after the training period, the gastrocnemius muscles of the hind limbs were68

removed and washed with ice-cold saline. After removing the connective tissue, fat, and nerve,69

the muscles were weighed, clamp-frozen in liquid nitrogen, and then stored at -80 ˚C until

70

subsequent analyses.

71

The deep portion of the gastrocnemius muscle was homogenized and fractionated72

according to a modified method described in previous studies 13-14). Briefly, the tissue was73

homogenized in 17 volumes of ice-cold Solution A (250 mM sucrose, 5 mM NaN3, 2 mM

74

EGTA, 20 mM HEPES-Na, pH 7.4) with the strokes of a Teflon pestle in a Potter-Elvehjem

75

glass tissue homogenizer at 1,000 rpm. The homogenate was centrifuged at 600 × g for 10 min

76

at 4 ˚C to remove nuclei and debris. Part of the supernatant was stored as a whole fraction

77

solution. The remaining supernatant was centrifuged at 10,000 g for 30 min at 4 ˚C to precipitate78

crude mitochondrial pellets. The crude mitochondrial pellet was washed twice with Solution A

79

re-suspended in Solution A. The mitochondrial pellet was re-pelleted by centrifugation at80

10,000 g for 30 min at 4 ˚C. This pellet was washed in Solution C (1 mM EDTA, 10 mM Tris,81

pH 7.4) and was resuspended in 200 µl of Solution C containing 1% NP-40 and centrifuged at82

1,100 g for 20 min at room temperature; the supernatant was used for immunoblots as a

83

mitochondrial fraction. The protein concentration in the resuspension was determined using the

84

Bradford method with a protein assay kit (Bio-Rad Laboratories). All samples were adjusted to85

a constant protein concentration of 2.0 µg/µl with Solution A or C, and then equal amounts of86

2X SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-87mercaptoethanol, 0.002% bromophenol blue) were added. The final concentration of the sample

88

was adjusted to 1.0 µg/µl.

8990Western blotting and citrate synthase activity in skeletal muscle

91

Western blot analysis was performed according to a modified version of the method

92

described by Yamada et al.14). Samples with equivalent protein contents were resolved by 15%

93

SDS polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically94transferred onto PVDF membranes (Clear Blot Membrane-P plus, ATTO, Japan) using a semi-95

dry system (WSE-4045, HorizeBLOT 4M, ATTO, Japan). Membranes were then incubated in96

blocking buffer followed by incubation with anti-Mfn1 (H-65, Santa Cruz Biotechnology,

97USA), anti-Fis1 (ALX-210-1037, Enzo Life Sciences, USA), anti-VDAC (ab15895, Abcam,

98USA), anti-OPA1 (612606, BD Biosciences, USA), anti-Drp1 (611112, BD Biosciences, USA),99

or anti-GAPDH (ab8245, Abcam, USA) antibodies at 4 ˚C overnight. After exposure to the100

secondary antibody for 1 h, the membranes were treated with ECL prime reagents (GE101

Healthcare, NJ, USA), and chemiluminescent signals were visualized using MicroChemi

102(Berthold Technologies, Baden Württemberg, Germany). Signal intensities were quantified

103

using ImageJ imaging software (NIH, USA).

104

Citrate synthase (CS) activity, a mitochondrial enzyme and marker of muscle oxidative105

potential, was measured in whole muscle homogenates using the spectrophotometric method

106

described by Srere 15).

107
108

Measurement of mitochondrial respiration

109

Mitochondria respiration was measured using an oxygen electrode via a modified

110

method reported in a previous study16). Briefly, soleus muscle was minced using stainless steel

111

scissors in the isolation medium and homogenized with Potter–Elvehjem tissue grinder in the112

isolation medium at pH 7.2 at 4°C (225 mM mannitol, 100 mM KCl, 20 mM Sucrose, 20 mM

113

Hepes-2Na, 1 mM EDTA (ethylenediaminetetraacetic acid), 0.2% BSA (bovine serum

114

albumin), 0.25 mg/mL proteinase, bacterial and 5 mM L-carnitine). The homogenized mixture

115

was centrifuged at 700 g for 10 min at 4°C to remove nuclei and debris, and the supernatant116

was further centrifuged at 10,000 g for 10 min at 4°C to collect the isolated mitochondria. The

117

mitochondrial pellet was washed once in the isolation medium and resuspended in a ratio of 0.4

118µm per mg initial muscle weight with resuspension buffer at pH 7.2 at 4°C (215 mM mannitol,

119

75 mM Sucrose, 20 mM Hepes-2Na, 1 mM EDTA, 0.2% BSA and 5 mM L-carnitine).

120

Mitochondrial respiration was monitored using a Clark-type oxygen electrode (Model-5300A,121

YSI Japan, Japan) at 37 °C in a thermostatically controlled chamber by adding 3 mL of reaction122

buffer (pH 7.2) at 37°C (225 mM mannitol, 75 mM Sucrose, 20 mM Hepes-2Na, 10 mM KCl,

123

10 mM K2HPO4, 1 mM MgCl2 6H2O, 1 mM EDTA, 0.1% BSA and 5 mM L-carnitine) to it.

124

After applying the substrate of Pyruvate (5 mM) and L-malate (2 mM), 60 µL of isolated

125

mitochondria under resuspension was added. After confirming that state 4 remained stable,

126

ADP at the final concentration of 200 µM was added to the medium to initiate state 3.

127
128

Statistical Analysis

129

All data are expressed as mean ± SD. Statistical differences were examined using a

130

two-way unpaired analysis of variance (ANOVA) (group × age). A simple main-effect test was131

applied if the interaction or main effect showed a significant difference. Pearson’s correlation132

coefficient was calculated when the relationship between two variables was evaluated. The

133

level of significance was set at p < < 0.05.

134

Results

135

Figure 1 shows the changes in CS activity and mitochondrial respiration rates for states

136

3 and 4 during endurance swimming training. CS activity differed significantly between groups137(F (1, 40) = 108.85, p < 0.05), ages (F (3, 40) = 27.05, p < 0.05), and with their interaction (F138(3, 40) = 2.88, p < 0.05). Comparing groups of the same age using simple main effects, CS

139

activity was significantly higher in the Tr group than in the Con group at all time points (p <1400.05). The simple main effects revealed significant differences between the ages (Con: 9 wks

141< 11-12 wks and 10-11 wks < 12 wks, Tr: 9 wks < 11-12 wks and 10-11 wks < 12wks, p <1420.05). The state 4 mitochondrial respiration rate differed significantly between groups (F (1,14334) = 7.67, p < 0.05), ages (F (3, 34) = 12.06, p < 0.05), and with their interaction (F (3, 34) =144

3.91, p < 0.05). Comparing groups of the same age using simple main effects, the state 4 rate145

was significantly higher in the Tr group than in the Con group at 11 and 12 wks (p < 0.05). The

146

simple main effects revealed significant differences in the state 4 rate between the ages (Tr: 9147

wks < 11-12 wks and 10 wks < 11-12 wks, p < 0.05). The state 3 respiration rate differed148

significantly between the ages (F (1, 34) = 8.96, p < 0.05) and with the interaction between age

149

and group (F (3, 34) = 3.31, p < 0.05), but not between groups (F (1, 40) = 2.64, n.s.). Comparing

150

groups of the same age using simple main effects, the state 3 rate was significantly higher in151

the training group than in the control group at 11 and 12 wks (p < 0.05), and differed152

significantly with age (Con: 9 wks < 11-12 wks, Tr: Tr: 9 wks < 11-12 wks and 10 wks < 11-153

12 wks, p < 0.05). *Please insert Figure 1 after this paragraph

154

Figure 2 shows the changes in the expression of mitochondrial fission proteins (Drp1155

and Fis1) over time. Drp1 expression in the whole fraction showed a statistical difference in the156

interaction factor (F (3, 40) = 3.23, p < 0.05), but not in the group factor (F (1, 40) = 0.19, n.s.)157

or age factor (F (3, 40) = 1.23, n.s.). The simple main effects showed that Drp1 expression was

158

significantly higher in the Tr group than in the Con group only at 9wks of age (p < 0.05), and159

significant differences were observed in age (Con: 9-10 wks < 12wks, Tr:10wks < 9wks, p <1600.05). Fis1 expression in the mitochondrial fraction did not show a significant change in the161

group factor (F (1, 40) = 0.04, n.s.), age (F (3, 40) = 0.42, n.s.), or interaction (F (3, 40) = 1.40,

162n.s.). The relationship between CS activity and Drp1 expression levels showed significant

163

positive correlations in Con group (r = 0.53, p < 0.05, n = 24), and significant negative

164

correlations in Tr group (Figure 3; r = -0.49, p < 0.05, n = 24). *Please insert Figure 2 & 3

165

after this paragraph

166

Figure 4 shows the changes in the expression of mitochondrial fusion proteins (OPA1

167

and Mfn1) in the whole fraction over time. OPA1 expression showed a statistically significant

168

difference in the group factor (F (1, 40) = 14.27, p < 0.05) and age factor (F (3, 40) = 4.82, p <1690.05), but not in the interaction factor (F (3, 40) = 0.15, n.s.). The simple main effects showed170

that OPA1 expression was significantly higher in the Tr group than in the Con group only at

171

12wks of age (p < 0.05), and there were significant differences in the age factor (Con:10wks <172

11wks, Tr:10 wks < 11-12 wks, p < 0.05). Mfn1 expression showed a statistical difference in173

the age factor (F (3, 40) = 3.97, p < 0.05), but not in the group factor (F (1, 40) = 3.70, n.s.) or

174

interaction factor (F (3, 40) = 1.18, n.s.). The simple main effects showed significant differences

175by age (Con: 10 wks < 11 wks, P < 0.05). *Please insert Figure 4 after this paragraph176

Discussion

177

Mitochondria continuously change shape in response to fission and fusion events,

178

which ultimately enables them to maintain proper function by forming a mitochondrial

179

network17). Mitochondrial dynamics are highly dependent on the nutrient availability and180

energy demand of the cell 18). Therefore, the mitochondrial morphology changes in response to181

exercise 19-21). In this study, we examined changes in the expression levels of mitochondrial

182

fusion/fission proteins in rats during four weeks of endurance swimming training. Drp1, a

183

mitochondrial fission protein, increased in the early phase of the training period, whereas OPA1,

184

a mitochondrial fusion protein, increased in the late phase. After the third week of the training

185

period, swimming training increased both mitochondria volume and mitochondrial respiratory

186

function in skeletal muscle.

187

The increase in Drp1 expression found in this study suggests that mitochondrial fission

188

is promoted during the early phase of an endurance swimming training program. Mitochondrial189

fission leads to an improved mitochondrial network as it separates dysfunctional mitochondrial

190

sections, thereby improving overall function 22). These fission events occur at an early stage in

191

response to continuous physiological stimuli to improve mitochondrial quality, and this192

adaptation precedes the enlargement of the mitochondrial network 17). The general research

193

consensus is that mitochondrial fission is regulated by Drp1 23). Drp1 is recruited to the194

mitochondrial outer membrane in response to changes in the intracellular environment, such as

195

decreased cellular energy, high cytosolic ADP levels, and acute hypoxia 24-26). Acute exercise

196

is likely to create such a cellular environment, especially during the early phases of swimming

197

training. In theory, the phosphorylation status of Drp1 should be measured to determine the198

degree of mitochondrial fission. We could not measure phosphorylated Drp1 in skeletal

199

muscles because Drpser616 levels returned to the basal phosphorylation status within a few hours

200of exercise stimuli

12). Nevertheless, based on the finding that the Ser616:Ser637

201

phosphorylation status of Drp1 correlates with Drp1 abundance on the outer mitochondrial

202

membrane 12,27), an increase in Drp1 expression in the whole fraction would indicate the203

promotion of mitochondrial fission if the degree of Drp1 activation and Drp1 abundance on the204

outer mitochondrial membrane relative to Drp1 content in the whole fraction remains constant.

205

In contrast, one of the receptor factors, Fis1, showed no change in expression throughout the206

training period, suggesting that the Fis1 protein expression level may not be as important for207

exercise-induced mitochondrial fission, as reported in recent studies 28).

208

OPA1 expression increased during the late phase of endurance swimming training in

209

this study. Mitochondrial fusion is essential for the maintenance of mitochondrial and cellular210

functions as it controls mtDNA replication, mitochondrial nucleoid distribution, and OXPHOS

211

capacity 29-30). Mitochondrial fusion is independently regulated at the outer and inner212

mitochondrial membranes 17). Outer mitochondrial membrane fusion is regulated by the213

activities of Mfn1 and Mfn2, whereas fusion of the inner mitochondrial membrane fusion is214

coordinated by the activity of OPA1 31-32). Our results showed an increase in expression of only

215

OPA1 and no change in Mfn1 expression, but we did not measure the expression level of Mfn2.

216

Moreover, in the previous study of Arribat et al. 10) Mfn1 expression was not altered in trained

217

muscle, whereas OPA1 and Mfn2 expression were elevated. These results suggest that218

mitochondrial fusion may be partially promoted during the late phase of the training period.

219

OPA1 plays a role in cristae remodeling and is considered an integral regulator of220

OXPHOS 33). Cristae membranes are highly dynamic and continuously undergo morphological221

changes mediated by OPA1, an i-AAA protease, ATP-dependent zinc metalloprotease 1

222(YME1L), metalloendopeptidase (OMA1), the mitochondrial contact site and cristae

223

organizing system (MICOS), and sorting and assembly machinery complex 50 (Sam50) 34).

224

Patten et al. 35) showed that OPA1 dynamically regulates cristae shape in healthy cells and that

225

OPA1-dependent modulation of cristae structure is necessary for cellular adaptation to energy

226

substrate availability. Cristae are invaginations of the inner mitochondrial membrane, where the227

essential machinery for mitochondrial respiration, the ETC, is located17). Therefore, oxidative228

phosphorylation activity is highly dependent on the cristae shape and ETC organization36). In

229

summary, the elevated OPA1 expression may have induced changes in the inner mitochondrial

230

membrane cristae in the late phase of the training period. Future studies will need to examine231

the effects of extended training periods on mitochondrial fusion.

232

In the training group, whole-fraction Drp1 expression was negatively correlated with233

CS activity, an indicator of mitochondrial volume, whereas in the control group, CS activity

234

and Drp1 expression were positively correlated (Figure 3). As Drp1 expression is also an

235

indicator of mitochondrial dynamics 12,27), these findings suggest that when mitochondria

236

volume is increased by exercise training, the intracellular environment is more likely to be237

conducive to mitochondrial fusion. Although the functional interaction between CS activity

238

levels and Drp1 expression remains unclear, exercise training is likely to alter the relationship239

between mitochondrial dynamics and mitochondrial biogenesis in skeletal muscle.

240

In this study, mitochondrial proteins and respiratory function were shown to increase241

and accelerate with age. Because CS activity in the soleus muscle was shown to increase from242

day 28 to 140 after birth in the previous study 37) and the breeding environment during the243

training period was similar between groups in the present study, the age-related changes in

244

respiratory function and CS activity in the control group were considered to be due to245development.

246

Our study reveals that the mitochondrial fission was promoted in the early phase of247

the training period, and the mitochondrial fusion was partially induced in the late phase of the248

training period. Such mitochondrial dynamics may properly manage mitochondrial249

functionality in response to changes in the intracellular environment caused by exercise training.

250
251

Acknowledgements
We would like to thank Editage (https://www.editage.jp) for English language editing.

252

Funding: This research was supported by Japan Society for the Promotion of Science

253(26282183, 25560336, KM; 24800074, HT).

254
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256

Conflict of Interest
There is no conflict of interest for this study.

257
258

Author Contribution

259

H.T., H.B., T.Y., and K.M. designed the study; H.B. and T.Y. conducted the260

experiments and analyzed the data; H.T. and H.B. wrote the manuscript; and K.M., R.K.,

261

T.S., and Y.N. edited the paper and helped with the experiments.

262

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385

Figure Legends

386

Figure 1. Change in CS activity, and mitochondrial respiration rates for states 3 and 4

387

over time from 9 to 12 wks old during endurance swimming training in the training and388

control groups.

389(A) A two-way ANOVA for CS activity revealed a statistical difference in the group factor (F390(1, 40) = 108.85, p < 0.05), age (F (3, 40) = 27.05, p < 0.05), and interaction (F (3, 40) = 2.88,

391

p < 0.05). The simple main effects showed that CS activity was significantly higher in the Tr

392

group than in the Con group at all time points (p < 0.05), and there were significant differences393

in age (p < 0.05). (B) A two-way ANOVA for the state 4 of respiration rate showed a394

statistically significant difference in the group factor (F (1, 34) = 7.67, p < 0.05), age (F (3, 34)395= 12.06, p < 0.05), and interaction (F (3, 34) = 3.91, p < 0.05). The simple main effects showed396that state 4 was significantly higher in the Tr group than in the Con group at 11 and 12 wks (p397< 0.05), and and there were significant differences in age (p < 0.05). (C) A two-way ANOVA

398

for the state 3 of respiration rate showed a statistically significant difference in the age factor

399(F (1, 34) = 8.96, p < 0.05) and interaction (F (3, 34) = 3.31, p < 0.05), but not in the group400

factor (F (1, 40) = 2.64, n.s.). The simple main effects showed that state 4 was significantly

401

higher in the Tr group than in the Con group at 11 and 12 wks (p < 0.05), and and there were402

significant differences in age (p < 0.05). Values are represented as mean ± SD (n = 6 for CS

403

activity in each group, n = 4-6 for mitochondrial respiration rate in each group). Abbreviations:404

Con, control; Tr, training; CS, citrate synthase. *: p < 0.05 vs. age-matched Con group, a: p <4050.05 vs. 9 wk-Con group, b: p < 0.05 vs. 9 wk-Tr group, c: p < 0.05 vs. 10 wks-Con group, d:406

p < 0.05 vs. 10 wks-Tr group, e: p < 0.05 vs. 11 wks-Con group.

407
408

Figure 2. Change in Drp1 and Fis1 over time from 9 to 12 wks old during endurance409

swimming training in the training and control groups.

410(A) Two-way ANOVA for Drp1 expression in the whole fraction revealed a statistical

411

difference in the interaction factor (F (3, 40) = 3.23, p < 0.05), but not in the group factor (F412(1, 40) = 0.19, n.s.) and the age factor (F (3, 40) = 1.23, n.s.). The simple main effects showed

413

that the Drp1 expression was significantly higher in Tr than in Con groups only in 9 wks old414(p < 0.05), and there were significant differences in the age factor (p < 0.05). (B) The Fis1

415

expression in the mitochondrial fraction did not show significant change in the group factor

416(F (1, 40) = 0.04, n.s.), the age factor (F (3, 40) = 0.42, n.s.), and the interaction (F (3, 40) =417

1.40, n.s.). Values are represented as mean ± SD (n = 6 in each group). Abbreviations: Con,

418

control group; Tr, training group; Drp1, dynamin-like protein 1; Fis1, fission 1. *: p < 0.05 vs.

419

age-matched Con group, a: p < 0.05 vs. 9 wks-Con group, b: p < 0.05 vs. 9 wks-Tr group, c: p420

< 0.05 vs. 10 wks-Con group.

421
422

Figure 3. Relationship between CS activity and Drp1 expression level in control and423

training groups.

424

The relationship between CS activity and Drp1 expression levels showed significant positive425

correlations in Con group (r = 0.53, p < 0.05, n = 24), and significant negative correlations in426

Tr group (r = -0.57, p < 0.05, n = 24). Values are represented as mean ± SD (n = 6 in each427

group). Abbreviations: Con, control group; Tr, training group; Drp1, dynamin-like protein 1;

428

CS, citrate synthase.

429
430

Figure 4. Change in Opa1 and Mfn1 over time from 9 to 12 wks of age during

431

endurance swimming training in the Tr and Con groups.

432(A) Two-way ANOVA for the Opa1 expression level in the whole fraction revealed a433

statistical difference in the group factor (F (1, 40) = 14.27, p < 0.05) and the age factor (F (3,43440) = 4.82, p < 0.05), but not in the interaction factor (F (3, 40) = 0.15, n.s.). The simple main435

effects showed that the Opa1 expression was significantly higher in Tr than in Con groups436

only in 12 wk-old rats (p < 0.05), and there were significant differences in the age factor (p <4370.05). (B) The Mfn1 expression in the whole fraction showed a statistical difference in the age

438

factor (F (3, 40) = 3.97, p < 0.05), but not in the group factor (F (1, 40) = 3.70, n.s.) or the439interaction factor (F (3, 40) = 1.18, n.s.). The simple main effects showed the significant

440

differences in the age factor (p < 0.05). Values are represented as mean ± SD (n = 6 in each441

group). Abbreviations: Con, control group; Tr, training group; Opa1, optic protein atrophy 1;442

Mfn1, mitofusion 1. *: p < 0.05 vs. age-matched Con group, c: p < 0.05 vs. 10 wks-Con

443

groups, d: p < 0.05 vs. 10 wks-Tr group.

(A)

(B)

(C)

Figure 1.

(A)

9 wk
Con

Tr

10 wk
Con

11 wk

Tr Con

Tr

12 wk
Con

(B)

Tr

9 wk
Con

Drp1

Fis1

GAPDH

VDAC

Tr

10 wk

11 wk

12 wk

Con

Con

Con TrTr

Tr

Figure 2.

Con group

Tr group

Figure 3.

(A)

9 wk
Con

10 wk

Tr Con

Tr

11 wk

12 wk

Con Tr Con

Tr

(B)

9 wk
Con

Opa1

Mfn1

GAPDH

GAPDH

10 wk

Tr Con

Tr

11 wk

12 wk

Con TrCon

Tr

Figure 4.