1 Full title - bioRxiv · 2020-08-26 · 28 60HT. CPT2 activity levels were markedly increased by...

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Full title: 1

Effects of hyperbaric environment on endurance and metabolism are exposure time-2

dependent in well-trained mice 3

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Short title: 5

Hyperbaric exposure and endurance performance 6

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Author: Junichi Suzuki * 8

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Address: Laboratory of Exercise Physiology, Health and Sports Sciences, Course of 10

Sports Education, Department of Education, Hokkaido University of Education, 11

Midorigaoka, Iwamizawa, Hokkaido, Japan 12

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* Corresponding author 14

E-mail: [email protected] 15

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 27, 2020. ; https://doi.org/10.1101/2020.08.26.268995doi: bioRxiv preprint

https://doi.org/10.1101/2020.08.26.268995

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Abstract 16

Hyperbaric exposure (1.3 atmospheres absolute with 20.9% O2) for 1 h a day was 17

shown to improve exercise capacity. The present study was designed to reveal whether 18

the daily exposure time affects exercise performance and metabolism in skeletal and 19

cardiac muscles. Male mice in the training group were housed in a cage with a wheel 20

activity device for 7 weeks from 5 weeks old. Trained mice were then subjected to 21

hybrid training (HT, endurance exercise for 30 min followed by sprint interval exercise 22

for 30 min). Hyperbaric exposure was applied following daily HT for 15 min (15HT), 23

30 min (30HT), or 60 min (60HT) for 4 weeks (each group, n = 10). In the endurance 24

capacity test, maximal work values were significantly increased by 30HT and 60HT. In 25

the left ventricle (LV), activity levels of 3-hydroxyacyl-CoA-dehydrogenase, citrate 26

synthase, and carnitine palmitoyl transferase (CPT) 2 were significantly increased by 27

60HT. CPT2 activity levels were markedly increased by 60HT in the plantaris muscle 28

(PL). Pyruvate dehydrogenase complex (PDHc) activity values in PL were enhanced 29

more by 30HT and 60HT than by HT. In both 30HT and 60HT groups, lactate 30

dehydrogenase activity levels were significantly increased and decreased, respectively, 31

in the gastrocnemius muscle and LV. These results indicate that hyperbaric exposure for 32

30 min or longer has beneficial effects on endurance, and 60-min exposure has the 33

potential to further increase performance by facilitating fatty acid metabolism in skeletal 34

and cardiac muscles in highly-trained mice. 35


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Introduction 36

Athletes with a broad range of performance levels use a commercial hyperbaric 37

apparatus, which functions at <1.5 atmospheres absolute (ATA) with room air (20.9% 38

O2). Acute hyperbaric exposure (1.3 ATA) for 1 h markedly up-regulated mRNA 39

expression levels of proliferator-activated receptor gamma coactivator 1-alpha (PGC-40

1α) and peroxisome proliferator-activated receptor alpha (PPARα) in hind-leg muscles 41

3 h after exposure [1]. Moreover, endurance exercise training followed by hyperbaric 42

exposure for daily 1 h markedly improved both fatty acid and glucose metabolism in 43

hind-leg muscles in highly trained mice [2]. To apply daily hyperbaric exposure to 44

human athletes, the duration of exposure should be shorter, and should not disturb the 45

daily training regimen. Acute hyperbaric exposure (1.3 ATA with room air) for 30 min 46

after maximal exercise markedly reduced the lactate concentration and heart rate in 47

humans [3]. However, an additive effect of hyperbaric exposure on exercise 48

performance has not yet been reported in terms of the daily exposure time. 49

High-intensity interval exercise (6 x 30-sec all-out cycling) followed by endurance 50

exercise (60% VO2max for 60 min) markedly enhanced mRNA levels of PGC-1! 51

compared with those observed after respective, single, uncombined regimens in trained 52

human muscle [4]. Hybrid exercise training (HT) consisting of interval and endurance 53

exercise may be beneficial to improve the endurance capacity of highly-trained 54

individuals. HT (endurance exercise for 30 min followed by sprint interval exercise (5-s 55

run-10-s rest) for 30 min) for 4 weeks enhanced the endurance capacity, but endurance 56

training did not, in mice that were trained from an early age [5]. 57

Although hyperbaric exposure for 1 h markedly facilitated exercise-induced muscle 58

hypertrophy in mice [2], mechanisms underlying these changes have yet to be clearly 59

determined. In previous studies, expression levels of heat shock protein (HSP) 70 were 60

upregulated by endurance training with hyperbaric exposure in hind-leg muscles [1, 2]. 61

HSP70 was shown to have an important role in the recovery of skeletal muscle after 62

intensive exercise [6]. AKT, also known as protein kinase B (PKB), is a serine/threonine 63

protein kinase with multiple regulatory functions, including the control of cell growth, 64

survival, apoptosis, proliferation, angiogenesis, and the metabolism of carbohydrates, 65

lipids and proteins [7]. AKT1 was shown to have a crucial role in regulating satellite 66

cell proliferation during skeletal muscle hypertrophy [8]. 67


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Expression levels of mitochondrial fusion and fission proteins were shown to be 68

affected by exercise with hyperbaric exposure [2]. In highly-trained mice, mitochondrial 69

fission marker dynamin-related protein (DRP)-1, but not mitofusion (MFN)-1, 70

expression levels were upregulated after endurance exercise training under normobaric 71

conditions with hyperbaric exposure for 1 h daily [2]. However, the effects of HT with 72

different durations of hyperbaric exposure on expression levels of these proteins remain 73

unknown. 74

Most ATP production in the heart depends on fatty acid oxidation [9]. Fat 75

metabolism in the heart was shown to decrease after chronic endurance training [10] 76

and interval training [11]. However, no study has observed enzyme activity levels 77

concerning fatty acid metabolism after chronic exercise training with hyperbaric 78

exposure. 79

In the present study, experiments were designed to elucidate the effects of HT with 80

different durations of hyperbaric exposure on exercise capacity as well as the expression 81

of proteins involved in muscle growth and mitochondrial biogenesis, and metabolic 82

enzyme activity levels in skeletal and cardiac muscles of well-trained mice. 83

84

Materials and methods 85

Ethical approval 86

All procedures were approved by the Animal Care and Use Committee of Hokkaido 87

University of Education (No. 3, approved on 2019/4/16) and performed in accordance 88

with the "Guiding Principles for the Care and Use of Animals in the Field of 89

Physiological Sciences" of the Physiological Society of Japan and the 'European 90

Convention for the Protection of Vertebrate Animals used for Experimental and other 91

Scientific Purposes' (Council of Europe No. 123, Strasbourg, 1985). 92

93

Animals, hyperbaric exposure, and exercise training 94

Fifty male ICR (MCH) mice (four weeks old) were purchased from Clea Japan Inc. 95

(Tokyo, Japan) and housed under the conditions of a controlled temperature (24±1˚C) 96

and relative humidity of approximately 50%. Lighting (7:00-19:00) was controlled 97

automatically. All mice were given commercial laboratory chow (solid CE-2, Clea 98

Japan) and tap water ad libitum. After mice had been fed for one week and allowed to 99


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adapt to the new environment, they were randomly assigned to a sedentary control 100

group (Sed, n=10) or training group (n=40). Mice in the training group were 101

individually housed in a cage with a wheel activity device (13 cm in diameter) for 7 102

weeks, as described previously [2]. Wheel activity (distance and running time) was 103

monitored and recorded using digital bike computers (CC-VL820, Cateye Co., Ltd., 104

Osaka, Japan). Mice in the Sed group were housed individually throughout the 105

experiment. To familiarize mice with the treadmill device, all mice including the Sed 106

group were subjected to treadmill walking once a week using a controlled treadmill 107

(Modular motor assay, Columbus Instruments Inc., Columbus, OH, USA) for 5 min per 108

day at 10-15 m min-1 with a 5-deg incline. 109

Running distance during voluntary wheel training is shown in Fig 1. Following 110

voluntary wheel training, mice were given a 48-h non-exercise period prior to the 111

maximal endurance capacity test. The test was performed with a graded ramp running 112

protocol using the controlled treadmill, as described previously [5]. Total work (joules) 113

was calculated as a product of body weight (kg), speed (m sec-1), time (sec), slope (%), 114

and 9.8 (m sec-2). Exhaustion was defined when the mouse stayed for more than 5 s on 115

the metal grid (no electrical shock) at the rear of the treadmill, despite external gentle 116

touch being applied to their tail with a conventional elastic bamboo stick (0.8 mm in 117

diameter). 118

Following the performance test, mice were given a 48-h non-exercise period prior to 119

treadmill training. Mice in the training group were divided into a hybrid-training group 120

(HT, n=10), hybrid-training group with hyperbaric exposure for 15 min daily (15HT, 121

n=10), 30 min daily (30HT, n=10), or 60 min daily (60HT, n=10), in order to match the 122

mean and SD values of total work (Fig 2A). Mice in the training groups were subjected 123

to HT in a normobaric environment 6 days per week for 4 weeks (within 2 hours from 124

approximately 5 AM) using a rodent treadmill (KN-73, Natsume Co., Tokyo, Japan). 125

Instead of using an electrical shock, the tali or feet of mice were touched with a 126

conventional test tube brush made of soft porcine bristles in order to motivate them to 127

run when they stayed on a metal grid for more than 2 s. HT consists of endurance 128

exercise for 30 min followed by an interval exercise regimen (5-sec run-10-sec rest) for 129

30 min interposed by a 5-min rest. For endurance exercise, mice ran at 20 m min-1 with 130

a 15-deg incline on the first and second days of training. The running speed was 131


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increased to 25 and 27.5 m min-1 on the first and fourth days of the second week, 132

respectively, and to 30 m min-1 on the second day of the third week. For the interval 133

exercise regimen, mice ran at 30 m min-1 with a 15-deg incline on the first day of 134

training. The running speed was increased to 35, 37.5, 40, and 42.5 m min-1 on the 4th, 135

7th, 10th, and 13th days of training, respectively. The maximal endurance capacity test 136

was performed 48 hours after the last run, as described above. 137

The hyperbaric exposure group was subjected to 1.3 ATA with room air (20.9% O2) 138

for 15, 30, or 60 min, once per day (started at approximately 30 min after daily 139

exercise), 6 days per week, for 4 weeks. The hyperbaric exposure was performed as 140

described previously [2]. The pressure of the chamber was gradually increased or 141

decreased at 0.136 ATA min-1 within 2.2 min. 142

Forty-eight hours after the last run, mice were anesthetized with α-chloralose (0.06 g 143

kg-1 i. p. Wako Pure Chemical Industries Ltd., Osaka, Japan) and urethane (0.7 g kg-1 i. 144

p. Wako). A toe pinch response was used to validate adequate anesthesia. The plantaris 145

(PL), and gastrocnemius muscles were excised and the deep red region (Gr) of the 146

gastrocnemius was isolated from the superficial white region (Gw). The diaphragm 147

(DIA) was excised. All samples were frozen in liquid nitrogen for biochemical analyses. 148

The remaining muscles, i.e., those on the right side, were excised and placed in 149

embedding medium, O.C.T. compound (Miles Inc., Elkhart, IN, USA), and then rapidly 150

frozen in isopentane cooled to its melting point (-160˚C) with liquid nitrogen. Mice 151

were killed by excision of the heart. The whole heart and left ventricle (LV) were 152

weighed and frozen in liquid nitrogen. All tissue samples were stored at -80˚C until for 153

analyses. 154

155

Histological analyses 156

Histochemical examinations of capillary profiles and muscle fiber phenotypes were 157

conducted as previously reported by the author with slight modifications [5]. Briefly, 158

ten-micrometer-thick serial cross-sections were obtained using a cryotome (CM-1500; 159

Leica Japan Inc., Tokyo, Japan) at -20˚C from the mid-belly portion of calf muscles. 160

These sections were air-dried, fixed with 100% ethanol at 4˚C for 15 min, and then 161

washed in 0.1 M phosphate-buffered saline (PBS) with 0.1% Triton X-100. Sections 162

were then blocked with 10% goat normal serum at room temperature for 30 min, 163


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washed in PBS for 5 min, and incubated at 4˚C overnight with a mixture of fluorescein-164

labelled Griffonia simplicifolia lectin (GSL I) (FL 1101 (1:100), Vector Laboratories 165

Inc., California, USA), an anti-type I myosin heavy chain (MHC) antibody (BA-F8, 166

mouse IgG2b, 1:80), and anti-type IIA MHC antibody (SC-71, mouse IgG1, 1:80) 167

diluted with PBS. Sections were then reacted with a secondary antibody mixture 168

containing Alexa Fluor 350-labeled anti-mouse IgG2b (1:100) and Alexa Fluor 647-169

labeled anti-mouse IgG1 (1:100) diluted with PBS at room temperature for 1 h. Sections 170

were coverslipped with Fluoromount/Plus (K048, Diagnostic BioSystems Co., 171

California, USA). Primary and secondary antibodies were purchased from the 172

Developmental Studies Hybridoma Bank (University of Iowa) and Thermo Fisher 173

Scientific Inc. (Tokyo, Japan), respectively. Fluorescent images of the incubated 174

sections were observed using a microscope (Axio Observer, Carl Zeiss Japan, Tokyo, 175

Japan). Muscle fiber phenotypes were classified as type I (blue), type I+IIA (faint blue 176

and faint red), type IIA (red), type IIAX (faint red), and type IIB+IIX (unstained). Non-177

overlapping microscopic fields were selected at random from each muscle sample. The 178

observer was blinded to the source (groups) of each slide during the measurements. 179

Fluorescent images were obtained from PL, the lateral (GrL) and medial (GrM) portions 180

of Gr, and Gw. Fiber cross-sectional area (FCSA) values were obtained using public 181

domain Image J software (NIH, Bethesda, MD, USA). The negative control without 182

primary antibodies was confirmed to show no fluorescent signal. 183

184

Biochemical analyses of enzyme activities 185

Frozen tissue powder was obtained using a frozen sample crusher (SK mill, Tokken 186

Inc., Chiba, Japan) and homogenized with ice-cold medium (10 mM HEPES buffer, pH 187

7.4; 0.1% Triton X-100; 11.5% (w/v) sucrose; and 5% (v/v) protease inhibitor cocktail 188

(P2714, Sigma-Aldrich)). After centrifugation at 1,500 x g at 4°C for 10 min, the 189

supernatant was used in enzyme activity analyses. The activities of 3-hydroxyacyl-CoA-190

dehydrogenase (HAD) and lactate dehydrogenase (LDH) were assayed according to the 191

method of Bass et al. [12]. The activities of citrate synthase (CS) and 192

phosphofructokinase (PFK) were assayed according to the method of Srere [13] and 193

Passonneau and Lowry [14], respectively. Pyruvate dehydrogenase complex (PDHc) 194

activity was measured by the phenazine methosulfate-3-(4,5-dimethylthiazol-2-yl)-2,5-195


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diphenyltetazolium bromide (PMS-MTT) assay [15]. For the carnitine palmitoyl 196

transferase (CPT) 2 assay, the tissue homogenate was added to the reaction medium 197

(200 mM HEPES, 10 mM EGTA, 0.2 M sucrose, 400 mM KCl, 2 mM DTNB (Sigma 198

O4126), 0.13% (w/v) bovine serum albumin (Wako 017-15146), 20 µM palmityl CoA 199

(Sigma P 9716), 10 µM malonyl CoA (Sigma M4263)) and incubated for 1 h in a dark 200

chamber at 25 ºC. After confirming there was no non-specific reaction, the reaction was 201

initiated by adding 15 mM L-carnitine (Sigma C0283). All measurements were 202

conducted at 25°C with a spectrophotometer (U-2001, Hitachi Co., Tokyo, Japan) and 203

enzyme activities were obtained as µmol hour -1 mg protein-1. Total protein 204

concentrations were measured using PRO-MEASURE protein measurement solution 205

(iNtRON Biotechnology Inc., Gyeonggi-do, Korea). 206

207

Western blot analyses 208

The tissue homogenates described above were used for Western blot analyses. A 209

sample (50 µg) was fractionated by SDS/PAGE on 12% (w/v) polyacrylamide gels 210

(TGX StainFree FastCast gel, Bio-Rad Inc., CA, USA), exposed to UV for 1 min and 211

total protein patterns were visualized using ChemiDoc MP Imager (Bio-Rad). The stain-212

free gel contains a trihalo compound which reacts with proteins during separation, 213

rendering them detectable using UV exposure [16, 17]. Then, gels were 214

electrophoretically transferred to a polyvinylidene fluoride membrane. The bands in 215

each lane on the membrane were detected with ChemiDoc MP and the images were 216

used for normalization process as described below. The blots were blocked with 3% 217

(w/v) bovine serum albumin (protease and IgG free, 010-25783 Fujifilm Wako Pure 218

Chemical, Osaka Japan), 1% (w/v) polyvinylpyrrolidone (PVP40, Sigma-Aldrich), and 219

0.3% (v/v) Tween-20 in PBS for 1 h, and then exposed to a specific primary antibody 220

(1:500, Santa Cruz Biotechnology Inc., California, USA) against AKT1 (sc-5298), 221

HSP70 (1:500, sc-66048), TFAM (sc-166965), MFN2 (sc-100560), FABP (sc-514208), 222

or DRP1 (1:500, sc-271583) diluted in PBS with 0.05% Tween-20 for 1 h. After the 223

blots had been incubated with a HRP-labeled mouse IgGκ light chain binding protein 224

(1:5000, sc-516102, Santa Cruz), they were reacted with Clarity Western ECL substrate 225

(Bio-Rad), Clarity Max Western ECL substrate (Bio Rad), or their mixture and the 226

required proteins were detected with ChemiDoc MP. The densities of the specific bands 227


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were quantified using Image Lab software (Bio Rad) and normalized to the densities of 228

all protein bands in each lane on the membrane [16, 17]. Then, the normalized densities 229

of the bands were normalized again to the same sample that was run on every gel and 230

transferred to every membrane. 231

232

Statistical analyses 233

Differences between the two groups were examined using Bayesian estimation with a 234

gamma prior distribution proposed by Kruschke [18]. The posterior distribution was 235

obtained using the Markov chain Monte Carlo (MCMC) methods. Public domain R, 236

RStudio, and JAGS programs were used for computing Bayesian inference. The MCMC 237

chains were considered to show a stationary distribution when the Gelman-Rubin values 238

(shrink factor) were less than 1.10 for all parameters. The significance of differences 239

was evaluated by the 95% highest density interval (HDI) and a region of practical 240

equivalence (ROPE) [19]. When the HDI value on the effect size fell outside of the 241

ROPE set at -0.1 to 0.1, the difference was regarded as significant [19, 20]. Bayesian 242

robust linear regression [18] was used to establish any correlation between the 243

parameters observed in the present study. The regression was considered significant 244

when HDI on the slope fell outside of the ROPE. Data are expressed as box and whisker 245

plots with 5th, 25th, 50th, 75th and 95th percentile in the figures and means ± standard 246

deviation (SD) in Table 1. 247

248

Results 249

Body and organ masses 250

Body weight values after HT were significantly lower in the four exercise-trained 251

groups than in Sed group (Table 1, each group, n = 10). However, body weight values 252

before HT and the amount of increase during HT were not significantly different among 253

groups. The absolute and relative weight values of the whole heart and LV were 254

significantly higher in the four exercise-trained groups than in the Sed group. The 255

relative weight values of GAS and PL were higher in the four exercise-trained groups 256

than in the Sed group, but the differences were not significant. Thus, hyperbaric 257

exposure did not affect the body weight or HT-induced cardiac and skeletal muscle 258

growth. 259


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260

Maximal exercise capacity 261

After voluntary wheel running for 7 weeks, total work values were significantly 262

greater in the training group by 6.9-fold (Fig 2A) than in Sed group (each group, n = 263

10). In 30HT and 60HT groups, total work values were significantly increased after 4 264

weeks of treadmill training. Moreover, total work values were significantly greater in 265

the 30HT group than in HT and 15HT groups. Thus, daily hyperbaric exposure longer 266

than 30 min had additive effects on HT-induced improvements in endurance capacity. 267

268

Metabolic enzyme activities 269

CPT2 activity values in Gr were significantly higher in the three exposure groups 270

than in the Sed group and, moreover, the values were significantly higher in 15HT and 271

60HT groups than in the HT group (Fig 3C, each group, n = 10). In PL and LV, CPT2 272

activity levels showed significantly higher values in the 60HT group than in the Sed 273

group. CPT2 activity values in Gw were significantly higher in 15HT and 30HT groups 274

than in the Sed group. In DIA, CPT2 values were positively correlated with maximal 275

work values (Table 2). 276

HAD activity values in LV were significantly higher in 60HT than in Sed, HT, and 277

15HT groups (Fig 3A, each group, n = 10). In PL, HAD levels were significantly lower 278

in 30HT and 60HT groups than in the HT group, while activity values showed 279

significantly greater values in the four training groups than in the Sed group. 280

CS activity values in LV were significantly higher in the 60HT than in Sed and 15HT 281

groups (Fig 3B, each group, n = 10). In DIA, a positive correlation was observed 282

between CS levels and maximal work values (Table 2). 283

PDHc activity values in PL were significantly higher in 30HT and 60HT groups than 284

in Sed, HT, and 15HT groups (Fig 4A, each group, n = 10). Moreover, in PL, a positive 285

correlation was found between PDHc levels and maximal work values (Table 2). In Gw, 286

PDHc activity levels showed significantly greater values in 15HT, 30HT, and 60HT 287

groups than in Sed and HT groups, and the levels were significantly greater in the 60HT 288

group than in the 15HT group. In DIA, PDHc activity values were significantly higher 289

in 15HT and 30HT groups than in the HT group. PDHc activity levels in LV showed 290

significantly lower values in the 15HT group than in Sed and HT groups. 291


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In 30HT and 60HT groups, LDH activity values were significantly higher and lower, 292

respectively, in Gr and LV, than in Sed, HT, and 15HT groups (Fig 4B, each group, n = 293

10). LDH activity values were positively correlated with maximal work values in Gr 294

(Table 2). 295

Thus, 60HT enhanced the activities of enzymes involved in mitochondrial fatty acid 296

as well as glucose metabolism in hindleg and cardiac muscles, whereas 30HT 297

predominantly enhanced glucose metabolism in the hindleg muscles. 298

299

Protein expression 300

TFAM expression in Gr was significantly stronger in the four training groups than in 301

the Sed group (Fig 5C, each group, n = 10). TFAM levels in PL were significantly 302

higher in HT and 60HT groups than in the Sed group. MFN2 protein expression in PL 303

was markedly stronger in the four training groups than in the Sed group (Fig 5B). 304

AKT1 protein expression in PL was significantly stronger in the four training groups 305

than in the Sed group (Fig 6B, each group, n = 10). AKT1 expression levels in PL were 306

positively correlated with values of total FCSA and FCSA of all fiber types (Table 2). A 307

significant positive correlation was observed between AKT1 expression levels and 308

values in PL (Table 2). 309

HSP70 expression levels in Gr were significantly stronger in the four training groups 310

than in the Sed group (Fig 6C, each group, n = 10). In Gw, HSP70 protein expression 311

was significantly stronger in HT, 15HT, and 30HT groups than in the Sed group. HSP70 312

levels in Gw tended to be greater (1.6-fold) in the 60HT group than in the Sed group. 313

HSP70 expression levels in DIA were significantly stronger in 15HT and 30HT groups 314

than in the Sed group. HSP70 levels were positively correlated with total FCSA values 315

in Gw and with FCSA of all fiber types in PL (Table 2). 316

317

Fiber type composition 318

HT with and without hyperbaric exposure significantly increased the proportion of 319

type IIA and IIAX fibers and decreased the proportion of type IIB+IIX fibers in PL (Fig 320

7C, each group, n = 10). In GrL, the proportion of type IIAX fibers was larger and that 321

of type IIB+IIX fibers was smaller in the four trained groups than in Sed group (Fig 322

7A). Thus, hyperbaric exposure did not affect the fiber proportion in hybrid-trained 323


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hindleg muscles. 324

325

Fiber cross-sectional area 326

Total FCSA values in Gw were higher in the 60HT group than in Sed (by 16%) and 327

30HT (by 11%) groups, but the difference was not significant (Fig 8B). The distribution 328

of total FCSA in the 60HT group was shifted to the larger fiber size in Gw (Fig 8A). 329

Thus, hyperbaric exposure for 1 h partially promoted muscle fiber hypertrophy in highly 330

glycolytic fibers. 331

332

Capillarization 333

Capillary-to-fiber (C:F) ratio values in PL were significantly higher in the four 334

trained groups than in the Sed group (Fig 9, each group, n = 10). C:F ratio values in GrL 335

and GrM were higher in the four training groups, but the differences were not 336

significant. C:F values in Gw were markedly higher in the 60HT group than in the Sed 337

group by 21%. Thus, daily hyperbaric exposure for 1 h markedly facilitated exercise-338

induced capillary growth in the muscle regions mainly composed of glycolytic fibers. 339

340

Discussion 341

In highly-trained mice, the present results showed that endurance exercise performance 342

was promoted by HT with daily hyperbaric exposure for 30 and 60 min, but not for 15 343

min. In both 30HT and 60HT groups, additive effects of muscle metabolism were noted 344

in PDHc activity values in PL and Gw, and in LDH activity values in Gr (Figs 3 and 4). 345

Thus, daily hyperbaric exposure for 30-60 min had additive effects on the training-346

induced increase in glucose metabolism in hindleg muscles. Endurance training with 60 347

min of hyperbaric exposure daily was shown to enhance PFK activity values, but did 348

not change LDH activity values in hindleg muscle of highly-trained mice [2], 349

suggesting that HT with hyperbaric exposure for at least 30 min daily may be beneficial 350

to improve glucose metabolism in hindleg muscles. 351

In addition to these beneficial changes, HT with daily hyperbaric exposure for 60 352

min markedly enhanced cardiac fatty acid metabolism, identified by marked increases 353

in HAD, CS, and CPT2 activity levels in LV (Fig 3). The present study is the first to 354

report the effects of exercise training with hyperbaric exposure on cardiac metabolism. 355


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Around 95% of ATP used by cardiomyocytes is generated by oxidative phosphorylation 356

in mitochondria [9]. Up to 70% of the oxidative metabolism principally relies on fatty 357

acid oxidation [9]. However, fatty acid metabolism in the heart was not shown to be 358

enhanced by chronic exercise training. After 5-6 weeks of endurance training, 359

palmitoylcarnitine oxidation was shown to be significantly reduced in the heart, 360

whereas it was unchanged in the gastrocnemius muscle of rats [10]. Endurance training 361

for 8 weeks did not increase palmitate oxidation while interval training (4-min run-2-362

min rest) decreased it in the mouse heart [11]. Consistent with these findings, in the 363

present study, HT itself did not affect enzyme levels concerning fatty acid metabolism 364

in LV. 365

Glycolytic energy production by active skeletal muscles increases in proportion to 366

exercise intensity, thereby increasing the plasma lactate concentration [21]. Myocardial 367

lactate oxidation is prominent and proportional to the exogenous concentration during 368

elevated workloads in rats [22]. In isolated working rat hearts, lactate was found to 369

contribute a relatively large proportion (up to 37%) of the total ATP supply under a low-370

fat condition [23]. In contrast, under a high-fat condition, the contribution of lactate 371

decreased to 13%, suggesting that fatty acid oxidation became a dominant source. This 372

was supported by a study reporting that, in the perfused rat heart, rate values of triacyl 373

glycerol degradation and total β-oxidation were markedly higher under high-fat and 374

high-lactate conditions than low-fat and low-lactate conditions [24]. Plasma fatty acid 375

levels have been shown to increase during exercise [25]. Thus, these findings may 376

indicate that fatty acid oxidation in cardiac muscle is facilitated during intensive 377

exercise, i.e., at high concentrations of blood lactate and free fatty acids. In the present 378

study, LDH activity values in LV were markedly reduced in 30HT and 60HT groups 379

(Fig 4B). This result may show that lactate oxidation in the heart was reduced and 380

thereby the plasma lactate concentration may have been higher in these two groups than 381

in the other groups during exercise. As mentioned above, in 60HT, but not 30HT, fatty 382

acid metabolism was markedly improved in LV, thereby increasing the capacity of the 383

heart to generate ATP via fatty acid oxidation during intensive exercise. Furthermore, 384

CPT2 activity levels were remarkably up-regulated by 60HT in Gr and PL (Fig 3C). 385

Thus, HT with daily hyperbaric exposure for 60 min has the potential to further increase 386

exercise performance by facilitating fatty acid metabolism in skeletal as well as cardiac 387


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muscles. 388

Although significant changes were not observed, HT with hyperbaric exposure 389

markedly upregulated DRP1 expression levels in LV (from 1.6- to 1.8-fold greater than 390

HT group, Fig. 5A). DRP1 inhibition was shown to suppress mitochondrial respiration 391

and reactive oxygen species without morphological changes in mitochondria, i.e. 392

fission, in rat cardiomyocytes [26]. After acute exercise, mitochondrial fission was 393

markedly observed in cardiac muscle [27]. In vascular smooth muscle cells, 394

mitochondrial fission induced by platelet-derived growth factor was shown to enhance 395

fatty acid oxidation and suppress glucose oxidation [28]. In the present study, 396

expression levels of DRP1 were significantly correlated with both CS and HAD activity 397

values in LV (Table 2). Thus, a modest increase in DRP1 levels may induce 398

mitochondrial fission and contribute to promoting fatty acid metabolism in cardiac 399

muscle. 400

In highly-trained mice as used in the present study, endurance training with 401

hyperbaric exposure for 1 h markedly increased TFAM and DRP1 expression in Gr [2]. 402

However, HT with hyperbaric exposure did not upregulate these protein expression 403

levels (Fig 5). Thus, endurance exercise rather than HT may stimulate the expression of 404

proteins concerning mitochondrial biogenesis in hind-leg muscles of highly-trained 405

mice. 406

While hyperbaric exposure did not cause an additive effect, AKT1 expression levels 407

(Fig 6B) were significantly increased in the four training groups in PL. A positive 408

correlation was observed between AKT1 expression levels and FCSA values in PL 409

(Table 2). Thus, hybrid training used in the present study caused muscle fiber 410

hypertrophy, possibly via AKT1 upregulation. AKT1 was shown to promote muscle 411

growth via facilitating satellite cell proliferation during skeletal muscle hypertrophy [8]. 412

In PL, HSP70 expression levels were also significantly correlated with FCSA values 413

(Table 2). In Gw, a significant correlation was observed between total FCSA and HSP70 414

expression levels, but not with AKT1 levels (Table 2). Mean values of HSP70 415

expression levels in Gw were markedly greater in 30HT (1.4-fold) and 60HT (1.6-fold) 416

groups than in the HT group. This is consistent with previous findings using untrained 417

[1] and highly-trained mice [2]. HSP70 was shown to play an important role in the 418

recovery of striated muscle after severe exercise [29]. Recovery from muscle damage 419


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15

induced by daily exercise may be facilitated by hyperbaric exposure-induced HSP70 up-420

regulation, resulting in the promotion of muscular adaptation to exercise training. 421

Muscle capillary geometry was markedly improved by HT irrespective of hyperbaric 422

exposure in PL (Fig 9). A positive correlation was noted between C:F ratios and AKT1 423

expression levels in PL (Table 2). In muscle-specific inducible AKT1 transgenic mice, 424

the C:F ratio was significantly increased in the soleus muscle [30]. Thus, promoted 425

expression of AKT1 by HT may contribute to improve muscle capillary network in 426

highly-trained mice. 427

428

Conclusions 429

The present study showed that hybrid training with daily hyperbaric exposure for longer 430

than 30 min augmented exercise capacity in well-trained mice. Further studies on 431

trained human subjects are needed to clarify whether exercise performance in athletes is 432

promoted by hyperbaric exposure. In humans, middle ear barotrauma (MEB) was 433

shown to be the most common side effect of hyperbaric oxygen therapy (approximately 434

3 ATA with 100% O2) [31, 32]. To reduce the risk of MEB, a compression rate may play 435

an important role. The risk of MEB was shown to increase not only at a high rate of 436

compression (0.28 ATA min-1) [31], but also at a low rate (0.068 ATA min-1) [32]. In the 437

present study, the compression rate was set at 0.136 ATA min-1, which was shown to be 438

the best rate for minimizing MEB and pain in humans [32]. However, commercial 439

hyperbaric apparatuses currently compress air at a rate of 0.03-0.05 ATA min-1. To apply 440

hyperbaric exposure to the daily training regimens of athletes, a new apparatus with a 441

safer compression rate needs to be developed in the future. 442

443

Funding 444

This work was supported by JSPS KAKENHI Grant Number 17K01749. 445

446

Acknowledgments 447

None 448

449

References 450


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31. Vahidova D, Sen P, Papesch M, Zein-Sanchez MP, Mueller PHJ. Does the slow 544

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551

Figure legends 552 Fig 1. Running distance per day during voluntary wheel training. Values are 553

expressed as box and whisker plots with 5th, 25th, 50th, 75th and 95th percentile. Dots 554

are individual data points. 555

556 Fig 2. Endurance exercise performance test. Total work capacity of the endurance 557

capacity test after 7 weeks of voluntary wheel running (A) and after 4 weeks of 558

treadmill exercise training (B). #, significantly different from pre-treadmill training 559

values of each group shown in the panel A. *, ∫, and §, significantly different from Sed, 560

HT, and 15HT groups, respectively. Values are expressed as box and whisker plots with 561

5th, 25th, 50th, 75th and 95th percentile. Dots are individual data points. 562

563 Fig 3. Enzyme activity values for HAD (A), CS (B), and CPT2 (C). Values are 564


are individual data points. *, ∫, and §, significantly different from Sed, HT, and 15HT 566

groups, respectively. 567

568 Fig 4. Enzyme activity values for PDHc (A), LDH (B), and PFK (C). Values are 569


are individual data points. *, ∫, and §, significantly different from Sed, HT, and 15HT 571

groups, respectively. 572

573 Fig 5. Expression of DRP1 (A), MFN2 (B), and TFAM (C) proteins. Values are 574



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20

are individual data points. *, significantly different from the Sed group. 576

577 Figure 6. Expression of FABP (A), AKT1 (B), and HSP70 (C) proteins. Values are 578



581 Fig 7. Fiber-type composition values in GrL (A), Grm (B), and PL (C) proteins. 582

Values are expressed as box and whisker plots with 5th, 25th, 50th, 75th and 95th 583

percentile. Dots are individual data points. *, significantly different from the Sed group. 584

585 Fig 8. Distribution of FCSA (A) and total FCSA (B) values. Values are expressed as 586

box and whisker plots with 5th, 25th, 50th, 75th and 95th percentile. Dots are individual 587

data points in the panel (B). 588

589 Fig 9. Capillary-to-fiber ratio (A) and capillary density (B) values. Values are 590




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Table 1. Body and organ masses

Sed (n=10) HT (n=10) 15HT (n=10) 30HT (n=10) 60HT (n=10)

Body mass (g)

Pre-HT 38.3 ± 1.7 36.1 ± 3.4 36.3 ± 0.95 35.0 ± 1.7 36.2 ± 1.1

Post-HT 41.0 ± 1.7 37.5 ± 2.0 * 38.1 ± 0.9 * 38.4 ± 1.7 * 38.4 ± 1.2 *

Post-Pre difference 2.71 ± 1.16 1.45 ± 1.77 1.81 ± 1.12 3.35 ± 1.15 2.19 ± 1.17

Organ mass (mg)

Gastrocnemius 178.6 ± 12.1 174.3 ± 8.9 171.4 ± 7.1 171.2 ± 8.1 175.2 ± 10.2

Plantaris 21.7 ± 1.1 23.1 ± 1.9 22.7 ± 2.2 23.7 ± 1.7 23.3 ± 3.1

Whole heart 158.1 ± 10.2 178.1 ± 12.3 * 182.6 ± 9.6 * 187.5 ± 10.6 * 183.3 ± 7.6 *

Left ventricle 114.3 ± 8.9 128.4 ± 9.9 * 133.3 ± 7.6 * 136.2 ± 8.7 * 137.1 ± 14.3 *

Organ mass-to-body mass ratio (mg g-1)

Gastrocnemius 4.36 ± 0.29 4.65 ± 0.26 4.50 ± 0.21 4.46 ± 0.21 4.56 ± 0.17

Plantaris 0.53 ± 0.04 0.62 ± 0.06 0.60 ± 0.06 0.62 ± 0.04 0.61 ± 0.08

Whole heart 3.86 ± 0.17 4.75 ± 0.22 * 4.79 ± 0.18 * 4.88 ± 0.21 * 4.78 ± 0.12 *

Left ventricle 2.79 ± 0.15 3.42 ± 0.16 * 3.49 ± 0.14 * 3.55 ± 0.18 * 3.56 ± 0.37 *

Values are presented as means ± SD. *, significantly different from the Sed group using

Bayesian estimation. Pre-HT and Post-HT, at the beginning and end, respectively, of the 4

weeks of HT.


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Table 2. Correlations

Explanatory

variable

Response

variable Gr Gw PL DIA LV

LDH maximal work ‡ ∗ ns ns ns ns

PDHc maximal work ‡ ns ns ∗ ns ns

CPT2 maximal work ‡ ns ns ns ∗ ns

CS maximal work ‡ ns ns ns ∗ ns

AKT1 Total FCSA ns ns ∗ ns ns

AKT1 C:F ratio ns ns ∗ ns ns

HSP70 Total FCSA ns ∗ ns ns ns

DRP1 CS ∗ ∗ ∗ ∗ ∗

DRP1 HAD ∗ ∗ ∗ ∗ ∗

DRP1 CPT2 ns ∗ ns ns ns

TFAM CS ∗ ∗ ns ns ∗

TFAM HAD ∗ ∗ ∗ ns ∗

MFN2 CS ∗ ns ns ns ns

MFN2 HAD ns ns ∗ ns ns

Correlations were established using Bayesian robust linear regression. *,

significant correlation; ns, non-significant correlation. ‡, Correlations were

calculated without Sed group.


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Figure 1

Dis

tanc

e ru

n pe

r day

(km

)

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0

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10

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20

1 2 3 4 5 6 7


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Figure 2

(A)

Tota

l wor

k (J)

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Figure 3

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)CS

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)

Gr Gw PL DIA LV

CPT2

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mg p

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in-1

[x10

-2])

(A)

(B)

(C)

Gr Gw PL DIA LV

** *

∫ *∫

**

* *§ *

§∫

* *∫

**

* **

**

* * * * * *§

*

**∫

**

§∫

*∫ *


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Figure 4

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**∫§

**

**

∫

*

§

* *

*∫§

∫§

** *

*

∫ § ∫ §

* *

∫∫∫

∫

∫§∫§


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Figure 5

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Gr Gw PL DIA LV(A)

Gr Gw PL DIA LV

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(B)

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** *

** *

* * * *

Figure 5


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Figure 6

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60HT

0.0

0.5

1.0

1.5

2.0

2.5

Sed HT

15HT

30HT

60HT

0.0

2.0

4.0

6.0

Sed HT

15HT

30HT

60HT

FABP

exp

ress

ion

(Sed

set t

o 1.

0)AK

T1 e

xpre

ssio

n (S

ed se

t to

1.0)

HSP7

0 ex

pres

sion

(Sed

set t

o 1.

0)

Gr Gw PL DIA LV(A)

Gr Gw PL DIA LV

Gr Gw PL DIA LV

(B)

(C)

undetected

*

*

**

* * * *

**

* *

* *

*

* *


https://doi.org/10.1101/2020.08.26.268995

5

10

15

20

25

30

Sed HT

15HT

30HT

60HT

30

40

50

60

70

Sed HT

15HT

30HT

60HT

0.0

0.5

1.0

1.5

2.0

Sed HT

15HT

30HT

60HT

0

5

10

15

20

25

Sed HT

15HT

30HT

60HT

0

10

20

30

40

50

60

Sed HT

15HT

30HT

60HT

20

30

40

50

60

Sed HT

15HT

30HT

60HT

30

40

50

60

70

Sed HT

15HT

30HT

60HT

0.01.02.03.04.05.06.07.0

Sed HT

15HT

30HT

60HT

0

5

10

15

20

Sed HT

15HT

30HT

60HT

05

101520253035

Sed HT

15HT

30HT

60HT

0

2

4

6

8

10

Sed HT

15HT

30HT

60HT

20

40

60

80

Sed HT

15HT

30HT

60HT

0.0

1.0

2.0

3.0

4.0

5.0

Sed HT

15HT

30HT

60HT

0

5

10

15

20

25

30

Sed HT

15HT

30HT

60HT

0

20

40

60

80

Sed HT

15HT

30HT

60HT

Figure 7

Type I Type IIA Type I+IIA Type IIAX Type IIB+IIX



(GrL)

Fiber

type

co

mpo

sitio

n (%

)Fib

er ty

pe

com

posit

ion

(%)

Fiber

type

co

mpo

sitio

n (%

)

(GrM)

(PL)

* **

*

** * *

* * * ** * * *

* * **


https://doi.org/10.1101/2020.08.26.268995

1.0

2.0

3.0

4.0

Sed HT

15HT

30HT

60HT

1.0

2.0

3.0

4.0

Sed HT

15HT

30HT

60HT

2.0

2.5

3.0

3.5

4.0

Sed HT

15HT

30HT

60HT

1.0

1.5

2.0

2.5

3.0

3.5

Sed HT

15HT

30HT

60HT

Figure 8

0

5

10

15

20

25

30

0 2000 4000 6000

SedHT15HT30HT60HT

0

5

10

15

20

0 2000 4000 6000

SedHT15HT30HT60HT

0

5

10

15

20

25

30

0 2000 4000 6000

SedHT15HT30HT60HT

0

5

10

15

20

25

30

0 2000 4000

SedHT15HT30HT60HT

Dist

ribut

ion

of F

CSA

(%)

(A)

(µm2) (µm2)(µm2) (µm2)

FCSA

(µm

2 [x

103 ])

GrL GrM Gw PL

GrL GrM Gw PL(B)


https://doi.org/10.1101/2020.08.26.268995

2.0

2.5

3.0

3.5

Sed HT

15HT

30HT

60HT

2.0

2.5

3.0

3.5

Sed HT

15HT

30HT

60HT

1.0

1.5

2.0

Sed HT

15HT

30HT

60HT

1.5

2.0

2.5

3.0

3.5

Sed HT

15HT

30HT

60HT

5

10

15

20

Sed HT

15HT

30HT

60HT

5

10

15

20

Sed HT

15HT

30HT

60HT

3.0

4.0

5.0

6.0

7.0

8.0

Sed HT

15HT

30HT

60HT

6

8

10

12

14

16

18

Sed HT

15HT

30HT

60HT

Figure 9

Capi

llary

den

sity

(num

ber µ

m-2

[x10

2 ])Ca

pilla

ry-to

-fibe

rra

tio

GrL GrM Gw PL

GrL GrM Gw PL

(A)

(B)

* * * *


https://doi.org/10.1101/2020.08.26.268995

1 Full title - bioRxiv · 2020-08-26 · 28 60HT. CPT2 activity levels were markedly increased by...

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