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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
4
Short title: 5
Hyperbaric exposure and endurance performance 6
7
Author: Junichi Suzuki * 8
9
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
13
* 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(which was not certified by peer review) is the author/funder.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
2
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
(which was not certified by peer review) is the author/funder.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
3
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
(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
4
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
(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
5
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
(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
6
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
(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
7
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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8
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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9
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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10
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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11
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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12
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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14
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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16
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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
expressed as box and whisker plots with 5th, 25th, 50th, 75th and 95th percentile. Dots 565
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
expressed as box and whisker plots with 5th, 25th, 50th, 75th and 95th percentile. Dots 570
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
expressed as box and whisker plots with 5th, 25th, 50th, 75th and 95th percentile. Dots 575
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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
expressed as box and whisker plots with 5th, 25th, 50th, 75th and 95th percentile. Dots 579
are individual data points. *, significantly different from the Sed group. 580
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
expressed as box and whisker plots with 5th, 25th, 50th, 75th and 95th percentile. Dots 591
are individual data points. *, significantly different from the Sed group. 592
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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
)
Weeks
0
5
10
15
20
1 2 3 4 5 6 7
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Figure 2
(A)
Tota
l wor
k (J)
Tota
l wor
k (J)
0
500
1000
1500
2000
2500
3000
Sed HT
15HT
30HT
60HT
* * * *
0
500
1000
1500
2000
2500
3000
HT
15HT
30HT
60HT
#∫§ #
(B)
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Figure 3
1.5
2.0
2.5
3.0
3.5
4.0
Sed HT
15HT
30HT
60HT
6
8
10
12
14
16
18
Sed HT
15HT
30HT
60HT
0
5
10
15
20
25
30
Sed HT
15HT
30HT
60HT
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Sed HT
15HT
30HT
60HT
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Sed HT
15HT
30HT
60HT
2
6
10
14
18
Sed HT
15HT
30HT
60HT
0.3
0.8
1.3
1.8
2.3
2.8
Sed HT
15HT
30HT
60HT
4
6
8
10
12
14
16
Sed HT
15HT
30HT
60HT
5
10
15
20
25
30Sed HT
15HT
30HT
60HT
5
6
7
8
9
10
11
12
Sed HT
15HT
30HT
60HT
12
14
16
18
20
22
24
Sed HT
15HT
30HT
60HT
0
5
10
15
20
25
30
Sed HT
15HT
30HT
60HT
5
6
7
8
9
10
11
Sed HT
15HT
30HT
60HT
22
24
26
28
30
Sed HT
15HT
30HT
60HT
0
4
8
12
16
Sed HT
15HT
30HT
60HT
Gr Gw PL DIA LV
HAD
activ
ity(µ
mol
h-1
mg p
rote
in-1
)CS
activ
ity(µ
mol
h-1
mg p
rote
in-1
)
Gr Gw PL DIA LV
CPT2
activ
ity(µ
mol
h-1
mg p
rote
in-1
[x10
-2])
(A)
(B)
(C)
Gr Gw PL DIA LV
** *
∫ *∫
**
* *§ *
§∫
* *∫
**
* **
**
* * * * * *§
*
**∫
**
§∫
*∫ *
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Figure 4
1.0
1.2
1.4
1.6
1.8
2.0
Sed HT
15HT
30HT
60HT
60
80
100
120
140
160
180
Sed HT
15HT
30HT
60HT
0
4
8
12
16
Sed HT
15HT
30HT
60HT
1.4
1.6
1.8
2.0
2.2
Sed HT
15HT
30HT
60HT
140
160
180
200
220
Sed HT
15HT
30HT
60HT
4
6
8
10
12
14
16
18
20
Sed HT
15HT
30HT
60HT
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Sed HT
15HT
30HT
60HT
100
120
140
160
180
200
Sed HT
15HT
30HT
60HT
0
5
10
15
20
25Sed HT
15HT
30HT
60HT
2.0
2.5
3.0
3.5
4.0
4.5
Sed HT
15HT
30HT
60HT
40
50
60
70
80
Sed HT
15HT
30HT
60HT
7
9
11
13
Sed HT
15HT
30HT
60HT
1.0
1.5
2.0
2.5
Sed HT
15HT
30HT
60HT
40
50
60
70
Sed HT
15HT
30HT
60HT
4
6
8
10
12
Sed HT
15HT
30HT
60HT
PDHc
activ
ity(µ
mol
h-1
mg p
rote
in-1
)
(A)
LDH
activ
ity(µ
mol
h-1
mg p
rote
in-1
)
(B)
PFK
activ
ity(µ
mol
h-1
mg p
rote
in-1
)
(C)
Gr Gw PL DIA LV
Gr Gw PL DIA LV
Gr Gw PL DIA LV
**∫§
**
**
∫
*
§
* *
*∫§
∫§
** *
*
∫ § ∫ §
* *
∫∫∫
∫
∫§∫§
(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
Figure 5
0.0
0.5
1.0
1.5
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
2.5
Sed HT
15HT
30HT
60HT
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Sed HT
15HT
30HT
60HT
0.00.51.01.52.02.53.03.5
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
2.5
Sed HT
15HT
30HT
60HT
0.0
1.0
2.0
3.0
4.0
Sed HT
15HT
30HT
60HT
0.5
1.0
1.5
2.0
2.5
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
2.5
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
2.5
Sed HT
15HT
30HT
60HT
0.40.60.81.01.21.41.61.8
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
2.5
Sed HT
15HT
30HT
60HT
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Sed HT
15HT
30HT
60HT
DRP1
exp
ress
ion
(Sed
set t
o 1.
0)M
FN2
expr
essio
n (S
ed se
t to
1.0)
TFAM
exp
ress
ion
(Sed
set t
o 1.
0)
Gr Gw PL DIA LV(A)
Gr Gw PL DIA LV
Gr Gw PL DIA LV
(B)
(C)
** *
** *
* * * *
Figure 5
(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
Figure 6
0.0
2.0
4.0
6.0
Sed HT
15HT
30HT
60HT
0.00.51.01.52.02.53.03.5
Sed HT
15HT
30HT
60HT
0
2
4
6
8
10
12
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Sed HT
15HT
30HT
60HT
0.00.51.01.52.02.53.03.5
Sed HT
15HT
30HT
60HT
0
5
10
15
20
25
Sed HT
15HT
30HT
60HT
0
2
4
6
8
10
12
Sed HT
15HT
30HT
60HT
0.5
1.0
1.5
2.0
2.5
Sed HT
15HT
30HT
60HT
0
2
4
6
8
Sed HT
15HT
30HT
60HT
0.0
1.0
2.0
3.0
Sed HT
15HT
30HT
60HT
0.5
1.0
1.5
2.0
Sed HT
15HT
30HT
60HT
0.0
1.0
2.0
3.0
4.0
5.0
Sed HT
15HT
30HT
60HT
0.0
0.5
1.0
1.5
2.0
Sed HT
15HT
30HT
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
*
*
**
* * * *
**
* *
* *
*
* *
(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
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
Type I Type IIA Type I+IIA Type IIAX Type IIB+IIX
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)
* **
*
** * *
* * * ** * * *
* * **
(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
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)
(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
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)
* * * *
(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