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Increase in carbon dioxide accelerates the performance of endurance exercise in rats

Increase in carbon dioxide accelerates the performance of endurance exercise in rats J Physiol Sci (2018) 68:463–470 https://doi.org/10.1007/s12576-017-0548-6 ORIGINAL PAPER Increase in carbon dioxide accelerates the performance of endurance exercise in rats 1,2 3,4 3 5 • • • • Takeshi Ueha Keisuke Oe Masahiko Miwa Takumi Hasegawa 3 3 3 3 • • • • Akihiro Koh Hanako Nishimoto Sang Yang Lee Takahiro Niikura 1,3 1,3 1 • • Masahiro Kurosaka Ryosuke Kuroda Yoshitada Sakai Received: 16 December 2016 / Accepted: 31 May 2017 / Published online: 10 June 2017 The Physiological Society of Japan and Springer Japan KK 2017 Abstract Endurance exercise generates CO via aerobic Keywords Activity wheel  Carbon dioxide  Endurance metabolism; however, its role remains unclear. Exogenous exercise  Running performance CO by transcutaneous delivery promotes muscle fibre-type switching to increase endurance power in skeletal muscles. Here we determined the performance of rats running in Introduction activity wheels with/without transcutaneous CO exposure to clarify its effect on endurance exercise and recovery from All animals generate energy by consuming oxygen (O ) muscle fatigue. Rats were randomised to control, training and and releasing carbon dioxide (CO ). Aerobic metabolism is CO groups. Endurance exercise included activity-wheel performed with O for energy production in the mito- 2 2 running with/without transcutaneous CO delivery. Running chondria [1, 2] using not only carbohydrates but also amino performance was measured after exercise initiation. We also acids and fatty acids [1, 3]. CO generated during aerobic analysed changes in muscle weight and muscle fibres in the metabolism is not merely a waste product but serves other tibialis anterior muscle. Running performance improved over important functions. the treatment period in the CO group, with a concomitant One role of CO is vasodilation, with important impli- 2 2 switch in muscle fibres to slow-type. The mitochondrial DNA cations in central nervous system function [4] and physi- content and capillary density in the CO group increased. CO ology [5]. CO also exerts the Bohr effect on haemoglobin 2 2 2 was beneficial for performance and muscle development in red blood cells [6, 7], which have high levels of carbonic during endurance exercise: it may enhance recovery from anhydrase that ionises CO to carbonate and hydrogen ions fatigue and support anabolic metabolism in skeletal muscles. [8]. Accumulating hydrogen ions dissociate O from oxy- haemoglobin. Thus, tissues with increased CO content can acquire necessary O via the Bohr effect. Importantly, the Mr. Takeshi Ueha and Dr. Keisuke Oe contributed equally to this Bohr effect can be achieved via exogenous CO [9]. work. 2 Moreover, we previously showed that transcutaneous CO & Yoshitada Sakai delivery to the hind limbs of rats upregulated peroxisome [email protected] proliferator-activated receptor-gamma coactivator-1a (PGC-1a) and vascular endothelial growth factor (VEGF) Division of Rehabilitation Medicine, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo- expression as well as the number of mitochondria. Fur- ku, Kobe 650-0017, Japan thermore, this approach promoted a change in muscle fibre NeoChemir Inc, Kobe, Japan type from IIB to IIA, similar to that observed in tibialis anterior (TA) muscles after aerobic exercise [10]. A recent Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan study also suggested that carbonate ion-mediated PGC-1a upregulation might promote a fast-to-slow fibre-type shift Department of Orthopaedic Surgery, Hyogo Prefectural Awaji Hospital, Awaji, Japan in muscle cells [11]. Aerobic exercise is an actively performed exercise Department of Oral and Maxillofacial Surgery, Kobe that utilises aerobic metabolism [2, 12]; endurance University Graduate School of Medicine, Kobe, Japan 123 464 J Physiol Sci (2018) 68:463–470 exercise is typically referred to as aerobic exercise Measurement of running distance [12, 13]. Repeated performance of endurance exercise increases aerobic metabolism via a positive-feedback We measured the total running distance during exercise loop along with transformation of muscle fibres and every day to analyse running performance. Data for the running distance were expressed as a percentage of running subsequent muscle endurance [12]. Studies show that the upregulation of PGC-1a gene expression plays a major distance during pre-training. To evaluate time-dependent change, we recorded the role in muscle fibre transformation [14–16]. PGC-1a in muscles not only increases the transcriptional activity of distance run at 5-min intervals on the final day (day 28). Data were presented as average in the CO group compared the mitochondrial transcriptional factor A (TFAM) but also induces capillary density by activating VEGF. As a with those in the training group. result, slow-twitch muscle fibres shift, in order, to type IIB, IID, IIA and I fibres; consequently, muscle endur- Activity wheels ance is increased and endurance exercise performance is enhanced [17, 18]. Activity wheels were purchased from Hoei Kinzoku Kogyo The responses to exogenous CO and endurance (Aichi, Japan), as previously described [19, 20]. The wheels measured 1 m in circumference and 14 cm in exercise are similar, and include changes, such as mitochondrial biogenesis, in muscle fibers following depth. We measured the initial torques using torque-mea- suring equipment (Sugisaki Meter Co., Ltd, Ibaraki, Japan); increase in PGC-1a, and increase in capillary density by activating VEGF. However, the relationship between the initial torque was less than 1 mN (Fig. 1b). The wheels endurance exercise and exogenous CO remains unclear. had a magnetic counting system that recorded the number Therefore, we hypothesised that the application of of rotations to determine the running distance. exogenous CO may support exercise endurance and evaluated whether transcutaneous CO application after CO treatment endurance exercise increased endurance performance and aerobic capacity in rats. Supplementing CO therapy Transcutaneous CO absorption-enhancing hydrogel was 2 2 during endurance exercise may be beneficial for long- provided by NeoChemir Inc. (Kobe, Japan) as previously described [9, 10, 21]. Briefly, all animals in the three distance runners as well as patients with reduced muscle capacity for endurance who might require postoperative groups were anaesthetised with diethyl ether (Wako Pure Chemical Industries, Osaka, Japan), hair on their hind rehabilitation. limbs was shaved, and CO hydrogel was applied to their hind limbs. The area of skin covered with the CO hydrogel Materials and methods was sealed with an adaptor. In only the CO group rats, 100% CO gas (Kobe Sanso KK, Kobe, Japan) was flowed Animal care and experimental design into the area for 10 min, as previously described [10, 21]. The CO group underwent this treatment every time after The use of animals was approved by the Animal Care and the training. Use Committee of Kobe University Graduate School of The expected mechanism of the transcutaneous CO application was as follows. Because of its relatively high Medicine (approval number, P100408). A total of 24 male Wistar rats that were 5 weeks old (CLEA Japan, Tokyo, solubility, CO gas dissolves in the hydrogel. The dissolved CO then permeates the skin, moving down its concentra- Japan) were used for all experiments. Animals were fed ad libitum and housed in a thermostatic environment at tion gradient when the maximum solubility in the hydrogel 21 C under a 12-h light/12-h dark cycle. is reached. Our previous study showed that CO penetrated As depicted in Fig. 1a, rats were initially divided into human tissue by this system [9]. two groups, exercise (n = 16) and no exercise (control group; n = 8), and further divided according to average Muscle isolation and preparation body weight. All rats were pre-trained using a 5-day pro- gramme that comprised activity-wheel running for 30 min Animals were sacrificed with an overdose of the anaes- every day. On the 6th day, rats in the exercise group were thetic pentobarbital at 24 h after final exercise and/or treatment, followed by decapitation and dissection of intact then randomly assigned to one of the two experimental groups: CO treatment (CO group, n = 8) and no treat- TA muscles in order to analyse changes in muscle fibre 2 2 types. Muscles were then weighed with an electronic bal- ment (training group, n = 8). Both experimental groups were subdivided according to average running distance and ance (A&D, Japan) after removing excess connective tis- body weight. sue, immediately frozen in isopentane precooled with 123 J Physiol Sci (2018) 68:463–470 465 (a) Four weeks After first week The first week Measuring Body weight Anesthesia n = 8 Control group n = 8 Pre- n = 8 Training group training n = 16 CO group n = 8 CO Activity wheel 30 min 10 min Magnetic sensor (b) Closing door with lock Activity wheel Fig. 1 a Experimental design to test the impact of transcutaneous CO (left). The initial torques measured by using torque-measuring equipment delivery on endurance exercise performance. b Photograph of a male (right) Wistar rat running in an activity wheel equipped with a magnetic sensor liquid nitrogen and stored at -80 C. Serial 10-lm-thick electrophoresis, as previously described [10, 22, 23]. After transverse sections were prepared from each block. Muscle electrophoresis, gels were stained using the Wako Silver weight ratio was calculated by the following equation: Stain Kit (Wako Pure Chemical Industries), and images of muscle weight ratio = muscle weight/body weight 9 100. MHC bands were captured with a LAS-3000 imager to quantify band intensities using ImageJ software (https:// ATPase staining imagej.nih.gov/ij/). Changes in MHC isoform expression among the three groups were presented as percent changes Unfixed frozen TA muscles were sectioned into 10-lm- from pre-training values. thick slices with a cryostat, and ATPase immunostaining at pH 4.6 was performed as previously described [10, 22]. Mitochondrial DNA copy number Isolation of myosin heavy chain Genomic DNA was isolated from 10-mg transverse slices TM of mid-belly TA muscles using a GenElute Mammalian Protein was isolated from 10-mg transverse slices of the Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, mid-belly of TA muscles using urea buffer, and 5–20 llof MO, USA). Mitochondrial DNA (mtDNA) content relative homogenates were analysed for the expression of myosin to the b-actin gene copy number was measured using real- heavy chain (MHC) isoforms by two-dimensional time PCR, as previously described [10, 23]. Torque (N·m) 466 J Physiol Sci (2018) 68:463–470 Fig. 2 a Percent change in (a) (b) 300% running distance among the Training Training groups by week. *p \ 0.05 CO CO b Changes in running distances on the final day in the CO and 200% training groups. *p \ 0.05 100% 0% Before 1 week 2 week 3 week 4 week 0 5 10 15 20 25 30 Endurance running Time (min) Quantitative real-time PCR Statistical analysis Quantification of mtDNA was performed using a Ste- The level of statistical significance was set at p \ 0.05. TM pOne Real-Time PCR System (Applied Biosystems). Data were expressed as mean ± standard deviation. Sig- Real-time PCR reactions (20 ll) contained 0.5 lMeach nificance was determined via analysis of variance of forward and reverse primers, 1 ll genomic DNA (ANOVA) and the two-tailed Mann–Whitney U test. template from the reverse transcription reaction and 10 ll Power SYBER Green PCR Master Mix (Applied Biosystems). Reaction conditions were 10 min at 95 C, Results followed by 40 cycles at 95 C(15 s) and60 C(1 min). mtDNA copy numbers were normalised to nuclear DNA Running distance copy numbers and compared among groups (DDCt method; Applied Biosystems), as previously described As shown in Fig. 2a, although the running distance was [10, 24]. The following forward and reverse primers longer in the CO group than in the training group after were used to determine mtDNA copy numbers: 5 -ACA 1 week, the difference was not statistically significant. 0 0 CCA AAA GGA CGA ACC TG-3 and 5 -ATG GGG However, rats that received transcutaneous CO ran sig- AAG AAG CCC TAG AA-3 , respectively. Nuclear nificantly longer distances than those in the training group DNA (b-actin) forward and reverse primers were 5 - at 2, 3 and 4 weeks after treatment (p \ 0.05, n = 8in 0 0 GCG GTG ACC ATA GCC CTC TTT-3 and 5 -TGC each group). CAC TCC CAA AGT AAA GGG TCA-3 , respectively. Figure 2b shows the running distances and speeds at Primers were purchased from Invitrogen (Carlsbad, CA, 5-min intervals over 30 min on the final day. In the first USA). stage (0–10 min), the running performance did not differ between the training and CO groups. However, Morphometric evaluation of capillary density the CO group had progressively increased speed and ran a longer distance, and the running performance of Skeletal muscle tissues cut into slices along the axial plane this group was significantly higher than that of the were washed in phosphate-buffered saline and incubated in training group in the last stage (25–30 min, p \ 0.05, MitoTracker Deep Red FM (Molecular Probes, Thermo n = 8 in each group). Fisher Scientific, Carlsbad, CA, USA) and isolectin B4 (Vector Laboratories, Burlingame, CA, USA) at 37 C for Muscle weight 1 h according to the manufacturer’s protocol. Images from stained slices were captured using a BZ-8100 microscope The muscle weight and muscle weight ratio tended to be (Keyence, Osaka, Japan), as previously described [25, 26]. higher in both exercise groups than those in the control All morphometric studies were performed by an examiner group. After the 4 weeks of training, the TA muscle weight blind to the treatments. Capillary density was measured by and muscle weight ratio in the CO , training and control quantification of isolectin B4 staining (green fluorescence) groups were as follows: 0.464 ± 0.026 g and in randomly selected fields (250 9 250 lm) of muscle 0.171 ± 0.007, 0.464 ± 0.056 g and 0.173 ± 0.008, specimens using Image J software, as previously described 0.443 ± 0.016 g and 0.161 ± 0.005, respectively. There- [25, 26]. fore, activity-wheel running exercise for 30 min for 5 days/ Change of running distance Distance covered (m) J Physiol Sci (2018) 68:463–470 467 Fig. 3 a ATPase staining in the deep layer of the tibialis anterior muscles of the control, training, and CO groups Capillary density and mitochondrial number week for 4 weeks led to increased muscle weight; however, there were no significant differences between the groups in muscle (p = 0.08, n = 8 in each group). The mean capillary density in the CO group was signifi- Muscle fibre type cantly higher than that in the control group (Fig. 5a, b; p \ 0.05, n = 4 in each group). Moreover, the amount of The mean percentage of type IIB fibres decreased while mitochondrial DNA in the CO group was significantly higher than that in the control group, as determined by real- that of type IID and/or IIA fibres increased in both the CO and training groups. There were significant differences in time PCR (Fig. 6; p \ 0.05, n = 4 in each group). The results showed that although there were differences in the the percentages of IIA, IID and IIB fibres between the control and CO groups (Figs. 3, 4a, b; p\ 0.01, n = 6in number of muscle mitochondria and the capillary density each group). These results indicated that in comparison between the training and the control groups as well as with non-transcutaneous CO delivery, transcutaneous CO 2 2 delivery following pre-training led to the change of a sig- (a) nificantly higher percentage of muscle fibres from type IIB to type IIA. (a) (b) 125% (b) ** Training Control ** CO 100% 75% ** 50% ** ** 25% ** Control Training CO 0% IIB IID IIA Fig. 5 a Staining for mitochondria and capillaries in the deep layer of Fig. 4 a Electrophoretic analysis of myosin heavy chain isoforms. the tibialis anterior muscles of the control, training, and CO groups. b The control group primarily expresses IIB fibres, as determined by b Neovascularisation in the CO group, as determined by the the semi-quantification of the band, whereas the CO group mainly quantification of capillary density, was significantly higher than that expresses IIB, IIA, and IID fibres. *p \ 0.05; **p \ 0.01 in the control group. *p \ 0.05 Percentage of muscle fibers Capillary Density (%) 468 J Physiol Sci (2018) 68:463–470 ischaemic diseases [31, 32], and Raynaud’s phenomenon [33]. However, the mechanism and potential of CO as a therapeutic modality are not well known. Moreover, CO concentration of saturated CO -enriched water is only 0.1%, and there is little evidence of CO absorption into the human body. Therefore, we previously used near-infrared spectroscopy to demonstrate that a novel transcutaneous 4 application of CO upregulated O pressure in the local 2 2 tissue and showed, for the first time, that our transcuta- neous CO system could cause absorption of CO and the 2 2 Bohr effect in the human body [9] and suggested that these results might be applicable in human cases. Our findings suggest that CO enhances the performance Control Training CO of endurance exercise in rats. Moreover, we previously demonstrated that CO activated PGC-1a gene expression Fig. 6 Real-time PCR analysis of mitochondrial DNA copy number. and increased the endurance potential in skeletal muscles The mitochondrial number was higher in the CO group than in the control group. *p \ 0.05 [10]. Together with the results presented herein, these findings strongly suggest that CO therapy might improve between the CO and the training groups, these were not endurance. Moreover, several studies demonstrated that statistically significant. endurance exercise induced PGC-1a gene expression via 2? Ca influx and AMPK pathway activation [11, 34]. Based on our previous study showing that transcutaneous CO 2? Discussion delivery induced Ca influx into cells, these results alto- gether suggest that endurance exercise improves perfor- 2? In the present study, running performance evaluated at 2, 3 mance by CO -mediated PGC-1a upregulation and Ca and 4 weeks after intervention was significantly higher in influx. rats that received transcutaneous CO than that in rats that Aerobic metabolism occurs in mitochondria [1]. The were trained but did not receive treatment. This difference number of mitochondria increases as a result of upregula- might be due to an increase in muscle weight, change in tion of PGC-1a and skeletal muscle activity during aerobic muscle fibre type from IIB to IIA and/or IID and increase exercise or recovery from muscle injury [35, 36]. Skeletal in capillary density and mitochondrial number within the muscle includes fast (white) muscle fibres that provide muscle as a result of CO treatment. Muscle fibres usually instant power and slow (red) muscle fibres that provide switch from type IIB to type IID and/or IIA fibres as a endurance. Exercise training was extensively shown to result of training [10]. However, in the present study, increase the mitochondrial content of skeletal muscles training after transcutaneous CO treatment resulted in a 2 [11, 35, 36]; however, the underlying mechanisms direct- higher percentage of TA muscle fibres changing to type IIA ing this adaptive response are incompletely understood. and/or IID when compared with animals that were trained Increases in mitochondrial function require complex without CO application. Type IIA, IID and IIB fibres coordination of genes encoded by nuclear and mitochon- differ in ATP production, mitochondrial number and cap- drial DNA [37, 38]. PGC-1a appears to play an integral illary density. In general, in skeletal muscles, type IIA and role in regulating the transcription of mitochondrial and IID fibres have a higher capacity for ATP production and nuclear genes [37, 38]. Our previous results suggested that higher mitochondrial numbers and capillary density com- transcutaneous CO application increased nuclear PGC-1a pared with type IIB fibres [22, 23]. Transcutaneous CO protein and induced mitochondrial biogenesis [10, 38]. delivery led to increased mitochondrial number and cap- Therefore, an increase in mitochondrial number by tran- illary density, providing evidence that this therapeutic scutaneous CO delivery might lead to a significant approach might potentially aid in the performance of increase in aerobic capacity. endurance exercise. CO therapy is supported by some scientific evidence. Gaseous molecules control various cellular signalling One is that CO increases blood flow, and our transcuta- pathways in the body [27–29]. CO is an important gaseous neous CO application has shown increased blood flow in molecule with well-defined roles [6, 30], including those the deep tissue [9, 21]. There is a relationship between related to nitric oxide [30]. In addition, CO therapies, blood flow volume and muscle fatigue, and increased blood including carbonate spa and artificial CO -enriched water, flow reduced muscle fatigue [39]. Therefore, there may be are used in many medical conditions, such as cardiac, a possibility that transcutaneous CO application might Relative mitochondrial DNA levels J Physiol Sci (2018) 68:463–470 469 Compliance with ethical standards increase the performance of endurance exercise via the reduction of muscle fatigue. Another piece of evidence is Conflict of interest The CO hydro-gel used in this study was pro- that this model of CO therapy accelerates muscle injury vided by NeoChemir Inc.; it is patented by NeoChemir Inc. (inter- repair in rat models [40]. This research was able to explain national publication number WO2004/002393; publication date, 8 January, 2004). In addition, the use of CO delivery for muscle that this form of CO therapy may increase next-day per- 2 strengthening is patented by National University Corporation Kobe formance via muscle injury repair. University and NeoChemir Inc. (international publication number This study includes several limitations. First, our WO2009/054501; date, 30 April 2009). experimental design did not include a group that received Funding This study was supported by grants from the Division of CO but no training. However, in our previous study, we Rehabilitation Medicine and Department of Orthopaedic Surgery, demonstrated that transcutaneous CO in the absence of Kobe University Graduate School of Medicine, and a Grant-in-Aid exercise training upregulated PGC-1a gene expression and for Scientific Research (C) from the Japan Society for the Promotion promoted muscle fibre changing to increase endurance in of Science (25350814 to YS). skeletal muscles [10]. Therefore, the present study focused Ethical approval All procedures performed in studies involving on whether CO enhanced endurance exercise perfor- animals were in accordance with the ethical standards of the institu- mance. Second, only TA muscle was analysed at the end of tion or practice at which the studies were conducted. the 4-week experiment. Most studies investigating endur- ance exercise in rat models use longer durations such as 45 days [41] and 10 weeks [42]. Because the duration of References our study was shorter than those of previous studies, there was little change in muscle fibres of the training group. 1. Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P (2014) Molecular biology of the cell. Garland Science, However, this research aimed to assess the role of the CO New York, pp 753–812 effect. Compared with the control group, the CO group 2. 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Increase in carbon dioxide accelerates the performance of endurance exercise in rats

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Springer Journals
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Copyright © 2017 by The Physiological Society of Japan and Springer Japan KK
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Biomedicine; Human Physiology; Neurosciences; Animal Biochemistry; Animal Physiology; Cell Physiology; Neurobiology
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10.1007/s12576-017-0548-6
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Abstract

J Physiol Sci (2018) 68:463–470 https://doi.org/10.1007/s12576-017-0548-6 ORIGINAL PAPER Increase in carbon dioxide accelerates the performance of endurance exercise in rats 1,2 3,4 3 5 • • • • Takeshi Ueha Keisuke Oe Masahiko Miwa Takumi Hasegawa 3 3 3 3 • • • • Akihiro Koh Hanako Nishimoto Sang Yang Lee Takahiro Niikura 1,3 1,3 1 • • Masahiro Kurosaka Ryosuke Kuroda Yoshitada Sakai Received: 16 December 2016 / Accepted: 31 May 2017 / Published online: 10 June 2017 The Physiological Society of Japan and Springer Japan KK 2017 Abstract Endurance exercise generates CO via aerobic Keywords Activity wheel  Carbon dioxide  Endurance metabolism; however, its role remains unclear. Exogenous exercise  Running performance CO by transcutaneous delivery promotes muscle fibre-type switching to increase endurance power in skeletal muscles. Here we determined the performance of rats running in Introduction activity wheels with/without transcutaneous CO exposure to clarify its effect on endurance exercise and recovery from All animals generate energy by consuming oxygen (O ) muscle fatigue. Rats were randomised to control, training and and releasing carbon dioxide (CO ). Aerobic metabolism is CO groups. Endurance exercise included activity-wheel performed with O for energy production in the mito- 2 2 running with/without transcutaneous CO delivery. Running chondria [1, 2] using not only carbohydrates but also amino performance was measured after exercise initiation. We also acids and fatty acids [1, 3]. CO generated during aerobic analysed changes in muscle weight and muscle fibres in the metabolism is not merely a waste product but serves other tibialis anterior muscle. Running performance improved over important functions. the treatment period in the CO group, with a concomitant One role of CO is vasodilation, with important impli- 2 2 switch in muscle fibres to slow-type. The mitochondrial DNA cations in central nervous system function [4] and physi- content and capillary density in the CO group increased. CO ology [5]. CO also exerts the Bohr effect on haemoglobin 2 2 2 was beneficial for performance and muscle development in red blood cells [6, 7], which have high levels of carbonic during endurance exercise: it may enhance recovery from anhydrase that ionises CO to carbonate and hydrogen ions fatigue and support anabolic metabolism in skeletal muscles. [8]. Accumulating hydrogen ions dissociate O from oxy- haemoglobin. Thus, tissues with increased CO content can acquire necessary O via the Bohr effect. Importantly, the Mr. Takeshi Ueha and Dr. Keisuke Oe contributed equally to this Bohr effect can be achieved via exogenous CO [9]. work. 2 Moreover, we previously showed that transcutaneous CO & Yoshitada Sakai delivery to the hind limbs of rats upregulated peroxisome [email protected] proliferator-activated receptor-gamma coactivator-1a (PGC-1a) and vascular endothelial growth factor (VEGF) Division of Rehabilitation Medicine, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo- expression as well as the number of mitochondria. Fur- ku, Kobe 650-0017, Japan thermore, this approach promoted a change in muscle fibre NeoChemir Inc, Kobe, Japan type from IIB to IIA, similar to that observed in tibialis anterior (TA) muscles after aerobic exercise [10]. A recent Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan study also suggested that carbonate ion-mediated PGC-1a upregulation might promote a fast-to-slow fibre-type shift Department of Orthopaedic Surgery, Hyogo Prefectural Awaji Hospital, Awaji, Japan in muscle cells [11]. Aerobic exercise is an actively performed exercise Department of Oral and Maxillofacial Surgery, Kobe that utilises aerobic metabolism [2, 12]; endurance University Graduate School of Medicine, Kobe, Japan 123 464 J Physiol Sci (2018) 68:463–470 exercise is typically referred to as aerobic exercise Measurement of running distance [12, 13]. Repeated performance of endurance exercise increases aerobic metabolism via a positive-feedback We measured the total running distance during exercise loop along with transformation of muscle fibres and every day to analyse running performance. Data for the running distance were expressed as a percentage of running subsequent muscle endurance [12]. Studies show that the upregulation of PGC-1a gene expression plays a major distance during pre-training. To evaluate time-dependent change, we recorded the role in muscle fibre transformation [14–16]. PGC-1a in muscles not only increases the transcriptional activity of distance run at 5-min intervals on the final day (day 28). Data were presented as average in the CO group compared the mitochondrial transcriptional factor A (TFAM) but also induces capillary density by activating VEGF. As a with those in the training group. result, slow-twitch muscle fibres shift, in order, to type IIB, IID, IIA and I fibres; consequently, muscle endur- Activity wheels ance is increased and endurance exercise performance is enhanced [17, 18]. Activity wheels were purchased from Hoei Kinzoku Kogyo The responses to exogenous CO and endurance (Aichi, Japan), as previously described [19, 20]. The wheels measured 1 m in circumference and 14 cm in exercise are similar, and include changes, such as mitochondrial biogenesis, in muscle fibers following depth. We measured the initial torques using torque-mea- suring equipment (Sugisaki Meter Co., Ltd, Ibaraki, Japan); increase in PGC-1a, and increase in capillary density by activating VEGF. However, the relationship between the initial torque was less than 1 mN (Fig. 1b). The wheels endurance exercise and exogenous CO remains unclear. had a magnetic counting system that recorded the number Therefore, we hypothesised that the application of of rotations to determine the running distance. exogenous CO may support exercise endurance and evaluated whether transcutaneous CO application after CO treatment endurance exercise increased endurance performance and aerobic capacity in rats. Supplementing CO therapy Transcutaneous CO absorption-enhancing hydrogel was 2 2 during endurance exercise may be beneficial for long- provided by NeoChemir Inc. (Kobe, Japan) as previously described [9, 10, 21]. Briefly, all animals in the three distance runners as well as patients with reduced muscle capacity for endurance who might require postoperative groups were anaesthetised with diethyl ether (Wako Pure Chemical Industries, Osaka, Japan), hair on their hind rehabilitation. limbs was shaved, and CO hydrogel was applied to their hind limbs. The area of skin covered with the CO hydrogel Materials and methods was sealed with an adaptor. In only the CO group rats, 100% CO gas (Kobe Sanso KK, Kobe, Japan) was flowed Animal care and experimental design into the area for 10 min, as previously described [10, 21]. The CO group underwent this treatment every time after The use of animals was approved by the Animal Care and the training. Use Committee of Kobe University Graduate School of The expected mechanism of the transcutaneous CO application was as follows. Because of its relatively high Medicine (approval number, P100408). A total of 24 male Wistar rats that were 5 weeks old (CLEA Japan, Tokyo, solubility, CO gas dissolves in the hydrogel. The dissolved CO then permeates the skin, moving down its concentra- Japan) were used for all experiments. Animals were fed ad libitum and housed in a thermostatic environment at tion gradient when the maximum solubility in the hydrogel 21 C under a 12-h light/12-h dark cycle. is reached. Our previous study showed that CO penetrated As depicted in Fig. 1a, rats were initially divided into human tissue by this system [9]. two groups, exercise (n = 16) and no exercise (control group; n = 8), and further divided according to average Muscle isolation and preparation body weight. All rats were pre-trained using a 5-day pro- gramme that comprised activity-wheel running for 30 min Animals were sacrificed with an overdose of the anaes- every day. On the 6th day, rats in the exercise group were thetic pentobarbital at 24 h after final exercise and/or treatment, followed by decapitation and dissection of intact then randomly assigned to one of the two experimental groups: CO treatment (CO group, n = 8) and no treat- TA muscles in order to analyse changes in muscle fibre 2 2 types. Muscles were then weighed with an electronic bal- ment (training group, n = 8). Both experimental groups were subdivided according to average running distance and ance (A&D, Japan) after removing excess connective tis- body weight. sue, immediately frozen in isopentane precooled with 123 J Physiol Sci (2018) 68:463–470 465 (a) Four weeks After first week The first week Measuring Body weight Anesthesia n = 8 Control group n = 8 Pre- n = 8 Training group training n = 16 CO group n = 8 CO Activity wheel 30 min 10 min Magnetic sensor (b) Closing door with lock Activity wheel Fig. 1 a Experimental design to test the impact of transcutaneous CO (left). The initial torques measured by using torque-measuring equipment delivery on endurance exercise performance. b Photograph of a male (right) Wistar rat running in an activity wheel equipped with a magnetic sensor liquid nitrogen and stored at -80 C. Serial 10-lm-thick electrophoresis, as previously described [10, 22, 23]. After transverse sections were prepared from each block. Muscle electrophoresis, gels were stained using the Wako Silver weight ratio was calculated by the following equation: Stain Kit (Wako Pure Chemical Industries), and images of muscle weight ratio = muscle weight/body weight 9 100. MHC bands were captured with a LAS-3000 imager to quantify band intensities using ImageJ software (https:// ATPase staining imagej.nih.gov/ij/). Changes in MHC isoform expression among the three groups were presented as percent changes Unfixed frozen TA muscles were sectioned into 10-lm- from pre-training values. thick slices with a cryostat, and ATPase immunostaining at pH 4.6 was performed as previously described [10, 22]. Mitochondrial DNA copy number Isolation of myosin heavy chain Genomic DNA was isolated from 10-mg transverse slices TM of mid-belly TA muscles using a GenElute Mammalian Protein was isolated from 10-mg transverse slices of the Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, mid-belly of TA muscles using urea buffer, and 5–20 llof MO, USA). Mitochondrial DNA (mtDNA) content relative homogenates were analysed for the expression of myosin to the b-actin gene copy number was measured using real- heavy chain (MHC) isoforms by two-dimensional time PCR, as previously described [10, 23]. Torque (N·m) 466 J Physiol Sci (2018) 68:463–470 Fig. 2 a Percent change in (a) (b) 300% running distance among the Training Training groups by week. *p \ 0.05 CO CO b Changes in running distances on the final day in the CO and 200% training groups. *p \ 0.05 100% 0% Before 1 week 2 week 3 week 4 week 0 5 10 15 20 25 30 Endurance running Time (min) Quantitative real-time PCR Statistical analysis Quantification of mtDNA was performed using a Ste- The level of statistical significance was set at p \ 0.05. TM pOne Real-Time PCR System (Applied Biosystems). Data were expressed as mean ± standard deviation. Sig- Real-time PCR reactions (20 ll) contained 0.5 lMeach nificance was determined via analysis of variance of forward and reverse primers, 1 ll genomic DNA (ANOVA) and the two-tailed Mann–Whitney U test. template from the reverse transcription reaction and 10 ll Power SYBER Green PCR Master Mix (Applied Biosystems). Reaction conditions were 10 min at 95 C, Results followed by 40 cycles at 95 C(15 s) and60 C(1 min). mtDNA copy numbers were normalised to nuclear DNA Running distance copy numbers and compared among groups (DDCt method; Applied Biosystems), as previously described As shown in Fig. 2a, although the running distance was [10, 24]. The following forward and reverse primers longer in the CO group than in the training group after were used to determine mtDNA copy numbers: 5 -ACA 1 week, the difference was not statistically significant. 0 0 CCA AAA GGA CGA ACC TG-3 and 5 -ATG GGG However, rats that received transcutaneous CO ran sig- AAG AAG CCC TAG AA-3 , respectively. Nuclear nificantly longer distances than those in the training group DNA (b-actin) forward and reverse primers were 5 - at 2, 3 and 4 weeks after treatment (p \ 0.05, n = 8in 0 0 GCG GTG ACC ATA GCC CTC TTT-3 and 5 -TGC each group). CAC TCC CAA AGT AAA GGG TCA-3 , respectively. Figure 2b shows the running distances and speeds at Primers were purchased from Invitrogen (Carlsbad, CA, 5-min intervals over 30 min on the final day. In the first USA). stage (0–10 min), the running performance did not differ between the training and CO groups. However, Morphometric evaluation of capillary density the CO group had progressively increased speed and ran a longer distance, and the running performance of Skeletal muscle tissues cut into slices along the axial plane this group was significantly higher than that of the were washed in phosphate-buffered saline and incubated in training group in the last stage (25–30 min, p \ 0.05, MitoTracker Deep Red FM (Molecular Probes, Thermo n = 8 in each group). Fisher Scientific, Carlsbad, CA, USA) and isolectin B4 (Vector Laboratories, Burlingame, CA, USA) at 37 C for Muscle weight 1 h according to the manufacturer’s protocol. Images from stained slices were captured using a BZ-8100 microscope The muscle weight and muscle weight ratio tended to be (Keyence, Osaka, Japan), as previously described [25, 26]. higher in both exercise groups than those in the control All morphometric studies were performed by an examiner group. After the 4 weeks of training, the TA muscle weight blind to the treatments. Capillary density was measured by and muscle weight ratio in the CO , training and control quantification of isolectin B4 staining (green fluorescence) groups were as follows: 0.464 ± 0.026 g and in randomly selected fields (250 9 250 lm) of muscle 0.171 ± 0.007, 0.464 ± 0.056 g and 0.173 ± 0.008, specimens using Image J software, as previously described 0.443 ± 0.016 g and 0.161 ± 0.005, respectively. There- [25, 26]. fore, activity-wheel running exercise for 30 min for 5 days/ Change of running distance Distance covered (m) J Physiol Sci (2018) 68:463–470 467 Fig. 3 a ATPase staining in the deep layer of the tibialis anterior muscles of the control, training, and CO groups Capillary density and mitochondrial number week for 4 weeks led to increased muscle weight; however, there were no significant differences between the groups in muscle (p = 0.08, n = 8 in each group). The mean capillary density in the CO group was signifi- Muscle fibre type cantly higher than that in the control group (Fig. 5a, b; p \ 0.05, n = 4 in each group). Moreover, the amount of The mean percentage of type IIB fibres decreased while mitochondrial DNA in the CO group was significantly higher than that in the control group, as determined by real- that of type IID and/or IIA fibres increased in both the CO and training groups. There were significant differences in time PCR (Fig. 6; p \ 0.05, n = 4 in each group). The results showed that although there were differences in the the percentages of IIA, IID and IIB fibres between the control and CO groups (Figs. 3, 4a, b; p\ 0.01, n = 6in number of muscle mitochondria and the capillary density each group). These results indicated that in comparison between the training and the control groups as well as with non-transcutaneous CO delivery, transcutaneous CO 2 2 delivery following pre-training led to the change of a sig- (a) nificantly higher percentage of muscle fibres from type IIB to type IIA. (a) (b) 125% (b) ** Training Control ** CO 100% 75% ** 50% ** ** 25% ** Control Training CO 0% IIB IID IIA Fig. 5 a Staining for mitochondria and capillaries in the deep layer of Fig. 4 a Electrophoretic analysis of myosin heavy chain isoforms. the tibialis anterior muscles of the control, training, and CO groups. b The control group primarily expresses IIB fibres, as determined by b Neovascularisation in the CO group, as determined by the the semi-quantification of the band, whereas the CO group mainly quantification of capillary density, was significantly higher than that expresses IIB, IIA, and IID fibres. *p \ 0.05; **p \ 0.01 in the control group. *p \ 0.05 Percentage of muscle fibers Capillary Density (%) 468 J Physiol Sci (2018) 68:463–470 ischaemic diseases [31, 32], and Raynaud’s phenomenon [33]. However, the mechanism and potential of CO as a therapeutic modality are not well known. Moreover, CO concentration of saturated CO -enriched water is only 0.1%, and there is little evidence of CO absorption into the human body. Therefore, we previously used near-infrared spectroscopy to demonstrate that a novel transcutaneous 4 application of CO upregulated O pressure in the local 2 2 tissue and showed, for the first time, that our transcuta- neous CO system could cause absorption of CO and the 2 2 Bohr effect in the human body [9] and suggested that these results might be applicable in human cases. Our findings suggest that CO enhances the performance Control Training CO of endurance exercise in rats. Moreover, we previously demonstrated that CO activated PGC-1a gene expression Fig. 6 Real-time PCR analysis of mitochondrial DNA copy number. and increased the endurance potential in skeletal muscles The mitochondrial number was higher in the CO group than in the control group. *p \ 0.05 [10]. Together with the results presented herein, these findings strongly suggest that CO therapy might improve between the CO and the training groups, these were not endurance. Moreover, several studies demonstrated that statistically significant. endurance exercise induced PGC-1a gene expression via 2? Ca influx and AMPK pathway activation [11, 34]. Based on our previous study showing that transcutaneous CO 2? Discussion delivery induced Ca influx into cells, these results alto- gether suggest that endurance exercise improves perfor- 2? In the present study, running performance evaluated at 2, 3 mance by CO -mediated PGC-1a upregulation and Ca and 4 weeks after intervention was significantly higher in influx. rats that received transcutaneous CO than that in rats that Aerobic metabolism occurs in mitochondria [1]. The were trained but did not receive treatment. This difference number of mitochondria increases as a result of upregula- might be due to an increase in muscle weight, change in tion of PGC-1a and skeletal muscle activity during aerobic muscle fibre type from IIB to IIA and/or IID and increase exercise or recovery from muscle injury [35, 36]. Skeletal in capillary density and mitochondrial number within the muscle includes fast (white) muscle fibres that provide muscle as a result of CO treatment. Muscle fibres usually instant power and slow (red) muscle fibres that provide switch from type IIB to type IID and/or IIA fibres as a endurance. Exercise training was extensively shown to result of training [10]. However, in the present study, increase the mitochondrial content of skeletal muscles training after transcutaneous CO treatment resulted in a 2 [11, 35, 36]; however, the underlying mechanisms direct- higher percentage of TA muscle fibres changing to type IIA ing this adaptive response are incompletely understood. and/or IID when compared with animals that were trained Increases in mitochondrial function require complex without CO application. Type IIA, IID and IIB fibres coordination of genes encoded by nuclear and mitochon- differ in ATP production, mitochondrial number and cap- drial DNA [37, 38]. PGC-1a appears to play an integral illary density. In general, in skeletal muscles, type IIA and role in regulating the transcription of mitochondrial and IID fibres have a higher capacity for ATP production and nuclear genes [37, 38]. Our previous results suggested that higher mitochondrial numbers and capillary density com- transcutaneous CO application increased nuclear PGC-1a pared with type IIB fibres [22, 23]. Transcutaneous CO protein and induced mitochondrial biogenesis [10, 38]. delivery led to increased mitochondrial number and cap- Therefore, an increase in mitochondrial number by tran- illary density, providing evidence that this therapeutic scutaneous CO delivery might lead to a significant approach might potentially aid in the performance of increase in aerobic capacity. endurance exercise. CO therapy is supported by some scientific evidence. Gaseous molecules control various cellular signalling One is that CO increases blood flow, and our transcuta- pathways in the body [27–29]. CO is an important gaseous neous CO application has shown increased blood flow in molecule with well-defined roles [6, 30], including those the deep tissue [9, 21]. There is a relationship between related to nitric oxide [30]. In addition, CO therapies, blood flow volume and muscle fatigue, and increased blood including carbonate spa and artificial CO -enriched water, flow reduced muscle fatigue [39]. Therefore, there may be are used in many medical conditions, such as cardiac, a possibility that transcutaneous CO application might Relative mitochondrial DNA levels J Physiol Sci (2018) 68:463–470 469 Compliance with ethical standards increase the performance of endurance exercise via the reduction of muscle fatigue. Another piece of evidence is Conflict of interest The CO hydro-gel used in this study was pro- that this model of CO therapy accelerates muscle injury vided by NeoChemir Inc.; it is patented by NeoChemir Inc. (inter- repair in rat models [40]. This research was able to explain national publication number WO2004/002393; publication date, 8 January, 2004). In addition, the use of CO delivery for muscle that this form of CO therapy may increase next-day per- 2 strengthening is patented by National University Corporation Kobe formance via muscle injury repair. University and NeoChemir Inc. (international publication number This study includes several limitations. First, our WO2009/054501; date, 30 April 2009). experimental design did not include a group that received Funding This study was supported by grants from the Division of CO but no training. However, in our previous study, we Rehabilitation Medicine and Department of Orthopaedic Surgery, demonstrated that transcutaneous CO in the absence of Kobe University Graduate School of Medicine, and a Grant-in-Aid exercise training upregulated PGC-1a gene expression and for Scientific Research (C) from the Japan Society for the Promotion promoted muscle fibre changing to increase endurance in of Science (25350814 to YS). skeletal muscles [10]. Therefore, the present study focused Ethical approval All procedures performed in studies involving on whether CO enhanced endurance exercise perfor- animals were in accordance with the ethical standards of the institu- mance. Second, only TA muscle was analysed at the end of tion or practice at which the studies were conducted. the 4-week experiment. Most studies investigating endur- ance exercise in rat models use longer durations such as 45 days [41] and 10 weeks [42]. Because the duration of References our study was shorter than those of previous studies, there was little change in muscle fibres of the training group. 1. Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P (2014) Molecular biology of the cell. Garland Science, However, this research aimed to assess the role of the CO New York, pp 753–812 effect. Compared with the control group, the CO group 2. 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The Journal of Physiological Sciences – Springer Journals

Published: Jun 10, 2017

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