The Journal of Physiological Sciences

, Volume 68, Issue 4, pp 463–470 | Cite as

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

  • Takeshi Ueha
  • Keisuke Oe
  • Masahiko Miwa
  • Takumi Hasegawa
  • Akihiro Koh
  • Hanako Nishimoto
  • Sang Yang Lee
  • Takahiro Niikura
  • Masahiro Kurosaka
  • Ryosuke Kuroda
  • Yoshitada SakaiEmail author
Original Paper


Endurance exercise generates CO2 via aerobic metabolism; however, its role remains unclear. Exogenous CO2 by transcutaneous delivery promotes muscle fibre-type switching to increase endurance power in skeletal muscles. Here we determined the performance of rats running in activity wheels with/without transcutaneous CO2 exposure to clarify its effect on endurance exercise and recovery from muscle fatigue. Rats were randomised to control, training and CO2 groups. Endurance exercise included activity-wheel running with/without transcutaneous CO2 delivery. Running performance was measured after exercise initiation. We also analysed changes in muscle weight and muscle fibres in the tibialis anterior muscle. Running performance improved over the treatment period in the CO2 group, with a concomitant switch in muscle fibres to slow-type. The mitochondrial DNA content and capillary density in the CO2 group increased. CO2 was beneficial for performance and muscle development during endurance exercise: it may enhance recovery from fatigue and support anabolic metabolism in skeletal muscles.


Activity wheel Carbon dioxide Endurance exercise Running performance 



The authors wish to express their sincere gratitude to Professor Masanobu Wada (Graduate School of Integrated Arts and Sciences, Hiroshima University) for his excellent technical assistance in ATPase staining and myosin heavy chain isolation, and Professor Sadahiko Nakajima and Dr. Takahisa Masaki (Graduate School of Department of Literature, Psychology, Kanseigakuin University) for their excellent technical assistance in the activity-wheel running programme.

Author Contributions

YS and MM conducted all experiments. TU, KO and TH contributed to the animal experiments. TU, KO, AK and HN contributed to the biological and histological analyses. YS and SYL analysed the data. TN, RK and MK supervised all aspects of this study. TU, KO and YS wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The CO2 hydro-gel used in this study was provided by NeoChemir Inc.; it is patented by NeoChemir Inc. (international publication number WO2004/002393; publication date, 8 January, 2004). In addition, the use of CO2 delivery for muscle strengthening is patented by National University Corporation Kobe University and NeoChemir Inc. (international publication number WO2009/054501; date, 30 April 2009).


This study was supported by grants from the Division of Rehabilitation Medicine and Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, and a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (25350814 to YS).

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.


  1. 1.
    Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P (2014) Molecular biology of the cell. Garland Science, New York, pp 753–812Google Scholar
  2. 2.
    Boyadjiev N, Delchev S, Hristova M (1997) Changes in the aerobic capacity and ultrastructure of skeletal muscles and myocardium of endurance training rats induced by specialized food supplements with different compositions. Folia Med 39:20–30Google Scholar
  3. 3.
    Alberts B, Bray D, Hopkin K, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2013) Essential cell biology. Garland Science, New York, pp 425–494Google Scholar
  4. 4.
    Sato K, Sadamoto T, Hirasawa A, Oue A, Subudhi AW, Miyazawa T, Ogoh S (2012) Differential blood flow responses to CO2 in human internal and external carotid and vertebral arteries. J Physiol 590:3277–3290CrossRefGoogle Scholar
  5. 5.
    Mador MJ, Tobin MJ (1992) The effect of inspiratory muscle fatigue on breathing pattern and ventilatory response to CO2. J Physiol 455:17–32CrossRefGoogle Scholar
  6. 6.
    Bohr C, Hasselbach K, Krogh A (1904) Concerning a biologically important relationship—the influence of the carbon dioxide content of blood on its oxygen binding. Skand Arch Physiol 16:402–412CrossRefGoogle Scholar
  7. 7.
    Jensen FB (2004) Red blood cell pH, the Bohr effect, and other oxygenation-linked phenomena in blood O2 and CO2 transport. Acta Physiol Scand 182:215–227CrossRefGoogle Scholar
  8. 8.
    Swietach P, Tiffert T, Mauritz JM, Seear R, Esposito A, Kaminski CF, Lew VL, Vaughan-Jones RD (2010) Hydrogen ion dynamics in human red blood cells. J Physiol 588:4995–5014CrossRefGoogle Scholar
  9. 9.
    Sakai Y, Miwa M, Oe K, Ueha T, Koh A, Niikura T, Iwakura T, Lee SY, Tanaka M, Kurosaka M (2011) A novel system for transcutaneous application of carbon dioxide causing an “artificial Bohr effect” in the human body. PLoS One 6:e24137CrossRefGoogle Scholar
  10. 10.
    Oe K, Ueha T, Sakai Y, Niikura T, Lee SY, Koh A, Hasegawa T, Tanaka M, Miwa M, Kurosaka M (2011) The effect of transcutaneous application of carbon dioxide (CO2) on skeletal muscle. Biochem Biophys Res Commun 407:148–152CrossRefGoogle Scholar
  11. 11.
    Yamaguchi T, Suzuki T, Arai H, Tanabe S, Atomi Y (2010) Continuous mild heat stress induces differentiation of mammalian myoblasts, shifting fiber type from fast to slow. Am J Physiol Cell Physiol 298:C140–C148CrossRefGoogle Scholar
  12. 12.
    Rowe GC, Safdar A, Arany Z (2014) Running forward: new frontiers in endurance exercise biology. Circulation 129:798–810CrossRefGoogle Scholar
  13. 13.
    Ju JS, Jeon SI, Park JY, Lee JY, Lee SC, Cho KJ, Jeong JM (2016) Autophagy plays a role in skeletal muscle mitochondrial biogenesis in an endurance exercise-trained condition. J Physiol Sci 66:417–430CrossRefGoogle Scholar
  14. 14.
    Popov DV, Bachinin AV, Lysenko EA, Miller TF, Vinogradova OL (2014) Exercise-induced expression of peroxisome proliferator-activated receptor γ coactivator-1α isoforms in skeletal muscle of endurance-trained males. J Physiol Sci 64:317–323CrossRefGoogle Scholar
  15. 15.
    Iwabu M, Yamauchi T, Okada-Iwabu M, Sato K, Nakagawa T, Funata M, Yamaguchi M, Namiki S, Nakayama R, Tabata M, Ogata H, Kubota N, Takamoto I, Hayashi YK, Yamauchi N, Waki H, Fukayama M, Nishino I, Tokuyama K, Ueki K, Oike Y, Ishii S, Hirose K, Shimizu T, Touhara K, Kadowaki T (2010) Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca2+ and AMPK/SIRT1. Nature 464:1313–1319CrossRefGoogle Scholar
  16. 16.
    Scarpulla RC (2008) Nuclear control of respiratory chain expression by nuclear respiratory factors and PGC-1-related coactivator. Ann N Y Acad Sci 1147:321–334CrossRefGoogle Scholar
  17. 17.
    Yan Z, Lira VA, Greene NP (2012) Exercise training-induced regulation of mitochondrial quality. Exerc Sport Sci Rev 40:159–164PubMedPubMedCentralGoogle Scholar
  18. 18.
    Baar K (2014) Nutrition and the adaptation to endurance training. Sports Med 44:5–12CrossRefGoogle Scholar
  19. 19.
    Masaki T, Nakajima S (2008) Forward conditioning with wheel running causes place aversion in rats. Behav Processes 79:43–47CrossRefGoogle Scholar
  20. 20.
    Masaki T, Nakajima S (2006) Taste aversion in rats induced by forced swimming, voluntary running, forced running, and lithium chloride injection treatments. Physiol Behav 88:411–416CrossRefGoogle Scholar
  21. 21.
    Koga T, Niikura T, Lee SY, Okumachi E, Ueha T, Iwakura T, Sakai Y, Miwa M, Kuroda R, Kurosaka M (2014) Topical cutaneous CO2 application by means of a novel hydrogel accelerates fracture repair in rats. J Bone Joint Surg Am 96:2077–2084CrossRefGoogle Scholar
  22. 22.
    Wada M, Inashima S, Yamada T, Matsunaga S (1985) Endurance training-induced changes in alkali light chain patterns in type IIB fibers of the rat. J Appl Physiol 94:923–929CrossRefGoogle Scholar
  23. 23.
    Ennion S, Sant’ana Pereira J, Sargeant AJ, Young A, Goldspink G (1995) Characterization of human skeletal muscle fibres according to the myosin heavy chains they express. J Muscle Res Cell Motil 16:35–43CrossRefGoogle Scholar
  24. 24.
    Onishi Y, Ueha T, Kawamoto T, Hara H, Toda M, Harada R, Minoda M, Kurosaka M, Akisue T (2014) Regulation of mitochondrial proliferation by PGC-1α induces cellular apoptosis in musculoskeletal malignancies. Sci Rep 4:3916CrossRefGoogle Scholar
  25. 25.
    Tei K, Matsumoto T, Mifune Y, Ishida K, Sasaki K, Shoji T, Kubo S, Kawamoto A, Asahara T, Kurosaka M, Kuroda R (2008) Administrations of peripheral blood CD34-positive cells contribute to medial collateral ligament healing via vasculogenesis. Stem Cells 26:819–830CrossRefGoogle Scholar
  26. 26.
    Okumachi E, Lee SY, Niikura T, Iwakura T, Dogaki Y, Waki T, Takahara S, Ueha T, Sakai Y, Kuroda R, Kurosaka M (2015) Comparative analysis of rat mesenchymal stem cells derived from slow and fast skeletal muscle in vitro. Int Orthop 39:569–576CrossRefGoogle Scholar
  27. 27.
    Kajimura M, Fukuda R, Bateman RM, Yamamoto T, Suematsu M (2010) Interactions of multiple gas-transducing systems: hallmarks and uncertainties of CO, NO, and H2S gas biology. Antioxid Redox Signal 13:157–192CrossRefGoogle Scholar
  28. 28.
    Kajimura M, Nakanishi T, Takenouchi T, Morikawa T, Hishiki T, Yukutake Y, Suematsu M (2012) Gas biology: tiny molecules controlling metabolic systems. Respir Physiol Neurobiol 184:139–148CrossRefGoogle Scholar
  29. 29.
    Kashiba M, Kajimura M, Goda N, Suematsu M (2002) From O2 to H2S: a landscape view of gas biology. Keio J Med 51:1–10CrossRefGoogle Scholar
  30. 30.
    Irie H, Tatsumi T, Takamiya M, Zen K, Takahashi T, Azuma A, Tateishi K, Nomura T, Hayashi H, Nakajima N, Okigaki M, Matsubara H (2005) Carbon dioxide-rich water bathing enhances collateral blood flow in ischemic hindlimb via mobilization of endothelial progenitor cells and activation of NO-cGMP system. Circulation 111:1523–1529CrossRefGoogle Scholar
  31. 31.
    Savin E, Bailliart O, Bonnin P, Bedu M, Cheynel J, Coudert J, Martineaud JP (1995) Vasomotor effects of transcutaneous CO2 in stage II peripheral occlusive arterial disease. Angiology 46:785–791CrossRefGoogle Scholar
  32. 32.
    Toriyama T, Kumada Y, Matsubara T, Murata A, Ogino A, Hayashi H, Nakashima H, Takahashi H, Matsuo H, Kawahara H (2002) Effect of artificial carbon dioxide foot bathing on critical limb ischemia (Fontaine IV) in peripheral arterial disease patients. Int Angiol 21:367–373PubMedGoogle Scholar
  33. 33.
    Calegari VC, Zoppi CC, Rezende LF, Silveira LR, Carneiro EM, Boschero AC (2011) Endurance training activates AMP-activated protein kinase, increases expression of uncoupling protein 2 and reduces insulin secretion from rat pancreatic islets. J Endocrinol 208:257–264PubMedGoogle Scholar
  34. 34.
    Holloszy JO (1975) Adaptation of skeletal muscle to endurance exercise. Med Sci Sports 7:155–164PubMedGoogle Scholar
  35. 35.
    Holloszy JO, Booth FW (1976) Biochemical adaptations to endurance exercise in muscle. Annu Rev Physiol 38:273–291CrossRefGoogle Scholar
  36. 36.
    Scarpulla RC (2006) Nuclear control of respiratory gene expression in mammalian cells. J Cell Biochem 97:673–683CrossRefGoogle Scholar
  37. 37.
    Onishi Y, Kawamoto T, Ueha T, Kishimoto K, Hara H, Fukase N, Toda M, Harada R, Minoda M, Sakai Y, Miwa M, Kurosaka M, Akisue T (2012) Transcutaneous application of carbon dioxide (CO2) induces mitochondrial apoptosis in human malignant fibrous histiocytoma in vivo. PLoS One 7:e49189CrossRefGoogle Scholar
  38. 38.
    Legerlotz K, Elliott B, Guillemin Smith HK (2008) Voluntary resistance running wheel activity pattern and skeletal muscle growth in rats. Exp Physiol 93:754–762CrossRefGoogle Scholar
  39. 39.
    Sugaya M, Sakamaki M, Ozaki H, Ogasawara R, Sato Y, Yasuda T, Abe T (2011) Influence of decreased muscular blood flow induced by external compression on muscle activation and maximal strength after a single bout of low-intensity exercise. Jpn J Phys Educ Health Sport Sci 56:481–489. CrossRefGoogle Scholar
  40. 40.
    Akahane S, Sakai Y, Ueha T, Nishimoto H, Inoue M, Niikura T, Kuroda R (2017) Transcutaneous carbon dioxide application accelerates muscle injury repair in rat models. Int Orthop 41(5):1007–1015 (Epub ahead of print) CrossRefGoogle Scholar
  41. 41.
    Malek MH, Olfert IM, Esposito F (2010) Detraining losses of skeletal muscle capillarization are associated with vascular endothelial growth factor protein expression in rats. Exp Physiol 95:359–368CrossRefGoogle Scholar
  42. 42.
    Termin A, Staron RS, Pette D (1989) Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle. Histochemistry 92:453–457CrossRefGoogle Scholar
  43. 43.
    Ishida K, Hiruta S, Miyamura M (1988) Effects of CO2 inhalation prior to maximal exercise on physical performance. Jpn J Physiol 38:929–933CrossRefGoogle Scholar
  44. 44.
    Graham T, Wilson BA, Sample M, Van Dijk J, Bonen A (1980) The effects of hypercapnia on metabolic responses to progressive exhaustive work. Med Sci Sports Exerc 12:278–284CrossRefGoogle Scholar

Copyright information

© The Physiological Society of Japan and Springer Japan KK 2017

Authors and Affiliations

  • Takeshi Ueha
    • 1
    • 2
  • Keisuke Oe
    • 3
    • 4
  • Masahiko Miwa
    • 3
  • Takumi Hasegawa
    • 5
  • Akihiro Koh
    • 3
  • Hanako Nishimoto
    • 3
  • Sang Yang Lee
    • 3
  • Takahiro Niikura
    • 3
  • Masahiro Kurosaka
    • 1
    • 3
  • Ryosuke Kuroda
    • 1
    • 3
  • Yoshitada Sakai
    • 1
    Email author
  1. 1.Division of Rehabilitation MedicineKobe University Graduate School of MedicineKobeJapan
  2. 2.NeoChemir IncKobeJapan
  3. 3.Department of Orthopaedic SurgeryKobe University Graduate School of MedicineKobeJapan
  4. 4.Department of Orthopaedic SurgeryHyogo Prefectural Awaji HospitalAwajiJapan
  5. 5.Department of Oral and Maxillofacial SurgeryKobe University Graduate School of MedicineKobeJapan

Personalised recommendations