[Purpose] We reported that carbon dioxide (CO
2) water bathing accelerates skeletal muscle regeneration; however, the underlying mechanism was unclear. MyoD and myogenin play roles in muscle regeneration, and the purpose of this study was to determine changes in MyoD and myogenin caused by CO
2 water bathing after injury. [Subjects] Sixteen female Wistar rats (n = 4 per group) were used. [Methods] The rats were divided into four groups: no-injury (NI), injury (IC), injury + tap water bathing (ITW), and injury + CO
2 water bathing (ICO
2). Muscle injury was induced by injection of bupivacaine hydrochloride into the left tibial anterior (TA) muscles. Tap water and CO
2 (1,000 ppm) water bathing were performed at 37 °C for 30 minutes once a day. The left TA muscles were removed 4 days after injury, and the expressions of MyoD and myogenin were measured. [Results] MyoD and myogenin were increased in the IC, ITW, and ICO
2 groups compared with the NI group. Although the MyoD level was similar in the IC, ITW, and ICO
2 groups, myogenin increased more in the ICO
2 group than in the IC and ITW groups. [Conclusion] CO
2 water bathing after muscle injury appears to induce an increase in the expression of myogenin.
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