In vivo dynamics of hydrogen peroxide and regulation of antioxidant gene expression in mouse skeletal muscle under cooling stimulus
Authors: Kano Ryotaro , Takeda Reo , Shirakawa Hideki , C Poole David , Kano Yutaka , Hoshino Daisuke 
Published: May 2024

Introduction: The dynamics of reactive oxygen species (ROS) in the cytosol and mitochondria function as signaling factors that regulates diverse cellular adaptations. Hydrogen peroxide (H2O2) is considered one of the most influential ROS in regulating physiological function; being a ubiquitous molecule with a long half-life and high diffusing capacity compared to other ROS (e.g., superoxide and hydroxyl radicals). Temperature changes within skeletal muscle myocytes constitute an environmental factor that modulates the expression of antioxidant-related genes. Although muscle temperature reduction by icing is a typical physical therapy modality, the relationship between cooling stimuli and in vivo H2O2 dynamics remains unclear. The purpose of this investigation was to elucidate the H2O2 dynamics in the cytosol and mitochondria induced by cooling stimuli and to determine the relationship between these dynamics and the expression of antioxidant-related genes. Specifically, we tested the hypothesis that cooling would reduce [H2O2] in intracellular compartments (cytosol and mitochondria) and suppress antioxidant-related gene expression levels. Methods: Experiment 1. in vivo H2O2 dynamics observation: Cytosolic and mitochondrial H2O2-sensitive fluorescent proteins (HyPer7, n = 6 and MLS-HyPer7, n = 9) were expressed by electroporation in the anterior tibialis muscle (TA) of male C57/BL6 mice (9-14 week of age). The TA was exposed under anesthesia, and cytosolic and mitochondrial [H2O2] were measured when the muscle temperature was reduced from 35 °C to 0 °C using in vivo bioimaging techniques. Experiment 2. Mice (9-12 week of age) were divided into an icing (ICE) group that received cooling stimulation (muscle temperature reduced from 35 °C to 13 °C and back to 35°C, total 6 sets for 60 min, n = 6) and a control (CONT) group (muscle temperature 35 °C for 60 min, n = 6). At 3 hours after temperature intervention, the TA was removed and mRNA levels were measured by Real-time PCR. Results: Experiment 1. In vivo [H2O2] in each compartment were significantly lower in the cytosol (over the range 28-6°C) and mitochondria (over the range 29-1°C) compared to 35 °C conditions. A decreasing phase of [H2O2] in both compartments was observed from 35 °C to around 13 °C, but, subsequently increasing [H2O2] were observed in the lower temperature range (i.e., 12 to 0 °C). Experiment 2: The ICE group significantly increased mRNA levels of antioxidant enzyme-related genes Nfe2l2 and HSF1 compared to CONT (1.2 ± 0.1-fold and 1.1 ± 0.1-fold, respectively, P < 0.01). On the other hand, mRNA levels of mitochondria-related genes such as PGC1α and COX4 were unchanged. Conclusions: This investigation is the first to reveal the H2O2 and related gene expression impact of cooling stimuli in an in vivo model. Cooling mouse skeletal muscle produced temperature-dependent decreases in [H2O2] that reversed with more severe cooling. In both cytosolic and mitochondrial compartments, the lowest point for [H2O2] was found to be 13°C. Repeated cooling cycles (between 35 and 13 °C) with their attendant decreasing [H2O2] elevated antioxidant-related mRNA levels. These results support that icing therapy may improve the antioxidant capacity of muscle despite decreasing muscle ROS levels. This study was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) for Grant-in-Aid for JSPS Fellows (no. JP23KJ0967) and JSPS KAKENHI Grant (No. JP20H04074). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.