Redox Modulation of Breathing Control: Lessons from The Effects Of The Phenothiazinium Chromophore Methylene Blue In Spontaneously Breathing Rats.
Authors: Haouzi Philippe , Sonobe Takashi 
Published: November 2023

Studies performed on the carotid bodies as well as on neurons of the central pattern generator have shown that changing their local redox environment can affect the peripheral chemoreceptor discharge and the central production of breaths. However, it is not clear how these effects can translate in vivo and whether they could be used as a way to pharmacologically manipulate breathing control.To clarify this question, we investigated the effects of a redox compound, currently approved for the treatment of methemoglobinemia, methylene Blue (MB). MB, a phenothiazinium dye which is extremely diffusible across cellular and mitochondrial membranes, is able to increase the mitochondrial activity, an observation that we have recently confirmed in vitro. indeed, MB increases VO2 in various cell culture types by more than threefold. In addition, MB can restore, within seconds, O2 consumption after poisoning with blockers of the mitochondrial complex I (Rotenone) or complex III (Antimycine). Indeed, MB has a redox potential higher than that of the most abundant reducing molecules present in cells and mitochondria (E0= 0.01 mv). It is readily reduced by NADH or NAPDH to form leucomethylene blue (LMB). As a result LMB can directly transfer up to 2 electron(s) to molecules with higher redox potentials such as the complexes of the mitochondrial electron chain. LMB is being re‐oxidized back during this process allowing a new cycle of reduction of MB into LMB.We found that infusion of MB in 9 tracheostomized spontaneously breathing urethane sedated rats provoked an increase in minute ventilation at dose as low as 1 mg/kg. Minute ventilation was stimulated in direct proportion to the dose administered. This increase in breathing was associated with a tenfold increase in the production of augmented breaths (from 0.1± 0.1/min to 0.93±0.15/min). These effects were not abolished by 100% O2. As soon as the dose of 40 mg/kg was reached, breathing was abruptly inhibited with rapid reduction in both breathing frequency and tidal volume leading to a central apnea.In all instances, the stimulation in breathing was associated with a reduction in lactate reflecting the decrease in NADH during the reduction of MB into LMB. This effect was abolished when the apnea occurred in parallel to a shift towards a more oxidative milieu.These data suggest that breathing and the production of augmented breaths are stimulated by the cyclic redox dye MB/LMB due to its effects on the mitochondrial complexes at the central level via an increase in cell/neuronal metabolism. At high dose, when the oxidative properties seems to dominate a depression of breathing is always observed. A mechanistic model on how the redox effects of MB on the mitochondrial activity could affect breathing control in presented. This observation offers an intriguing possibility of new pharmacological interventions on breathing control.Support or Funding InformationThis work has been supported by the National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), Grant Number UO1 NS097162 and 1R21NS090017‐02