Aquaporin transport of hydrogen peroxide in skeletal muscle
Authors: Staunton Caroline Amy , Vasilaki Aphrodite , McArdle Anne , Jackson Malcolm J , Barrett‐Jolley Richard 
Published: June 2021

In addition to the established intracellular production of H202 by skeletal muscle it is also produced extracellularly and studies have shown that H202 levels contribute to the pathophysiology of the age‐related loss of skeletal muscle mass and function [1, 2]. Furthermore, externally applied H2O2, inhibits skeletal neurotransmission by an undefined intracellular mechanism [3]. Despite considerable advancement of our understanding of the sources, sinks and functions of H202 within skeletal muscle fibres, there have been no previous reports of how H202 is transported into skeletal muscle fibres. Recent evidence in other systems have implicated aquaporin (AQP) channels in this role. These are a family of integral membrane proteins that are often described as “diffusion facilitators” and have been shown to aid the movement of H202 across membranes in yeast, plants and some mammalian cells [3]. In this study we investigated whether AQP are also a conduit for H2O2 in muscle.To measure unbiased AQP transcript abundance we used RNAseq (150bp‐read depth >280 M clusters per lane, n=20 subjects) on muscles from young and old mice (approx. 6 and 26 months) both control and peroneal nerve crushed TA muscle. To measure H2O2 permeability, we used an AAV6‐HyPer2 transfection method that allows measurement of intracellular H202 in response to extracellular H2O2 exposure. We also used the well‐established swell assay to assess H20 permeability. The peroneal crush model was used to induce motor unit turnover as seen in ageing. RNAseq studies used TA muscle, but for in vitro assays FDB preparations were used as they are more amenable to isolation of single fibres. Primary muscle fibres were isolated from FDB of young adult mice and once dissociated, plated onto matrigel coated dishes to enable permeability assays to be carried out.RNA deep seq studies identified significant expression of 8 AQP mRNA transcripts present within murine skeletal muscle. A decrease of AQP4 and AQP1, but a significant increase in AQP3 was detected with ageing. To verify functional expression of AQP's within the muscles we measured AQP permeability with a standard swell assay [4, 5]. Challenging FDB fibres with a low osmolality solution (80mOSm) led to volume increase that was inhibited by 5 mins pre‐treatment with the established AQP blocker HgCl2 and the selective AQP1 blocker AQP1b. Similarly, extracellular H2O2, applied at physiological levels (10uM), led to an increase in intracellular H2O2 levels, recorded in terms of AAV6‐Hyper2 signal intensity. This was significantly inhibited by 5 mins pre‐treatment with AQP blockers HgCl2 (30μM), TEA (3mM) and Bumetanide (5μM).These data clearly demonstrate, for the first time, that AQPs contribute to H2O2 membrane transport within skeletal muscle. Future work aims to examine changes of these transport mechanisms with age and injury using specific AQP shRNA and the role of AQP isoforms in neuromuscular transmission.Support or Funding InformationWork funded by British MRCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.