Genomic Profiling and Physiological Approaches to Understand Aquaporins and their Role in ROS Signalling within Skeletal Muscle

Abstract

Aquaporin (AQP) channels are a family of integral membrane proteins shown to aid the movement of hydrogen peroxide (H202) across membranes [1]. Their role in skeletal muscle is of key importance since intracellular generation of H202 and other reactive oxygen species (ROS) occurs in response to muscle contractions. These ROS initiate signalling processes and activate of a number of transcription factors [2, 3]. During ageing, excessive levels of ROS have pathological effects, inducing muscle degradation, loss of neuromuscular junction integrity and loss of neuromuscular transmission.Our studies [4], as well as those of others, has identified the sources, sinks and functions of H202 in muscle but exactly how H202 is transported from its site of origin within muscle has never been examined. We hypothesize that (AQPs) are the primary means of regulating H202 movement through skeletal muscle membranes. This study has therefore examined expression of AQP isoforms in muscle and how they may change with age.RNA transcripts from Tibialis Anterior (TA) muscle from control adult (6–8 months), and old (26month) mice, and from mice following peroneal nerve crush, were examined by RNAseq (150bp‐read depth >280 M clusters per lane, n=5). Individual muscle myotubes were also treated pharmacologically with aquaporin inhibitors and transfected with AAV6‐HyPer2 to determine the role of different AQP isoforms on intracellular H202 content and H20 permeability was determined using the swell assay [5, 6].RNAseq data identified 8 AQP mRNA transcripts present in TA muscle. A decrease in AQP4 and AQP1 and an increase in AQP3 contents were found following nerve crush (n=5, P<0.01) and this was confirmed by immunological techniques. The localisation and roles of these aquaporins is currently being investigated to determine whether they have protective functions or contribute to the age‐related decline skeletal muscle.An exponential increase in intracellular H2O2 levels was recorded when AAV6‐Hyper2 transfected muscle fibres were exposed to 10μM H2O2. Pre‐treatment of muscle cells with AQP blockers HgCl2 (30μM), TEA (3mM) or Bumetanide (5μM) substantially decreased the rate of increase in intracellular H202 content. These data demonstrate the ability of aquaporins to regulate skeletal muscle H2O2 permeability. Challenging cells with an 80mOsm solution in conjunction with pharmacological AQP1 inhibitors also demonstrated a significant reduction in the rate of increase in cell volume.In summary, these data demonstrate a potential important role for AQPs in muscle. Further work using AAV‐shRNA to knockdown specific AQPs is being utilised to identify the major AQPs that function in skeletal muscle.Support or Funding InformationThis study was supported by the MRC and NIA (AG051442).