23-24 – Aquaporins facilitate H2O2 transport at skeletal muscle membranes of the Neuromuscular Junction

Disruption of the neuromuscular junction (NMJ), loss of motor units and fewer muscle fibres is characteristic of sarcopenia. Excess production and aberrant regulation of reactive oxygen species (ROS), including hydrogen peroxide (H2O2) is to be key to this decline. We hypothesise that AQPs are a primary means of regulating H2O2 passage through skeletal muscle membranes at the NMJ, and that dysregulation of this transport mechanism contributes to dysregulation of neuromuscular function.
To investigate this, we have used RNA sequencing, Western blots and functional in vitro permeability assays in skeletal muscle fibres with the H2O2 reporter AAV6-cytoHyPer2 and AAV6-mitoHyPer2. Current work examines AQP8’s role in H2O2 signalling at the mitochondrial membrane, as well as utilising cell surface biotinylation assays to quantify changes in AQP surface localisation in response to exogenous changes in H2O2, to guide future studies into pharmacological modulation of AQP translocation as a novel approach for managing ROS signalling in sarcopenia.
A total of 7 AQP isoforms are present in skeletal muscle; AQP1 mRNA was significantly reduced with age and injury, and AQP4 significantly reduced post nerve injury. Pharmacological block of AQPs in isolated FDB skeletal muscle fibres significantly reduced H2O2 transport (using HgCl2, TEA and TGN020). After shRNA-AAV knock-down of AQP1 and AQP4 (91 and 97% respectively) H2O2 permeability was reduced both in terms of maximal intensity and slope.
This study emphasises the importance of AQP’s role in H2O2 permeability across the skeletal muscle plasma and mitochondrial membranes. Future studies aim to study perisynaptic Schwann cell and motor neuron cell involvement in redox signalling at the NMJ and we will use in vivo adult and aged models to assess skeletal muscle function post-AQP-shRNAs to determine the roles of AQPs on age-related neuromuscular dysfunction.