Aquaporins: A hole in our understanding of hydrogen peroxide regulation

Lay Summary:

There has been great interest in the idea that free radicals (and other chemicals that are collectively called reactive oxygen species "ROS") contribute to loss of muscle performance and indeed a whole range of age-related disorders. We have lots of evidence that excessive production of one of these reactive species, hydrogen peroxide, can be damaging to muscle structure and function. Since loss of muscle mass and strength is a leading cause of immobility in older people in the developed world it is surprising that there are no published studies of how hydrogen peroxide passes through skeletal muscle cell membranes (sarcolemma) and no previous attempts to see if blocking such transport can preserve muscle function. In this project, we will close this fundamental knowledge gap.
In the past few years, it has become clear that the major route of transport of hydrogen peroxide through membranes in various non-muscle cells is the family of membrane protein channels called "aquaporins".

Our pilot data confirm that aquaporins facilitate hydrogen peroxide membrane transport in skeletal muscle too.

The existence of aquaporin proteins has been known for about 25 years now, but they were first thought of simply as the route of passage of water through membranes. In fact, the characterisation of these so-called "water channels" gained Prof Agre a Nobel Prize in 2003. Surprisingly, therefore, there have been only a few studies of muscle aquaporins and no studies of their role in the regulation of muscle hydrogen peroxide at all. This is perhaps because previously there had been a lack of appropriate research tools to study hydrogen peroxide in fine detail.

Our group's unique combination of novel molecular tools and muscle degeneration models now allow us to answer a number of fundamental and important biological questions about hydrogen peroxide transport in skeletal muscle.
(a) Which of the aquaporins are most important for hydrogen peroxide spread in muscles?
(b) Does aquaporin regulation of hydrogen peroxide change with age or muscle injury?
(c) Does block of muscle aquaporins promote maintenance of muscle function after neuromuscular injury?

It is often assumed that free radicals are "bad" and "antioxidants" are good and that therefore block of aquaporins or elimination of peroxide would be beneficial to muscle function, however, this is a very simplistic view and needs validating. For example, some recent data support the possibility that hydrogen peroxide transport is critical to high-performance muscle function. Furthermore, there are dozens of common dietary substances that can block some of the aquaporin channels, many of these are so-called plant "polyphenols" and, coincidentally, better known for their antioxidant properties. So we need to answer these widely important biological questions urgently so that dieticians and those in medical research can start to develop more informed treatment strategies for age and injury-related muscle loss.

Physical frailty is a common problem in older people since there is an inexorable decline in skeletal muscle mass and strength in people from the age of 50 years old. There are no interventions available that can arrest this decline; exercise increases muscle mass in people of all ages, but it does not prevent the decline in muscle mass and strength that occurs with ageing.

The biology of many aspects of this decline is well established, such as neuromuscular dysfunction, loss of motor units, dysregulation of reactive oxygen species (ROS, including H2O2), activation of NFkB signalling pathways and activation of protein degradation. Many established models of skeletal muscle ageing are built around a fundamental hole in our knowledge of skeletal muscle ROS signalling; how does H2O2 pass through both the skeletal muscle mitochondrial and sarcolemma membranes? We have shown that in common with other cell types, such transport is facilitated by aquaporin membrane channels, but this has never been studied in detail in skeletal muscle. In this proposal we can now close this knowledge gap using the newly created molecular tools and in vivo imaging techniques we have, uniquely, at our disposal including novel adeno-associated viral vectors for transfection of mature muscle, state-of-the-art fluorescent reporters and inhibitors and in vivo confocal microscopy that, together, allow real-time visualisation of H2O2 spread in mouse skeletal muscle beds.

We will quantify the mitochondrial and sarcolemmal transport of H2O2 through a range of aquaporins and determine the fraction which passes independently of aquaporins. We will then examine the effect of aquaporin block on muscle recovery from neuromuscular injury in our in vivo model.

Further details INCLUDING timelines are included in our Impact Statement attachment.

*Industry*
This is a proposal addressing some fundamental mechanistic questions in biology, but the ultimate output of this project is discovery of whether modulation of aquaporin channels can potentially be used as a means to improve recovery from neuromuscular injury and slow its decline with ageing in the mouse model. In our "Pathways to Impact" document we have therefore set-out a timeline for follow-on pre-clinical trials in association with Nordic Biosciences, collaborating with Icagen to develop novel inhibitors and enlisting our Business Gateway to begin a further partner search and draw-up confidentiality agreements.

*Training*
Training is a big part of this project. Dr Caroline Staunton is the Researcher Investigator on this proposal because she had the original concept and has assisted with the experimental design and writing of this proposal. If successful Dr Staunton would assist with the day to day running of the project and gain considerable experience and confidence in project management. We would assist her to move to independent funding by the end of this project. Integrative Physiology is one of the key skill shortages listed in the RCUK documentation and this project includes a logical progression of in vitro to in vivo ("integrative") experiments. This project is training rich; Dr Staunton already has many in vivo skills but these will be developed and allowed to flourish in the current work and have costed two additional training courses. Furthermore, we would expect to run an Institute-funded PhD studentship alongside in which Dr Staunton would be a co-supervisor. In training others she will develop her own managerial skills. Furthermore, our group will train many undergraduate students (our group typically hosts 5 to 10 undergraduate student projects every year) in areas of mathematical and integrative biology that are key skill shortages. The value of this project to the undergraduate skills base should not be overlooked since both physiology and mathematical biology are both listed as endangered biological skills by the BBSRC.

*3Rs*
Our intra-vital (in vivo) methodology allows us to track the degeneration and recovery of the same individual muscle fibres in an animal over the course of several weeks. As detailed in the Animal Ethics sections of this proposal, this design dramatically decreases the numbers of animal required and, of course it should be noted that muscle wasting with age, immobility and injury is a problem for domestic animals too so our research could be of welfare benefit to, particularly, horses, cats and dogs.

*Public Dissemination*
We will disseminate through our disadvantaged school children initiative (KIND) and the University of Liverpool Meet-The-Scientists event in Liverpool World Museum. This was set-up initially by RBJ and CS working together with other members of our group. With older children (and adults) we will run a series of demonstrations of muscle functional loss with ageing and have posters behind explaining about free radicals and aquaporins. For younger children, we typically teach them what a muscle is and how strength can be built with regular exercise. We would also link this work to our other projects, such as the experiments we are launching up to the International Space Station in 2021.