Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Water channels called aquaporins (AQPs) are membrane proteins that conduct water molecules in and out of the cell. AQP4 is selectively expressed at the muscle membrane of fast twitch (type II) fibres, and one of the most abundant proteins in human brain. However, its roles in muscle and brain have remained elusive. We previously identified that AQP4 is localized by alpha-syntrophin at the cell membrane in muscle and brain (Yokota et al. 2000). More recently, we have shown that alpha-syntrophin deficient muscles exhibit impaired muscle volume regulation and muscle force recovery after osmotic shocks or treadmill exercise (Yokota et al. 2014, Nichols et al. 2015). We hypothesize that 1) AQP4 plays important roles in recovery from muscle fatigue during and after exercise, and 2) AQP4 is involved in increased cerebrospinal fluid (CSF) flow during and after exercise. The goal of this study is to decipher novel roles of AQP4 by analyzing mutant mouse models.

1. Osmotic Regulation in AQP4 Deficient Muscles
   Increased activity of fast twitch skeletal muscle fibres results in hyper-osmolality due to increased intracellular fluid lactate concentration. AQPs in other tissues are involved in cell volume regulation called regulatory volume increase (RVI) or regulatory volume decrease (RVD) upon osmotic shocks. It is still controversial whether the skeletal muscles are capable of volume regulation such as RVI and RVD in response to changes in osmotic conditions. To answer this question, we will examine if muscle volume regulation and force recovery against osmotic changes are impaired in the AQP4 deficient muscles. We will isolate muscle fibre bundles from the mutant mice, and examine the water contents by measuring the specific gravity ex-vivo. In addition, we will measure the force generated by muscle bundles after osmotic shock. We anticipate that the deficient mice show impaired muscle volume regulation and decreased force generation against osmotic shocks.

2. The role of AQP4 upon exercise in vivo
   We recently demonstrated that alpha-syntrophin deficient mice exhibit delayed recovery from treadmill exercise (Yokota et al. Muscle Nerve, 2014). First, we will examine if these changes are observed in AQP4 deficient skeletal muscles. Second, we will monitor the CSF formation in brain in the mutant mice using non-invasive near-infrared (NIR) light-scattering method, as exercises are known to naturally enhance CSF flow and AQP4 is involved in the formation of CSF in brain. In addition, we will examine the changes of AQP4 expression in muscle and brain in wild-type (WT) mice during and after exercise.
   Successful completion of these aims will clearly demonstrate that AQP4 is involved in recovery from muscle fatigue and maintenance of brain function during and after exercise. These results will provide valuable knowledge of the basic muscle physiology and brain science.