P&F Grant Awards

Year 11

Grant # 25

SWELL1-LRRC8 regulation of skeletal muscle metabolism and function

PI: Rajan Sah, MD, PhD


Specific Aims

Regular exercise and maintenance of muscle endurance and lean muscle mass are known to be beneficial in the prevention of obesity and obesity-related diseases such as diabetes and heart disease, in addition to promoting overall health of our aging population. Skeletal muscle atrophy is associated with cancer (cachexia), heart failure, chronic corticosteroid use, paralysis or denervation (disuse atrophy) and aging1 and can contribute to poor metabolic health. Accordingly, a deeper understanding of the molecular mechanisms that regulate skeletal muscle maintenance, growth and function is critical for human health.
It is well established that insulin/IGF1-PI3K-AKT-mTOR signaling is a critical regulator of skeletal muscle differentiation and growth1. In addition, mechanical loading of muscle, as occurs with regular activity, exercise, and resistance training is well-known to also induce mTOR-mediated skeletal muscle growth, however the cellular mechanosensor(s) responsible remain unclear. b1-integrin and focal adhesion kinase signaling has been proposed as a candidate mechanosensory complex that contributes the skeletal muscle hypertrophic signaling. Similarly, ion channels are putative mechanosensory membrane proteins that may regulate intracellular signaling.
We recently identified SWELL1 (LRRC8a) as a swell or stretchactivated volume sensor in adipocytes that regulates glucose uptake, lipid content, and adipocyte growth via a novel SWELL1-GRB2-PI3KAKT signaling pathway – providing a putative feed-forward amplifier to enhance adipocyte growth and insulin signaling during caloric excess. Our preliminary data reveal that SWELL1-LRRC8 channels are expressed and active in skeletal muscle cells, and that SWELL1 is required for intact skeletal muscle insulin-PI3K-AKT signaling. SWELL1-/- C2C12 myotubes and skeletal muscle primary muscle cells exhibit markedly impaired differentiation. Genome-wide RNA sequencing of WT compared to SWELL1-/- C2C12 myotubes reveal significant down-regulation of integrin, mTOR, IGF1, PI3K and hypertrophy signaling pathways. Moreover, Seahorse measurements reveal that cellular oxygen consumption and glycolysis is abrogated in SWELL1-/- primary skeletal muscle cells. Consistent with these cellular studies, post-developmental deletion of SWELL1 in skeletal muscle in Myl1-Cre;SWELL1fl/fl mice exhibit impaired exercise capacity upon treadmill testing. Our overarching hypothesis is that SWELL1 is a component of a mechanosensitive complex that “tunes” insulin/IGF1-PI3K-AKT-mTOR signaling to regulate skeletal muscle differentiation, growth and function. Our objective for this Pilot and Feasibility Grant is to test two potential pathways by which SWELL1 may affect muscle function in vivo: a). SWELL1 regulation of the response to aerobic training; b) SWELL1 regulation of the response to strength training. Although we hypothesize that both pathways will be SWELL1 dependent,their mechanisms of action and relevance to muscle physiology differ. The findings of these studies will guide future mechanistic exploration of the relevant pathways with the intent to ultimately potentiate muscle endurance and strength by targeting the action of SWELL1. To accomplish this, we will make use of the facilities available in the Cores to more deeply phenotype both skeletal muscle function (Musculoskeletal Structure and Strength Core) and morphology (Musculoskeletal Histology and Morphometry) in skeletal muscle-specific SWELL1 genetic loss and gain-of-function models: (1) early SWELL1 deletion (Myf5-Cre;SWELL1fl/fl), (2) late SWELL1 deletion (Myl1-Cre;SWELL1fl/fl) and (3) SWELL1 overexpression (Myf5-Cre;CAG-LSL-SWELL1-3xFlag). We have partnered with Dr. Gretchen Meyer, an expert in measuring skeletal muscle function, both in vivo and ex-vivo to provide expertise in these techniques. To achieve these goals, we propose the following two aims:

Aim 1: Determine the contribution of SWELL1 signaling to skeletal muscle growth, endurance and composition upon aerobic training. In the three genetic models described above we will perform: (a) Treadmill exercise capacity: pre- and post-treadmill training and then assess for skeletal muscle hypertrophy, fiber-type composition (histologically, transcriptionally), induction of AKT/mTOR signaling and mitochondrial biogenesis.
Aim 2: Determine the contribution of SWELL1 to skeletal muscle force generation and intracellular signaling upon strength training. As in AIM#1, we will use the skeletal muscle-targeted SWELL1 loss- and gain-of-function mice to perform in vivo strength testing pre-, immediately post and 48 hour recovery from an eccentric contraction bout. We will measure fiber size and fiber type changes (histologically, transcriptionally), and expression of genes involved in myogenic differentiation, and activation of AKT/mTOR signaling pathways.