β-catenin Regulates Membrane Potential In Skeletal Muscle Cells By Regulating The α2Subunit Of Na,K-ATPase

Objective:β-catenin cytoplasmic protein is a key downstream component of the canonical (β-catenin-dependent) Wnt signaling pathway, and its function is very extensive in all tissues and systems. Muscle β-catenin has been shown to play a role in the formation of the neuromuscular junction (NMJ). Our previous studies showed that muscle-specific conditional knockout of B-catenin (HSA-β-cat-/-) results in early postnatal death in mice. Recently, the previous studies considered that early postnatal death of skeletal muscle-specific B-catenin-deficient mice mainly involved in development of NMJ. To understand the underlying mechanisms, we investigated the electrophysiological properties of muscle cells from HSA-B-cat-/-and the control mice. Furthermore, to confirm the difference above, we combined the method in vitro by transfecting a primary line of mouse myoblasts (C2C12) with small-interfering RNAs targeting B-catenin. We explored a possible cause underlying the early death and a possible mechanism underlying the depolarized RMP in the absence of muscle B-catenin, and provided additional evidence supporting a role for B-catenin in the development of the NMJs.Methods: 1. Groups:1) HSA-B-caf-/-P0mice group and6-catloxp/loxp P0mice control group, n=3-6mice per group.2) C2C12cells transfected with β-catenin siRNA259, β-catenin siRNA577, B-catenin siRNA1312and scrambled siRNA (control);2. Research methods: We generated the β-catenin mutant mice and control mice, and investigated the electrophysiological properties of muscle cells from the diaphragm, gastrocnemius, and extensor digitorum longus muscles by intracellular recording; We transfected C2C12cells and detected the RMP by whole-cell clamp recording; We detected the expression levels of the a2subunit of sodium/potassium adenosine triphosphatase (a2NKA), the a1subunit of sodium/potassium adenosine triphosphatase (a1NKA), and Kv3.4by Western blotting; We investigated the a2NKA mRNA level of muscle cells from the two group mice by real-time quantitative reverse-transcription PCR.Results:1. We found that in the absence of muscle B-catenin, the resting membrane potential (RMP) depolarized in muscle cells from the diaphragm, gastrocnemius, and extensor digitorum longus muscles;2. Furthermore, C2C12cells transfected with small-interfering RNAs targeting B-catenin, the RMP depolarized as well;3. We found that the expression levels of the a2NKA were reduced by B-catenin knockdown in vitro or deletion in vivo;4. We further confirmed that a deletion of B-catenin affects the transcriptional activity of a2NKA (mRNA).Conclusion:1. Skeletal muscle B-catenin is crucial for the maintenance of the RMP of postsynaptic skeletal muscle cells of the NMJ;2. Skeletal muscle cells lacking B-catenin induces a decreased expression of a2NKA; 3. We suggested a likely mechanism by which β-catenin regulates the RMP and affects the normal function of skeletal muscle.