β2 Adrenoceptor Signaling Modulates Myoblast Differentiation via miR‐374b‐5p/GSK3β and miR‐326‐3p/Tnfrsf11a Axes

ABSTRACT Myoblast differentiation is a crucial step of skeletal muscle regeneration and is impaired in the absence of the β2 adrenoceptor (ADRβ2). Since miR‐374b and miR‐326 regulate cell differentiation, this study investigated if ADRβ2 regulates myoblast differentiation via miR‐374b‐5p and miR‐326‐3p. Primary myoblasts from ADRβ2 knockout (β2KO) mice were cultured in proliferation and differentiation media. In silico, cellular and molecular analyses were performed to evaluate NF‐κB signaling activation and validate mRNAs as miR‐374b‐5p and miR‐326‐3p targets in β2KO differentiating myoblasts. Differentiating myoblasts from β2KO mice had decreased mRNA levels of Nr4a1, Myog, Myh3, Cdkn1c, and Tnni2 and expression of miR‐374b‐5p and miR‐326‐3p. Overexpression of these miRs increased fusion index and gene expression of myogenic markers of these cells. In silico analyses indicated that GSK‐3β and receptor activator of NF‐kB (Tnfrsf11a) had potential binding sites for miR‐374b‐5p and miR‐326‐3p, respectively. The dual‐luciferase reporter assay showed that GSK‐3β and Tnfrsf11a were targets of miR‐374b‐5p and miR‐326‐3p, respectively. Regarding miR‐326‐3p, there was a reduction of NF‐κB signaling and NF‐κB mRNA levels in differentiating myoblasts from β2KO mice, and an upregulation of Tnfrsf11a and a downregulation of Tnfrsf11 . In summary, ADRβ2 modulates myoblast differentiation via miR‐374b‐5p/ GSK‐3β and miR‐326‐3p/ Tnfrsf11a axes.