Yes-Associated Protein is up-regulated by mechanical overload and is sufficient to induce skeletal muscle hypertrophy

Abstract

Mechanically-induced skeletal muscle growth is regulated by mammalian/mechanistic target of rapamycin complex 1 (mTORC1). Yes-Associated Protein (YAP) is a mechanically-sensitive, and growth-related, transcriptional co-activator that can regulate mTORC1. Here we show that, in skeletal muscle, mechanical overload promotes an increase in YAP expression; however, the time course of YAP expression is markedly different from that of mTORC1 activation. We also show that the overexpression of YAP induces hypertrophy via an mTORC1-independent mechanism. Finally, we provide preliminary evidence of possible mediators of YAP-induced hypertrophy (e.g. increased MyoD and c-Myc expression, and decreased Smad2/3 activity and muscle ring finger 1 (MuRF1) expression).

Keywords: Hippo pathway; Mammalian/mechanistic target of rapamycin complex 1; Mechanotransduction; Synergist ablation; TEA domain; Yes-Associated Protein.

Figure 1. The effect of synergist ablation on YAP, Akt and c-Myc, and mTORC1 signaling

Plantaris muscles were subjected to synergist ablation (SA) and then collected either immediately (0 day) or up 14 days after the onset of SA. (A) The muscles were subjected to Western blot analysis with the indicated antibodies and then the Western blot membranes were stained with Coomassie blue to verify equal loading of protein in all lanes. (B) The mean time course data for the phosphorylated to total ratio (P/T) of p70^S6k1, and the amounts of phosphorylated Akt [P-Akt(T308)] and YAP. (C) Correlation between the SA-induced changes in YAP and P-Akt(T308). All values are expressed as a percentage of the mean value obtained in the control (0 day) samples and are reported as the mean + SEM, n = 3-8 / group. * Significantly different from 0 day, P ≤ 0.05.

Figure 2. Overexpressed YAP is transcriptionally active

Mouse TA muscles were cotransfected with HA-tagged YAP, or GFP as a control, as well as a TEAD binding element firefly (FF) luciferase reporter containing 8 GGAATG binding motifs (GT-IIC FF), and the pRL-SV40 Renilla (Ren) luciferase reporter. At 72 hours post-transfection, the muscles were collected and FF and Ren luciferase activities were measured with a dual-luciferase assay. Measurements of the relative light units produced by FF luciferase were normalized to that produced by Ren luciferase and this ratio is expressed as a percentage of the values obtained from the GFP transfected muscles. Values are reported as the mean + SEM, n = 3-5 / group. * Significantly different from the values obtained in GFP-transfected muscles.

Figure 3. The overexpression of YAP induces hypertrophy through an mTORC1-independent mechanism

Mouse TA muscles were transfected with LacZ as a control (A, B), HA-tagged YAP (C, D) or YFP-tagged Rheb (E, F). Immediately after transfection, the mice were given daily injections of a solvent vehicle (A, C, and E) or 1.5 mg/kg rapamycin (RAP) (B, D, and F). Muscles were collected at 7 days post-transfection and subjected to immunohistochemistry for laminin (red) and LacZ, HA or YFP (green). (A-F) Representative merged images of Laminin and (A, B) LacZ, (C, D) HA-YAP, or (E, F) YFP-Rheb. The CSA of the transfected fibers and non-transfected fibers were measured and expressed relative to the mean value obtained for the non-transfected fibers in each muscle. Histograms represent the relative CSA for the transfected fibers (green bars) and non-transfected fibers (black bars) obtained in each group (n = 363-645 transfected and n = 440-645 non-transfected fibers/group). (G) The graph represents the average relative CSA of the transfected fibers that was obtained in each group. Values are reported as the mean + SEM, n = 363-645 fibers (4-5 muscles) / group. * Significantly different from the drug-matched (vehicle or RAP) LacZ transfected fibers, P ≤ 0.05. Scale bars in the images represent a length of 50 μm.

Figure 4. YAP activates the MyoD and c-Myc promoters, inhibits the MuRF1 promoter and inhibits Smad 2/3 transcriptional activity

Mouse TA muscles were co-transfected with HA-tagged YAP, or GFP as a control, as well as the pRL-SV40 Renilla (Ren) luciferase reporter, and either the (A) MyoD (B) c-Myc or (C) MuRF1 promoter, or a (D) Smad Binding Element (SBE) firefly (FF) luciferase reporter. At 72 hours post-transfection, the muscles were collected and FF and Ren luciferase activities were measured with a dual-luciferase assay. Measurements of the relative light units produced by FF luciferase were normalized to that produced by Ren luciferase and this ratio was expressed as a percentage of the values obtained in the GFP transfected muscles. Values are reported as the mean + SEM, n = 3-4 / group. * Significantly different from the GFP-transfected muscles, P ≤ 0.05.