Constitutive expression of Yes-associated protein (Yap) in adult skeletal muscle fibres induces muscle atrophy and myopathy

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Weight loss, kyphosis and muscle wasting in MCK-tTA-hYAP1 S127A mice following transgene activation.

<p>A) Changes in body weight of transgenic mice following doxycycline withdrawal. Dotted red lines indicate the weight of MCK-tTA-hYAP1 S127A mice after doxycycline withdrawal. The black circles represent the mean of mice that remained on doxycycline. B) Gross phenotype of MCK-tTA-hYAP1 S127A versus control mice. C) Skeletal muscle weights of control and transgenic mice following 3 weeks (n = 7) and 5–7 weeks (n = 15) of hYAP1 S127A transgene expression. D) Organ weights taken from control and MCK-tTA-hYAP1 S127A mice (n = 8). All values present mean ±SEM and displayed as percentage change from time point 0 (A) or control (D) or displayed as raw values (C). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

No alteration in muscle fibre type proportions following over expression of hYAP1 S127A in vivo.

<p>A+B) hYAP1 S127A transgene expression was induced by doxycycline (dox) withdrawal for 5–7 weeks before TA muscles were collected, sectioned and subjected to ATPase histochemical staining following a pH10.5 pre-incubation. C+D) Quantification of the proportion of Type IIa/x (dark) and Type IIb (light/no stain) fibres in the deep and superficial portions of muscle sections. Values present mean ±SEM (n = 10) and displayed as percentage of total fibres per image from 3 representative images per mouse. Scale Bar = 200 µm.</p

Characterisation of MCK-tTA-hYAP1 S127A mouse model.

<p>A) Schematic outline of the inducible ‘Tet-Off’ transgenic system. B) Genotyping of MCK-tTA and TRE-hYAP1 S127A alleles in single and double transgenic mice. C) Yap protein levels in skeletal muscles of transgenic mice of indicated genotypes following 25 days with (+) or without (−) doxycycline (dox). Tibialis anterior (TA), gastrocnemius (Gas) and extensor digitorum longus (EDL). D) Muscle specific expression of hYAP mRNA in MCK-tTA-hYAP1 S127A mice 25 days after doxycycline withdrawal. E) Time course of Yap protein expression in tibialis anterior of MCK-tTA-hYAP1 S127A mice following doxycycline removal.</p

Re-administration of doxycycline partially rescues muscle phenotype induced by hYAP1 S127A expression.

<p>hYAP1 S127A transgene expression was induced for 5 weeks and then stopped by the re-administration of doxycycline (dox) for 5 weeks (hYAP1 S127A rescued). A) Weekly changes in mouse body weight throughout trial B) Western blot of Yap protein expression from control and hYAP1 S127A rescued mice. C) Quantification of TA, GAS, EDL muscle weights from control and hYAP1 S127A rescued mice. D+E) Representative images of NADH-TR stained TA cross sections of control and hYAP1 S127A rescued mice. F+G) Representative images of H+E staining from TA cross sections of control and hYAP1 S127A rescued mice. E) Quantification of centrally located nuclei. F) Western blot of Yap protein expression from control and hYAP1 S127A rescued mice. Values present mean ±SEM (n = 6). Weights are displayed as percentage change from control. Central nuclei are displayed as the percentage of muscle fibres that had central nuclei. Scale bars = 100 µm.</p

Up regulation in genes associated with muscle regeneration and atrophy in MCK-tTA-hYAP1 S127A mice.

<p>TA muscles of transgenic mice were harvested following doxycycline withdrawal at indicated time points and RNA processed for qRT-PCR analysis of A) hYAP1 mRNA, B) embryonic myosin heavy chain, C) Myf5 mRNA, D) Pax7 mRNA, E) myogenin mRNA, F) Caspase-3 mRNA, G) Atrogin-1 mRNA and H) MuRF-1 mRNA. Expression normalised to Gapdh. All values present mean ±SEM (n = 12) and are displayed as fold change relative to control. ***P<0.001, **P<0.01, *P<0.05. Mdx Samples from a mouse model of Duchenne muscular dystrophy.</p

Constitutive expression of Yap induces skeletal muscle degeneration, necrosis, atrophy and features of myopathy.

<p>hYAP1 S127A transgene expression was induced by doxycycline (dox) withdrawal for 3 weeks or 5–7 weeks before TA muscles were collected. A & B) H+E staining of cross sections from control and MCK-tTA-hYAP1 S127A mice, revealing central nuclei (white arrows) and infiltrating cells (black arrows). C) Quantification of fibres with centrally located nuclei (n = 18). D) Quantification of muscle fibre cross sectional area (n = 16). E & F) Necrotic fibre staining of cross sections from control and MCK-tTA-hYAP1 S127A mice with fluorescently conjugated IgG. G) Quantification of necrotic fibres, displayed as % of IgG+ myofibres per section (n = 10) H) Serum creatine kinase (CK) activity in control and MCK-tTA-hYAP1 S127A mice (n = 8). I & J) Imunnofluorescent staining of embryonic myosin heavy chain (eMyHC) on cryosections from control and MCK-tTA-hYAP1 S127A mice. K, L & M) Nicotinamide adenine dinucleotide-reductionase (NADH-TR) staining of TA muscle cross sections from control and MCK-tTA-hYAP1 S127A mice (representative image from at least 10 transgenic mice, quantification based on n = 9). All values present mean ±SEM. *P<0.05 ***P<0.001. Scale Bars = 100 µm (H+E, NADH-RT), 100 µm (IgG+eMyHC).</p

A TNF receptor loop peptide mimic blocks RANK ligand–induced signaling, bone resorption, and bone loss

Activating receptor activator of NF-κB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-α–induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M-CSF–induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-α promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF

The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src−/− OCLs by Src inhibitors or by expressing dominant-negative SrcK295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization