Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C44
Overexpression of constitutively active Yes-associated protein (Yap) in muscle fibres of adult mice results in myopathy and is lethal after ≈40 days
R. Judson1, A. Carroll1, K. I. Watt2,1, C. Itzstein1, R. Jones1, K. Parikh1, A. Lionikas1, C. De Bari1, H. Wackerhage1
1. University of Aberdeen, Aberdeen, United Kingdom. 2. Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.
Yap is a transcriptional co-factor which regulates C2C12 myogenesis (Watt et al., 2010). To analyse its function in muscle fibres, MCK-tTA mice were crossed with TRE-hYAP1 S127A mice to obtain MCK-tTA-hYAP1 S127A mice where the expression of constitutively active human hYAP1 S127A in muscle fibres can be induced by doxycycline withdrawal. We have used these mice to test in vivo the hypotheses that overexpression of constitutively active hYAP1 S127A in muscle fibres either a) induces hypertrophy as in other organs or b) induces a fast-to-slow fibre type shift because Yap can co-activate Tead transcription factors which can promote slow fibre formation (Tsika et al., 2008). In a first experiment, MCK-tTA-hYAP1 S127A offspring (n=6) were compared to litter mate TRE-hYAP1 S127A controls (n=3) that lacked the MCK-tTA construct. 14-23 weeks after birth doxycycline was withdrawn. This increased Yap protein ≈8-12 fold above control in skeletal muscles. Mice were sacrificied, tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius and soleus muscles were dissected, frozen in melting isopentane and cryo-sections were cut at 10 and 5 µm. Sections were stained for haematoxylin and eosin, myosin ATPase after acid (pH 4.47) preincubation to stain type 1 fibres, NADH-tetrazolium reductase to stain for mitochondria and by immunohistochemistry for slow myosin heavy chain with DAPI as a nuclear counterstain. After doxycycline withdrawal MCK-tTA-hYAP1 S127A mice initially grew similar to control mice. After 34 days off doxycycline, however, one MCK-tTA-hYAP1 S127A mouse was found dead. Between 35 and 53 days off doxycycline the remaining MCK-tTA-hYAP1 S127A mice lost weight, appeared ill and were sacrificed whilst the MCK-tTA controls appeared normal. At the time of sacrifice body weight of the MCK-tTA-hYAP1 S127A mice was 68±17% of control and muscle weights were between 57±15% (EDL) and 83±13% (soleus) of control. In the TA of MCK-tTA-hYAP1 S127A but not control mice we observed areas with a) a high density of nuclei, b) fibres with centrally located nuclei, c) small fibres positive for NADH-tetrazolium reductase, slow myosin ATPase and slow myosin heavy chain. To conclude, long-term overexpression of hYAP1 S127A in skeletal muscles of adult mice does not promote hypertrophy but instead causes myopathy. Moreover, small, oxidative fibres positive for slow myosin heavy chain and regenerating myofibres with centrally located nuclei appear in areas of the TA where they are not normally present.
Where applicable, experiments conform with Society ethical requirements