Abstract
The mammalian homolog of Drosophila Hippo, Mst1, is a kinase which negatively regulates cardiomyocyte (CM) growth and survival. Mst1 promotes CM apoptosis in response to oxidative stress and mediates myocardial ischemia-reperfusion (I/R) injury. In Drosophila , the adaptor protein Salvador is required for activation and function of Hippo. We investigated the role of the mammalian homolog of Salvador, WW45, in the regulation of Mst1 activity in the heart. To address this issue, we studied the cardiac phenotype of cardiac-specific WW45 knock-out (W-cKO) mice. At baseline, W-cKO mice were characterized by a mild increase in LV weight/body weight compared to controls (3.44 ± 0.13 vs. 2.92 ± 0.10 mg/g; p<0.05) and normal cardiac function, suggesting that endogenous WW45 negatively regulates baseline growth of the heart. After I/R (30 minutes/24 hours), W-cKO mice exhibited a significantly smaller infarct size/area at risk than controls (20.4 ± 6.0 vs. 38.2 ± 4.5%; p<0.05). Interestingly, Mst1 activation, as evaluated by its phosphorylation status (Thr183), was significantly reduced in W-cKO mice both at baseline (0.5 ± 0.01 fold, p<0.05) and during reperfusion (0.37 ± 0.04 fold, p<0.05), indicating that WW45 is required for Mst1 activation. Co-immunoprecipitation analysis showed that WW45 physically interacts with Mst1 in CMs. We further evaluated whether downregulation of WW45 affects the phosphorylation status (Ser127) and the accumulation of Yes-associated protein (YAP), a transcription factor co-factor which promotes cellular growth and survival. YAP is phosphorylated by the Hippo pathway, and, upon its phosphorylation, YAP is exported from the nucleus and degraded. The level of YAP was increased in the hearts of W-cKO mice at baseline (2.6 ± 0.03 fold, p<0.05) and during reperfusion (4.2± 0.16 fold, p<0.05), and its relative phosphorylation status was decreased (-64% and -75%, p<0.05), indicating that WW45 positively regulates phosphorylation of YAP. There was a significant increase in nuclear localization of YAP during I/R in W-cKO CMs compared to in control CMs (2.2 ± 0.26 fold, p<0.05). In conclusion, our data suggest that endogenous WW45 is required for Mst1-dependent inactivation of YAP and promotion of myocardial damage during I/R.
Published Version
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