Background-E193, a heterozygous truncating mutation in the human transcription cofactor Eyes absent 4 (Eya4), causes hearing impairment followed by dilative cardiomyopathy. Methods and Results-In this study, we first show Eya4 and E193 alter the expression of p27 kip1 in vitro, suggesting Eya4 is a negative regulator of p27. Next, we generated transgenic mice with cardiac-specific overexpression of Eya4 or E193. Luciferase and chromatin immunoprecipitation assays confirmed Eya4 and E193 bind and regulate p27 expression in a contradictory manner. Activity and phosphorylation status of the downstream molecules casein kinase-2α and histone deacetylase 2 were significantly elevated in Eya4-but significantly reduced in E193-overexpressing animals compared with wild-type littermates. Magnetic resonance imaging and hemodynamic analysis indicate Eya4-overexpression results in an age-dependent development of hypertrophy already under baseline conditions with no obvious functional effects, whereas E193 animals develop onset of dilative cardiomyopathy as seen in human E193 patients. Both cardiac phenotypes were aggravated on pressure overload. Finally, we identified a new heterozygous truncating Eya4 mutation, E215, which leads to similar clinical features of disease and a stable myocardial expression of the mutant protein as seen with E193. Conclusions-Our

Introduction: E193, a truncating mutation in the transcription cofactor Eyes absent 4 (Eya4) causes hearing impairment followed by heart failure. Here we identified the Eya4 dependent molecular mechanisms leading to the cardiac phenotype in the E193 mutation. Methods and Results: First we showed in vitro that the cyclin-dependent kinase inhibitor protein p27kip1 is a direct target of Eya4/Six1 and is suppressed upon Eya4 overexpression, whereas E193 has a dominant negative effect, releasing Eya4 mediated suppression of p27. We next generated transgenic mice with cardiac specific constitutive overexpression of full-length Eya4 or the mutant form E193. While E193 transgenic mice developed age-dependent DCM, Eya4 mice displayed cardiac hypertrophy already under basal conditions as judged by increases in heart weight and cardiomyocyte cross-sectional areas along with increases in myocardial dimension and mass. These two distinct cardiac phenotypes were even more aggravated upon pressure overload suggesting Eya4 is a regulator of cardiac hypertrophy. We also observed that the activity of Casein Kinase 2-α and the phosphorylation status of HDAC2 were significantly upregulated in the Eya4 transgenic mice, while they were significantly reduced in E193 mice, under baseline conditions and pressure overload. We were also able to identify a new human mutation (E215) with a phenotype comparable to the one seen in E193 patients. Conclusion: Our results implicate that Eya4/Six1 regulates cardiac hypertrophic reactions via p27/CK2-α/HDAC2 and indicate that truncating mutations in Eya4 interfere with this newly established signalling pathway.

No abstract