Abstract 3588: The Eya4-Six1 Signalling Cascade is Crucial in Acquired Heart Disease
Introduction: We previously identified a mutation in human Eya4 (E193) as cause of DCM and heart failure. Eya proteins are recruited to target genes via interaction with transcription factors, including Six family members. So far only a few direct Eya4-Six1 targets are known, including the cyclin-dependent kinase inhibitor p27Kip1 (p27), which inhibits hypertrophic growth in adult cardiomyocytes. We hypothesize that the Eya-Six signalling cascade is also activated in acquired heart disease.
Methods and results: We examined the correlation of p27 phosphor-ylation and Eya4 in cryosections of failing and normal human hearts. Immunocytochemical analysis revealed Eya4 is distributed in the cytoplasm while p27 resides mainly in the nucleus. In sections of failing human hearts, Eya4 was accumulated in the perinuclear region; nuclear p27 levels were significantly diminished, phosphorylated p27 was evenly distributed in the cytoplasm. In a murine model of MI immunofluorescence staining showed Eya4 is translocated to the nucleus in a time-dependent manner. 16 min after experimental MI there is a strong perinuclear accumulation of Eya4 in cardiomyocytes; translocation to the nucleus was detectable 24h post infarction compared to the cytoplasmic distribution in control heart tissue. We studied the effect of Eya4 overexpression on nuclear translocation in permanent mammalian cell lines and cardiac myocytes by immunofluorescence microscopy. 48h post transfection EGFP-tagged WT Eya4 was located in the cytoplasm and translocated to the nucleus after co-expression with DsRed-tagged Six1. Promoter studies with a p27 promoter-luciferase plasmid including Six1 consensus sites also revealed that activated Eya-Six acts as suppressor on p27 expression. Further transfection experiments demonstrated that Eya-Six activation and subsequent p27 suppression increased expression of molecular markers for myocardial hypertrophy and increased protein synthesis.
Discussion: We identified a mutation in Eya4 to cause DCM. We now provide evidence that the Eya-Six signalling cascade is also activated in acquired heart disease. Eya-Six seems to suppress the expression of p27, an important inhibitor for the development of hypertrophy in postmitotic cardiomyocytes.