Abstract 16597: Eya4 is a Transcriptional Suppressor of P27kip1 in Acquired Heart Disease
Introduction: We previously identified a mutation in the human transcriptional cofactor Eya4 (E193) as cause of familial dilated cardiomyopathy (DCM) and heart failure. Eya4 is recruited to and interacts with target genes upon interaction with real transcription factors, including Six family members. The cyclin-dependent kinase inhibitor p27kip1, which inhibits hypertrophic growth in adult cardiomyocytes, is one of the few known Eya-Six targets expressed in the heart. We therefore hypothesize that Eya4/Six1 regulates targets relevant in normal cardiac function.
Methods and results: We examined the correlation of p27 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 while phosphorylated p27 was distributed in the cytoplasm. We then examined p27 expression in response to Eya4 in permanent mammalian cell lines and primary cardiac myocytes. Western blot analysis demonstrated that the overexpression of Eya4 led to a significant downregulation of p27, whereas overexpression of E193 had no effect on p27 levels. Studies using a p27 promoter fragment including Six1 consensus sites cloned in front of a Luciferase reporter gene revealed that Eya4 acts as a suppressor of p27 already at the transcriptional level, while E193 could not sufficiently inhibit p27 expression. Further transfection and knockdown experiments revealed that an Eya4 overexpression and subsequent p27 suppression increased the expression of molecular markers for myocardial hypertrophy and increased protein synthesis.
Conclusion: In summary, we identified a mutation in Eya4 to cause a rare version of inheritable DCM. We now provide evidence that the Eya4/Six1 signalling cascade is also relevant in more common forms of acquired heart disease. Eya4/Six1 seems to suppress the expression of p27kip1, an important inhibitor of the development of hypertrophy in postmitotic cardiomyocytes.
- © 2010 by American Heart Association, Inc.