Eya4 Induces Hypertrophy via Regulation of p27kip1.
BACKGROUND
E193, a heterozygous truncating mutation in the human transcription cofactor Eyes absent 4 (Eya4), causes hearing impairment followed by dilative cardiomyopathy.


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 results implicate Eya4/Six1 regulates normal cardiac function via p27/casein kinase-2α/histone deacetylase 2 and indicate that mutations within this transcriptional complex and signaling cascade lead to the development of cardiomyopathy.
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Tatjana
T
Williams
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Moritz
M
Hundertmark
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Peter
P
Nordbeck
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Sabine
S
Voll
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Paula Anahi
PA
Arias-Loza
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Daniel
D
Oppelt
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Melanie
M
Mühlfelder
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Susanna
S
Schraut
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Ines
I
Elsner
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Martin
M
Czolbe
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Lea
L
Seidlmayer
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Britta
B
Heinze
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Stefanie
S
Hahner
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Katrin
K
Heinze
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Jost
J
Schönberger
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.).
Peter
P
Jakob
From the Department of Medicine I, University Hospital Wuerzburg, Wuerzburg, Germany (T.W., M.H., P.N., P.A.A.-L., D.O., M.M., S.S., I.E., M.C., L.S., B.H., S.H., J.S., O.R.); Comprehensive Heart Failure Center Wuerzburg, Wuerzburg, Germany (T.W., M.C., O.R.); Experimental Physics V, University Wuerzburg, Wuerzburg, Germany (P.N., S.V., P.J.); DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany (K.H.); and Department of Cardiology and Pneumology, Medical University Brandenburg, Brandenburg, Germany (O.R.). o.ritter@klinikum-brandenburg.de Williams_T@ukw.de.
Oliver
O
Ritter
D000818:Animals; D001483:Base Sequence; D006332:Cardiomegaly / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D051381:Rats; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
C436291:CDKN1B protein, human; C493961:Cdkn1b protein, mouse; C117736:EYA4 protein, human; C527542:Eya4 protein, mouse; D015534:Trans-Activators; D050760:Cyclin-Dependent Kinase Inhibitor p27
Eya4; dilated cardiomyopathy; molecular biology; p27Kip1; signaling pathways