TEAD-1 overexpression in the mouse heart promotes an age-dependent heart dysfunction

Abstract

TEA domain transcription factor-1 (TEAD-1) is essential for proper heart development and is implicated in cardiac specific gene expression and the hypertrophic response of primary cardiomyocytes to hormonal and mechanical stimuli, and its activity increases in the pressure-overloaded hypertrophied rat heart. To investigate whether TEAD-1 is an in vivo modulator of cardiac specific gene expression and hypertrophy, we developed transgenic mice expressing hemagglutinin-tagged TEAD-1 under the control of the muscle creatine kinase promoter. We show that a sustained increase in TEAD-1 protein leads to an age-dependent dysfunction. Magnetic resonance imaging revealed decreases in cardiac output, stroke volume, ejection fraction, and fractional shortening. Isolated TEAD-1 hearts revealed decreased left ventricular power output that correlated with increased betaMyHC protein. Histological analysis showed altered alignment of cardiomyocytes, septal wall thickening, and fibrosis, although electrocardiography displayed a left axis shift of mean electrical axis. Transcripts representing most members of the fetal heart gene program remained elevated from fetal to adult life. Western blot analyses revealed decreases in p-phospholamban, SERCA2a, p-CX43, p-GSK-3alpha/beta, nuclear beta-catenin, GATA4, NFATc3/c4, and increased NCX1, nuclear DYKR1A, and Pur alpha/beta protein. TEAD-1 mice did not display cardiac hypertrophy. TEAD-1 mice do not tolerate stress as they die over a 4-day period after surgical induction of pressure overload. These data provide the first in vivo evidence that increased TEAD-1 can induce characteristics of cardiac remodeling associated with cardiomyopathy and heart failure.

FIGURE 1.

Expression pattern of transgenic and endogenous TEAD-1 at different ages. Western blot analysis was performed using 100 μg of total protein extract isolated from hearts of fetal, neonatal, and 5- and 10-month-old WT and TEAD-1 transgenic mice (line 12). Analysis using either anti-HA or anti-TEAD-1 antibody revealed the expression of the TEAD-1 transgene (HA-tagged TEAD-1 protein) at all ages.

FIGURE 2.

TEAD-1 induced changes in cardiac myosin heavy chain protein and LV power output of isolated working hearts. High resolution glycerol gel electrophoresis of protein extract (0.75 μg) isolated from the left ventricle of 5-month-old WT and Tg lines 4, 12, and 14 revealed that a sustained increase in TEAD-1 protein leads to an αMyHC to βMyHC transition in TEAD-1 transgenic mice (A). LV power output of isolated TEAD-1 transgenic hearts of 5-month-old mice confirm a contractile dysfunction (B). High resolution glycerol gel electrophoresis of protein extract (0.75 μg) isolated from LV, right ventricle (RV), and septum of 10-month-old WT and TEAD-1 Tg line 12 mice (C) display a significant induction of βMyHC protein (n = 3). *, p < 0.05.

FIGURE 3.

MRI. Short axis MRI images show a systolic dysfunction represented by a significant increase in end systolic (ES) volume and significantly decreased LV ejection fraction due to decreased % fractional shortening in the TEAD-1 transgenic heart (A). Long axis MRI images reveal a change in the morphology of 10-month-old TEAD-1 Tg line 12 hearts (B). Significantly decreased LV ejection fraction (EF) (C) and significantly increased end systolic (ES) volume (D) are shown. (n = 5). *, p < 0.05; **, p < 0.01. ED, end diastolic labeling.

FIGURE 4.

Western blot analysis of total Serca2a, phosphorylated phospholamban (p-PLB), and NCX1 in the heart. Membrane protein extracts (100 μg) obtained from 10-month-old wild type and TEAD-1 Tg L12 hearts revealed a significant decrease in the total level of Serca2a protein, the phosphorylation of phospholamban, and increased NCX1 protein (n = 4).

FIGURE 5.

Picrosirius red staining of heart sections. PSR staining of heart sections obtained from 10-month-old TEAD-1 transgenic mice (line 12, n = 3) revealed increased levels of fibrosis (collagen stained pink) in the septum, left and right ventricle free wall, and apex at ×20 magnification (A). Hematoxylin and eosin (H&E) staining of these heart sections showed misalignment of cardiomyocytes at ×40 magnification (B).

FIGURE 6.

ECG and Western blot analysis of total and phosphorylated connexin43 in the heart. ECG tracing of leads I, II, and III obtained from 10-month-old WT and TEAD-1 Tg (line 12, n = 10) mice show a mean electrical axis shift (A). Membrane protein extracts (100 μg) obtained from 10-month-old WT and TEAD-1 Tg (line 12, n = 3) hearts revealed a significant decrease in the phosphorylation of connexin43 (B).

FIGURE 7.

Western blot analysis of total and phosphorylated GSK-3α/β, total dual specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A), β-catenin, GATA-4, NFATc3, and NFATc4 in WT and TEAD-1 Tg hearts. Total protein extracts (100 μg) obtained from 10-month-old WT and TEAD-1 Tg (line 12) hearts revealed no change in total GSK-3α/β and a significant decrease in phospho-GSK-3α/β (A). TEAD-1 Tg hearts displayed a significant increase in DYRK1A protein in total and nuclear extract (B and C) and decreases in nuclear β-catenin, GATA-4, NFATc3, and NFATc4 (D) (n = 5–8). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 8.

Western blot analysis of total and phosphorylated GSK-3β, β-catenin, GATA-4, NFATc3, and NFATc4 in WT and TEAD-1 Tg hearts. Total protein extracts (100 μg) obtained from fetal, neonatal, and 5- and 10-month-old WT and TEAD-1 Tg (line 12) hearts revealed a significant decrease in phospho-GSK-3β at all ages and no change in total GSK-3β (A). TEAD-1 Tg neonatal (day 14) hearts displayed a significant decrease in nuclear β-catenin, GATA-4, NFATc3- and NFATc4 (B) (n = 4).

FIGURE 9.

Western blot analysis of phosphorylated and total Akt protein in WT and TEAD-1 Tg hearts. Total protein extracts (100 μg) from the hearts of 10-month-old WT and TEAD-1 Tg (line 12) mice revealed no significant changes in total Akt and phospho-Akt (n = 8).

FIGURE 10.

Western blot analysis of Purα and Purβ in the heart. Total protein extracts (A) and nuclear protein extract (B) obtained from 10-month-old WT and TEAD-1 Tg (line 12) hearts revealed a significant increase in Purα and Purβ (n = 4).

FIGURE 11.

Kaplan-Meier plot of survival. This plot illustrates a striking reduction in survival of TEAD-1 Tg mice as a function of time after surgical induction of pressure overload as compared with WT mice. Fifteen TEAD-1 Tg and 15 WT mice were used in this study.