Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart
Joerg Heineke, Mannix Auger-Messier, Jian Xu, Toru Oka, Michelle A. Sargent, Allen York, Raisa Klevitsky, Sachin Vaikunth, Stephen A. Duncan, Bruce J. Aronow, Jeffrey Robbins, Timothy M. Crombleholm, Jeffery D. Molkentin
Joerg Heineke, Mannix Auger-Messier, Jian Xu, Toru Oka, Michelle A. Sargent, Allen York, Raisa Klevitsky, Sachin Vaikunth, Stephen A. Duncan, Bruce J. Aronow, Jeffrey Robbins, Timothy M. Crombleholm, Jeffery D. Molkentin
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Research Article

Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart

Abstract

The transcription factor GATA4 is a critical regulator of cardiac gene expression, modulating cardiomyocyte differentiation and adaptive responses of the adult heart. We report what we believe to be a novel function for GATA4 in murine cardiomyocytes as a nodal regulator of cardiac angiogenesis. Conditional overexpression of GATA4 within adult cardiomyocytes increased myocardial capillary and small conducting vessel densities and increased coronary flow reserve and perfusion-dependent cardiac contractility. Coculture of HUVECs with either GATA4-expressing cardiomyocytes or with myocytes expressing a dominant-negative form of GATA4 enhanced or reduced HUVEC tube formation, respectively. Expression of GATA4 in skeletal muscle by adenoviral gene transfer enhanced capillary densities and hindlimb perfusion following femoral artery ablation. Deletion of Gata4 specifically from cardiomyocytes reduced myocardial capillary density and prevented pressure overload–augmented angiogenesis in vivo. GATA4 induced the angiogenic factor VEGF-A, directly binding the Vegf-A promoter and enhancing transcription. GATA4-overexpressing mice showed increased levels of cardiac VEGF-A, while Gata4-deleted mice demonstrated decreased VEGF-A levels. The induction of HUVEC tube formation in GATA4-overexpressing cocultured myocytes was blocked with a VEGF receptor antagonist. Pressure overload–induced dysfunction in Gata4-deleted hearts was partially rescued by adenoviral gene delivery of VEGF and angiopoietin-1. To our knowledge, these results demonstrate what is to our knowledge a previously unrecognized function for GATA4 as a regulator of cardiac angiogenesis through a nonhypoxic, load, and/or disease-responsive mechanism.

Authors

Joerg Heineke, Mannix Auger-Messier, Jian Xu, Toru Oka, Michelle A. Sargent, Allen York, Raisa Klevitsky, Sachin Vaikunth, Stephen A. Duncan, Bruce J. Aronow, Jeffrey Robbins, Timothy M. Crombleholm, Jeffery D. Molkentin

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Figure 8

GATA4 directly regulates the Vegfa gene promoter.

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GATA4 directly regulates the Vegfa gene promoter. (A) Sc...
(A) Schematic representation of the mouse VEGF promoter showing 3 putative GATA binding sites. The number of base pairs upstream of the transcription start site are denoted by –240 and –1300. (B) VEGF promoter activity shown as fold increase with Ad-GATA4 infection with Ad–β-gal infection set to 1. pGL2-basic is the backbone luciferase reporter vector with the VEGF promoter, while pGL2-control contains the SV40 promoter and is not induced by Ad-GATA4 infection. Results from a representative experiment are shown (n = 3 per promoter construct). The experiment was repeated twice with similar results. (C) Results from a similar experiment to that shown in B, except that Ad-ΔCnA (activated calcineurin) infection was used instead of Ad-GATA4, and a NFAT-luciferase reporter control adenovirus was used to show the effectiveness of Ad-ΔCnA. (D) EMSA to detect GATA4 binding to the 3 putative GATA binding sites in the Vegfa gene promoter. Oligonucleotides were incubated with unprogrammed or GATA4-programmed reticulocyte lysate. (E) Schematic of a portion of the Vegfa gene locus showing the position of the 2 primer pairs used for ChIP (arrows). (F) ChIP from GATA4 DTG hearts with GATA4 antibody or a nonspecific IgG to the promoter region or exon 8. (G) Schematic of the Vegfa gene promoter with sites 1 and 3 mutated. (H) Relative luciferase activity from cardiomyocytes transfected with the WT VEGF-1300 luciferase reporter or an identical reporter containing mutations in GATA sites 1 and 3. Myocytes were infected 24 hours prior with Ad–β-gal or Ad-GATA4. *P < 0.01 versus WT Ad–β-gal; #P < 0.01 versus WT Ad-GATA4.