GATA-4 Is a Nuclear Mediator of Mechanical Stretch-activated Hypertrophic Program

Sampsa Pikkarainen,Heikki Tokola,Theresa Majalahti-Palviainen,Risto Kerkelä,Nina Hautala,Suparna S Bhalla,Frédéric Charron,Mona Nemer,Olli Vuolteenaho,Heikki Ruskoaho

doi:10.1074/jbc.m302719200

Abstract

In overloaded heart the cardiomyocytes adapt to increased mechanical and neurohumoral stress by activation of hypertrophic program, resulting in morphological changes of individual cells and specific changes in gene expression. Accumulating evidence suggests an important role for the zinc finger transcription factor GATA-4 in hypertrophic agonist-induced cardiac hypertrophy. However, its role in stretch-induced cardiomyocyte hypertrophy is not known. We employed an in vitro mechanical stretch model of cultured cardiomyocytes and used rat B-type natriuretic peptide promoter as stretch-sensitive reporter gene. Stretch transiently increased GATA-4 DNA binding activity and transcript levels, which was followed by increases in the expression of B-type natriuretic peptide as well as atrial natriuretic peptide and skeletal alpha-actin genes. The stretch inducibility mapped primarily to the proximal 520 bp of the B-type natriuretic peptide promoter. Mutational studies showed that the tandem GATA consensus sites of the proximal promoter in combination with an Nkx-2.5 binding element are critical for stretch-activated B-type natriuretic peptide transcription. Inhibition of GATA-4 protein production by adenovirus-mediated transfer of GATA-4 antisense cDNA blocked stretch-induced increases in B-type natriuretic peptide transcript levels and the sarcomere reorganization. The proportion of myocytes with assembled sarcomeres in control adenovirus-infected cultures increased from 14 to 59% in response to stretch, whereas the values for GATA-4 antisense-treated cells were 6 and 13%, respectively. These results show that activation of GATA-4, in cooperation with a factor binding on Nkx-2.5 binding element, is essential for mechanical stretch-induced cardiomyocyte hypertrophy.

Highlights

Cardiac hypertrophy is the primary adaptive mechanism for terminally differentiated cardiac myocytes to increased hemodynamic load
The proportion of myocytes with assembled sarcomeres in control adenovirus-infected cultures increased from 14 to 59% in response to stretch, whereas the values for GATA-4 antisense-treated cells were 6 and 13%, respectively. These results show that activation of GATA-4, in cooperation with a factor binding on Nkx-2.5 binding element, is essential for mechanical stretchinduced cardiomyocyte hypertrophy
Mechanical Stretch Induces a Hypertrophic Phenotype—To validate the experimental system we first analyzed the effect of mechanical stretch on the gene expression of B-type natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and skeletal muscle ␣-actin (sk␣A), which represents genetic hallmarks of the stretch-induced cardiac hypertrophic program [23, 33,34,35]

Summary

EXPERIMENTAL PROCEDURES

Materials—Specific antibodies raised against GATA-4 (C-20 and H-112), GATA-5 (M-20), GATA-6 (C-20), components of AP-1 complex (c-Fos [4]-G, c-Fos (K-25)-G, c-Jun/AP-1 (N)-G, Jun B (N-17)-G, or JunD [329]-G), and Nkx-2.5 (N-19) were from Santa Cruz Biotechnology (San Diego, CA), and anti-rabbit IgG antibody linked to horseradish peroxidase was from New England Biolabs Ltd. (Hertfordshire, UK). Cells were plated onto 100-mm culture plates and transfected with 1 ␮g of pEF-FLAG-Nkx2.5 or pMT2 control plasmid with FuGENETM 6 reagent. The cDNA probes complementary to rat ANP, BNP, sk␣A, and GATA-4 mRNA or ribosomal 18 S RNA were random prime-labeled with RediprimeTM II (Amersham Biosciences). Binding reactions contained 3– 6 ␮g of nuclear protein and 2 ␮g of poly-(dI-dC)1⁄7(dI-dC) in a buffer containing 16 mM HEPES, pH 7.9, 120 mM NaCl, 1 mM MgCl2, 40 mM KCl, 1 mM dithiothreitol, 0.7 mM EDTA, 0.3 mM EGTA, 8% glycerol, 0.02% Nonidet P-40, 0.25 mM phenylmethylsulfonyl fluoride, and 1 ␮g/ml of each aprotinin, leupeptin, pepstatin, and when appropriate, various molar excesses of competitor DNAs. When designed, supershift experiments were performed by preincubating nuclear extract with 1 ␮g of appropriate antibody for 20 min at room temperature before performing the FIG. A value of p Ͻ 0.05 was considered statistically significant

RESULTS

Binding site

DISCUSSION