Serine 105 phosphorylation of transcription factor GATA4 is necessary for stress-induced cardiac hypertrophy in vivo

Abstract

Cardiac hypertrophy is an adaptive growth process that occurs in response to stress stimulation or injury wherein multiple signal transduction pathways are induced, culminating in transcription factor activation and the reprogramming of gene expression. GATA4 is a critical transcription factor in the heart that is known to induce/regulate the hypertrophic program, in part, by receiving signals from MAPKs. Here we generated knock-in mice in which a known MAPK phosphorylation site at serine 105 (S105) in Gata4 that augments activity was mutated to alanine. Homozygous Gata4-S105A mutant mice were viable as adults, although they showed a compromised stress response of the myocardium. For example, cardiac hypertrophy in response to phenylephrine agonist infusion for 2 wk was largely blunted in Gata4-S105A mice, as was the hypertrophic response to pressure overload at 1 and 2 wk of applied stimulation. Gata4-S105A mice were also more susceptible to heart failure and cardiac dilation after 2 wk of pressure overload. With respect to the upstream pathway, hearts from Gata4-S105A mice did not efficiently hypertrophy following direct ERK1/2 activation using an activated MEK1 transgene in vivo. Mechanistically, GATA4 mutant protein from these hearts failed to show enhanced DNA binding in response to hypertrophic stimulation. Moreover, hearts from Gata4-S105A mice had significant changes in the expression of hypertrophy-inducible, fetal, and remodeling-related genes.

Fig. 1.

Generation of Gata4–S105A knock-in mice. (A) Western blotting for phospho-GATA4 (S105) from nuclear extracts of sham- or TAC-treated hearts. (B) Schematic of the targeting strategy used to create Gata4–S105A knock-in mice. (C) DNA sequencing traces from WT (Top), S105A heterozygous (Middle), and S105A homozygous (Bottom) mice. Arrows indicate mutated bases resulting in change of S105 to alanine, which introduced a new KasI restriction site. (D) Western blotting of WT and homozygous knock-in mice (S105A mut) for total GATA4 protein and phospho-GATA4 (S105) from nuclear extracts. Lamin A/C was used as a loading control. (E) Ventricular to body weight ratio (VW/BW) from WT and S105A mut mice at 8 wk of age (*P < 0.05 vs. WT).

Fig. 2.

PE causes cardiac hypertrophy through phosphorylation of GATA4. (A) Western blotting for phosphorylated GATA4 (S105), total GATA4, and phosphorylated ERK1/2 from WT and S105A mut nuclear cardiac lysates, and for phosphorylated and total ERK1/2 and p38 from WT and S105A mut cytoplasmic cardiac lysates. Hearts were harvested at indicated time points after s.c. PE injection. (B) VW/BW ratio in WT and S105A mut mice after 2 wk of continuous infusion of vehicle or PE by osmotic minipump. The number of mice analyzed is shown in the graph (*P < 0.05 vs. vehicle, ^†P < 0.05 vs. WT PE). (C) Myocyte surface area (μm²) from cardiac histological sections of the mice shown in B. A total of 300 myocytes were counted across four or five hearts each (*P < 0.05 vs. vehicle, ^†P < 0.05 vs. WT PE). (D) Gel-shift assay shows GATA DNA binding activity from cardiac nuclear protein extracts from WT and S105A mut mice at baseline or 15 min after s.c. PE injection. Addition of probe only and cold competitor probe show specificity of GATA binding activity.

Fig. 3.

Phosphorylation of GATA4 is required for activated ERK-induced cardiac hypertrophy. (A) VW/BW ratio from the indicated genotypes of mice at 8 wk of age. The number of mice analyzed is shown in the graph (*P < 0.05 vs. nontransgenic, ^†P < 0.05 vs. Gata4^fl/fl × MEK1 transgenic). (B) VW/BW ratio from WT and S105A mut mice with or without the MEK1 transgene. The number of mice analyzed is shown in the graph (*P < 0.05 vs. nontransgenic, ^†P < 0.05 vs. WT × MEK1 transgenic). (C) Western blotting of protein extracts from hearts of the indicated mice for MEK1 and phospho- and total ERK. Western blotting with phospho-GATA4 (S105A) antibody was performed from cardiac nuclear extracts. GAPDH was a loading control.

Fig. 4.

Pressure overload results in blunted hypertrophy and progression to dilation in S105A mut mice. Echocardiographic assessment of interventricular septal (A) and posterior wall thicknesses (B) from sham- and TAC-operated mice measured at 1 and 2 wk after surgery (*P < 0.05 vs. sham, ^†P < 0.05 vs. WT TAC at 2 wk). (C) VW/BW ratio in the indicated groups of mice. The legend in A also applies to B–F (*P < 0.05 vs. sham, ^†P < 0.05 vs. WT TAC at 1 wk). Echocardiographic assessment of left ventricular end-diastolic (D) and end-systolic (E) chamber dimension from the indicated groups of mice (*P < 0.05 vs. sham, ^†P < 0.05 vs. WT TAC 2 wk). (F) Ventricular fractional shortening (FS) percentage in WT and S105A mut mice after sham operation or 1 to 2 wk after TAC surgery (*P < 0.05 vs. sham, ^†P < 0.05 vs. WT TAC at 2 wk). (G) Length of isolated cardiomyocytes from hearts after 10 d of TAC. (H) Width of isolated cardiomyocytes from hearts after 10 d of TAC. (I) Length-to-width ratio of isolated cardiomyocytes from hearts after 10 d of TAC. (J) Fibrosis quantification after 2 wk of TAC as a percentage of fibrotic area per section (n = 7 hearts; *P < 0.05 vs. WT TAC in G–J). The number of mice analyzed throughout is shown in the graphs in A–F. For G–I, 251 WT and 174 S105A mut cardiomyocytes were counted from three and four hearts, respectively.

Fig. 5.

Phosphorylation of GATA4 is important for expression of multiple genes in the heart. Normalized TaqMan RNA expression assays for (A) Nppa, (B) Nppb, (C) Myh7, (D) Myh6, and (E) Acta1 on RNA from WT and S105A mut hearts. Normalized SYBR green quantitative PCR for (F) Ephbi, (G) Fgf2, (H) Fgf16, (I) Spp1, (J) Cthrc1, (K) Col1a2, (L) Ctgf, (M) Thbs4, (N) Col3a1, (O) Ltbp2, (P) Tgfbi, (Q) Figf, (R) Timp1, (S) Timp4, and (T) Fstl4 on RNA from WT and S105A mut hearts. All graphs are plotted as relative to WT. All samples were analyzed in duplicate from three hearts each.