Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure

Abstract

An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiomyocytes, the transcription factor Gata4 is required for agonist-induced hypertrophy. We hypothesized that, in the intact organism, Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress. To test this hypothesis, we studied mice heterozygous for deletion of the second exon of Gata4 (G4D). At baseline, G4D mice had mild systolic and diastolic dysfunction associated with reduced heart weight and decreased cardiomyocyte number. After transverse aortic constriction (TAC), G4D mice developed overt heart failure and eccentric cardiac hypertrophy, associated with significantly increased fibrosis and cardiomyocyte apoptosis. Inhibition of apoptosis by overexpression of the insulin-like growth factor 1 receptor prevented TAC-induced heart failure in G4D mice. Unlike WT-TAC controls, G4D-TAC cardiomyocytes hypertrophied by increasing in length more than width. Gene expression profiling revealed up-regulation of genes associated with apoptosis and fibrosis, including members of the TGF-beta pathway. Our data demonstrate that Gata4 is essential for cardiac function in the postnatal heart. After pressure overload, Gata4 regulates the pattern of cardiomyocyte hypertrophy and protects the heart from load-induced failure.

Fig. 1.

Baseline characterization of G4D mice. (A) Relative Gata4 mRNA levels were measured by qRT-PCR and normalized to GAPDH. (B) Representative Western blotting for Gata4. (C) Relative Gata4 protein levels were measured by Western blotting and normalized to GAPDH. RNA (A) and protein (B and C) were prepared from adult heart ventricles. (D) Echocardiography of G4D mice showed mildly depressed FS. (E and F) Measurement of intraventricular pressure during Dob infusion showed decreased contractile reserve and impaired diastolic function in G4D mice. In Figs. 1–6, numbers inside bars indicate number of samples per group. For Dob infusion, n = 6 per group. ∗, P < 0.05.

Fig. 2.

Baseline morphology of G4D mice. (A) Representative long axis sections through WT and G4D adult hearts. G4D hearts were structurally normal. (Scale bar, 2 mm.) (B) HW/BW ratio was significantly decreased in G4D hearts. #, P = 0.002. (C) Representative dissociated cardiomyocytes from WT and G4D hearts. (Scale bar, 50 μm.) (D) Volume of G4D cardiomyocytes was significantly larger than WT cardiomyocytes. (E) Fetal cardiomyocyte number, measured by design-based stereology, was significantly decreased in G4D embryonic day 17.5 embryos compared with WT embryos. ∗, P < 0.05.

Fig. 3.

Heart failure and eccentric hypertrophy in G4D mice after TAC. (A) G4D hearts showed the same degree of hypertrophy after TAC as WT heart. (B) Systolic function of WT and G4D mice after TAC. WT mice compensated for increased afterload and maintained normal FS, whereas G4D mice developed severe systolic dysfunction after TAC. (C)TAC resulted in increased lung weight in G4D mice but did not alter lung weight in WT mice. (D) Representative images of dissociated WT and G4D cardiomyocytes after TAC. (Scale bar, 50 μm.) (E) G4D and WT cardiomyocytes increased in size after TAC. (F) In WT cardiomyocytes, length (L) and width (W) both increased, so that the length/weight ratio (L/W) was unchanged. In G4D cardiomyocytes, length increased out of proportion to width, so that L/W was increased. ∗, P < 0.05.

Fig. 4.

Increased apoptosis and fibrosis in G4D-TAC hearts. (A) Representative TUNEL staining of G4D myocardium. Apoptotic nuclei were labeled by TUNEL (red), counterstained to mark nuclei (Topro-3, blue) and cardiomyocytes (desmin, green), and imaged by confocal microscopy. (B) Frequency of TUNEL-positive cardiomyocyte nuclei was increased by TAC. G4D-TAC cardiomyocytes had significantly increased apoptosis compared with WT-TAC. (C) Post-TAC cardiac fibrosis, demonstrated by Masson's trichrome stain. Concentric hypertrophy of WT-TAC hearts and eccentric hypertrophy of G4D-TAC hearts were also evident. (Scale bar, 2.0 mm.) (D) The area fraction of fibrotic tissue was increased more in G4D hearts compared with WT hearts (n = 3). ∗, P < 0.05.

Fig. 5.

Gene expression analysis. Expression was determined by qRT-PCR (for BNP, Fhl1, Casp12, Tgfβ2, and Ctgf) or by Affymetrix microarray. (A) Relative expression of hypertrophy marker genes. Number of samples per group: WT-Sham, 4; G4D-Sham, 4; WT-Band, 6; G4D-TAC,6. (B) Relative expression of fibrosis and apoptosis associated genes. #, P < 0.001 for effect of operation type. ^, P < 0.001 for effect of genotype. ∗, P < 0.01 for pairwise comparisons.

Fig. 6.

Protective effects of IgfR overexpression. (A) TAC-induced increase in frequency of TUNEL-positive cardiomyocyte nuclei was blocked by IgfR and G4D-IgfR. (B) IgfR and G4D-IgfR also blocked the TAC-induced increase in fibrosis. (C) TAC-induced increase in the lung weight to BW ratio (LW/BW ratio) in G4D was not present in G4D-IgfR. (D) FS was significantly reduced in G4D but not in G4D-IgfR. ∗, P < 0.05. IgfR n = 4; G4D-IgfR n = 7.