Phosphorylation of GATA4 at serine 105 is required for left ventricular remodelling process in angiotensin II-induced hypertension in rats

Abstract

In this study, we investigated whether local intramyocardial GATA4 overexpression affects the left ventricular (LV) remodelling process and the importance of phosphorylation at serine 105 (S105) for the actions of GATA4 in an angiotensin II (AngII)-induced hypertension rat model. Adenoviral constructs overexpressing wild-type GATA4 or GATA4 mutated at S105 were delivered into the anterior LV free wall. AngII (33.3 µg/kg/h) was administered via subcutaneously implanted minipumps. Cardiac function and structure were examined by echocardiography, followed by histological immunostainings of LV sections and gene expression measurements by RT-qPCR. The effects of GATA4 on cultured neonatal rat ventricular fibroblasts were evaluated. In AngII-induced hypertension, GATA4 overexpression repressed fibrotic gene expression, reversed the hypertrophic adult-to-foetal isoform switch of myofibrillar genes and prevented apoptosis, whereas histological fibrosis was not affected. Overexpression of GATA4 mutated at S105 resulted in LV chamber dilatation, cardiac dysfunction and had minor effects on expression of myocardial remodelling genes. Fibrotic gene expression in cardiac fibroblasts was differently affected by overexpression of wild-type or mutated GATA4. Our results indicate that GATA4 reduces AngII-induced responses by interfering with pro-fibrotic and hypertrophic gene expressions. GATA4 actions on LV remodelling and fibroblasts are dependent on phosphorylation site S105.

Keywords: angiotensin II; fibrosis; hypertension; myocardial remodelling; transcription factor.

FIGURE 1

Timeline for the in vivo experiments. (A) Angiotensin II‐induced hypertension. Rats were implanted with osmotic minipumps loaded with AngII (33.3 µg/kg/h) and adenoviral constructs overexpressing GATA4 or GATA4‐S105A (1 × 10⁹ pfu and 2 × 10⁹ pfu, respectively, in 100 µL) or dose‐matched LacZ adenoviral constructs were injected into the LV anterior free wall, generating four adenovirus treatment groups followed for 7 and 14 d. (b) Myocardial infarction. LAD ligation or a sham intervention was performed in rats at day 0, followed by injection of adenoviral constructs overexpressing GATA4‐S105A (2 × 10⁹ pfu in 100 µL) or dose‐matched LacZ adenoviral constructs within the same surgical procedure. Due to different virus dose, the control LacZ groups are not comparable to each other. The groups were studied at 3, 7 or 14 d. At the end of experiments, the cardiac function and structure were analysed by echocardiography and the hearts were excised and processed for further histological and biochemical analyses

FIGURE 2

GATA4 overexpression by intramyocardial adenoviral gene delivery into the LV in AngII‐induced hypertension in rats. (A) RT‐qPCR was used to quantify the mRNA levels of GATA4 at 1 and 2 wks. The bars represent average values + SD (n = 6‐8/group). * P ≤ .05, ** P ≤ .01. (B) Protein levels were examined by immunoblotting for GATA4, GATA4 phosphorylation at S105 (GATA4‐pS105) and Lamin B. Figure shows a representative Western blot at 2 wks (full blot images in Fig. S1). (C‐E) Immunofluorescent study of GATA4 (red) for cellular localization using specific cell markers for myocytes (αAct, C), fibroblasts (P4H, D) and myofibroblasts (Acta2, E). Representative LV histological sections are shown, white arrows indicate co‐localization with the specific cell markers (LacZ control group representative images are shown in Fig. S2). The scale bars represent 25 µm

FIGURE 3

The effect of GATA4 overexpression on AngII‐induced fibrosis. (A and B) Masson's trichrome staining was used to quantify the percentage of fibrosis in LVs at 2 wks (A, n = 7‐8/group). The results are average values ± SD. Representative images (B) are shown where the collagenous fibres are stained blue. The scale bars represent 75 µm. (C‐K) The mRNA levels of fibrosis‐associated genes FN1, Col1a1, Col3a1, TGFβ, MMP2, MMP9, TIMP1, FGF2 and PEX1 at 1 and 2 wks were quantified by RT‐qPCR. The values were normalized to GAPDH and are expressed as relative to LacZ control groups (n = 6‐8/group). The results are represented as average + SD or median + interquartile range [FN1, 2 wks]. * P ≤ .05, ** P ≤ .01, *** P ≤ .005

FIGURE 4

Analysis of hypertrophy‐associated genes in LVs at 1 and 2 wks after intramyocardial adenoviral GATA4 gene delivery. (A‐I) ANP, BNP, FGF1, αMHC, βMHC, Actc1 and Acta1 mRNA levels were quantified by RT‐qPCR. The mRNA values were normalized to GAPDH and are expressed as relative to LacZ control groups (n = 6‐8/group). The results are represented as average + SD or median + interquartile range [Actc1/Acta1 ratio, 1 wk]. *P ≤ .05, **P ≤ .01

FIGURE 5

Effect of GATA4 overexpression on apoptosis, cell proliferation, stem cell‐like cells and angiogenesis in LVs of 2 wks AngII‐induced hypertension. Immunohistochemical staining for TUNEL‐positive cells was used to determine the number of apoptotic cells in LVs (A). Representative images are shown (B). Immunohistochemical staining against Ki‐67 was used to determine the effect on cellular proliferation (C), representative images are shown (D). (E) c‐kit antibody was used to quantify stem cell‐like cells. (F) Histological sections were stained with vWF to determine the number of capillaries. The whole LV was scanned, and positive cells were counted from high power fields (40×) selecting 6‐8 hotspots. The scale bars represent 100 µm. The results are represented as average + SD (n = 7‐8/group). * P ≤ .05

FIGURE 6

Effect of GATA4 and GATA4‐S105A overexpression on cardiac function and structure in AngII‐induced hypertension in rats. (A‐G) Structural and functional parameters were analysed at 1 wk by echocardiography in rats overexpressing GATA4 or GATA4‐S105A and their dose‐matched LacZ control groups. Due to different virus dose, the control LacZ groups are not comparable to each other. The results are represented as average + SD or median + interquartile range [LVPW, GATA4‐S105 and LacZ control] (n = 7‐10/group). LVID = left ventricular internal diameter; LVPW = left ventricular posterior wall thickness; IVS = interventricular septum; FAC = fractional area change; LV vol. = left ventricular volume; LVEF = left ventricular ejection fraction; LVFS = left ventricular fractional shortening. * P ≤ .05, ** P ≤ .01, *** P ≤ .005

FIGURE 7

Effect of GATA4‐S105A overexpression on fibrosis, apoptosis and cell proliferation in LVs of 1 wk AngII‐induced hypertension. Overexpression of GATA4 by local adenoviral gene transfer at 1 and 2 wks was confirmed by RT‐qPCR (A) and Western blotting (B). Representative blot of 1 wk samples is shown (full blot images in Fig. S3). Fibrosis was quantified by Masson's trichrome staining (C), representative images from LV histological sections are shown (D). The scale bar represents 75 µm. (E) The number of apoptotic cells was quantified from TUNEL staining. (F) Immunohistochemical staining against Ki‐67 was used to determine number of proliferating cells in LV histological sections. The results are represented as average ± SD (n = 6‐7/group). * P ≤ .05, *** P ≤ .005

FIGURE 8

Analysis of gene expression in LVs at 1 and 2 wks after GATA4‐S105A gene delivery in AngII‐induced hypertension. The mRNA levels of fibrosis‐associated genes (A‐I) and hypertrophic‐associated genes (J–R) were quantified by RT‐qPCR. The expression values were normalized to GAPDH and are expressed as relative to LacZ control groups (n = 6‐8/group). The results are represented as average + SD or median + interquartile range [Col1a1, MMP9, FGF2, βMHC, Acta1 and Actc1/Acta1 ratio, 1 wk]. * P ≤ .05, ** P ≤ .01, *** P ≤ .005

FIGURE 9

Assessment of fibrosis‐associated genes expressions in NRVFs after GATA4 and GATA4‐S105A adenovirus‐mediated overexpression for 48 hrs. The experiments are grouped into 3 sets according to the adenovirus concentration: 2 MOI (+; ++) and 4 MOI (+++). GATA4 overexpression (A) and the mRNA levels of fibrotic genes FN1, Col1a1, Col3a1, TGFβ1, MMP2, MMP9, TIMP1, FGF2 and PEX1 (B‐J) were quantified by RT‐qPCR. The expression values were normalized to GAPDH and are expressed as relative to LacZ control groups (n = 3‐4/group). The results are represented as average + SD. * P ≤ .05, ** P ≤ .01, *** P ≤ .005 vs LacZ. # P ≤ .05, ## P ≤ .01, ### P ≤ .005 vs GATA4

FIGURE 10

The effects of GATA4‐S105A overexpression by intramyocardial gene delivery after myocardial infarction in rats. (A) RT‐qPCR quantification of GATA4 mRNA levels in LVs of sham‐operated and MI animals at 3 d, 1 and 2 wks. The values were normalized to GAPDH and are expressed as relative to sham LacZ control groups at each time point. The bars represent average values + SD (n = 5‐8/group). * P ≤ .05, ** P ≤ .01, *** P ≤ .005 Sham GATA4‐S105A compared to LacZ group and # P ≤ .05 MI GATA4‐S105A compared to LacZ. (B‐E) Echocardiographic measurements were performed at 2 wks after infarction and gene transfer. LVEF = left ventricular ejection fraction; LVFS = left ventricular fractional shortening; LVPW = left ventricular posterior wall thickness; LVID = left ventricular internal diameter. (F‐I) Immunohistochemistry studies for apoptosis (TUNEL, F), c‐kit‐positive cells (G), fibrosis (Masson's trichrome, H) and angiogenesis (vWF, I) from LV histological sections at 2 wks after MI and injection of the adenoviral constructs. (J) The infarct area was measured from 3 d and 2 wks histological LV sections stained with Masson's trichrome. The results are represented as average ± SD (n = 5‐8/group)

FIGURE 11

Schematic illustration of GATA4 actions induced upon cardiac stress. Stress signals activate intracellular signalling cascades that lead to activation of GATA4 through post‐translational modifications, such as phosphorylation at serine 105. GATA4 then contributes to the adaptive response of the myocardium through modulation of genes involved in angiogenesis, hypertrophic growth, cell survival and the fibrotic process. ACTA1 = Skeletal muscle actin, alpha 1; AngII = angiotensin II; ANP = atrial natriuretic peptide; BCL = B‐cell lymphoma; BNP = B‐type natriuretic peptide; Col = collagen; ET‐1 = endothelin‐1; JAK/STAT = janus kinase/signal transducer and activator of transcription; MAPKs = mitogen‐activated protein kinases; MHC = myosin heavy chain; MMP = metalloproteinase; NFAT = nuclear factor of activated T cells; PE = phenylephrine; PKC = protein kinase C; TIMP = tissue inhibitor metalloproteinase; Snai1 = Snail family transcriptional repressor 1; VEGF = vascular endothelial growth factor