Pulmonary hypertension-induced GATA4 activation in the right ventricle

Abstract

The major cause of death among pulmonary hypertension patients is right heart failure, but the biology of right heart is not well understood. Previous studies showed that mechanisms of the activation of GATA4, a major regulator of cardiac hypertrophy, in response to pressure overload are different between left and right ventricles. In the left ventricle, aortic constriction triggers GATA4 activation via posttranslational modifications without influencing GATA4 expression, while pulmonary artery banding enhances GATA4 expression in the right ventricle. We found that GATA4 expression can also be increased in the right ventricle of rats treated with chronic hypoxia to induce pulmonary hypertension and investigated the mechanism of increased GATA4 expression. Examination of Gata4 promoter revealed that CCAAT box plays an important role in gene activation, and hypoxic pulmonary hypertension promoted the binding of CCAAT-binding factor/nuclear factor-Y (CBF/NF-Y) to CCAAT box in the right ventricle. We found that CBF/NF-Y forms a complex with annexin A1, which inhibits DNA binding activity. In response to hypoxic pulmonary hypertension, annexin A1 gets degraded, resulting in CBF/NF-Y-dependent activation of Gata4 gene transcription. The right ventricle contains a higher level of CBF/NF-Y compared to the left ventricle, and this may allow for efficient activation in response to annexin A1 degradation. Signaling via iron-catalyzed protein oxidation mediates hypoxic pulmonary hypertension-induced annexin A1 degradation, Gata4 gene transcription, and right ventricular hypertrophy. These results establish a right heart-specific signaling mechanism in response to pressure overload, which involves metal-catalyzed carbonylation and degradation of annexin A1 that liberates CBF/NF-Y to activate Gata4 gene transcription.

Fig. 1. Hypoxic PH promotes GATA4 activity and expression in the RV

Rats were subjected to CH. Nuclear extracts from (A) RV and (B) LV were subjected to EMSA to monitor GATA DNA binding activity +/− antibodies (Ab). ss denotes supershifted bands. (C) Values represent means ± SEM. (D) GATA4 protein levels in nuclear extracts were measured by Western blot. A non-specific (NS) band suggests no differences in loading. *significantly different from normoxia control (n = 5 – 6).

Fig. 2. Identification of Gata4 promoter region that is activated by hypoxic PH in the RV

Rats were subjected to CH. (A) RNA was isolated, and Gata4 mRNA and 28S rRNA levels were measured by RT-PCR. The bar graph represents means ± SEM (n = 4 – 5). (B) Basal DNA binding activities of RV nuclear extracts toward ³²P-labeled Probes #1 – 7 constructed from the 250 bp Gata4 promoter region. (C) DNA binding activities in nuclear extracts toward Probe #2 were monitored by EMSA. The bar graph represents means ± SEM (n = 6). *significantly different from no hypoxia.

Fig. 3. Role of the CCAAT Box

(A) Sequences of various double stranded EMSA probes. CCAAT box regions are indicated in the shaded area. (B) Nuclear extracts from rat RV (7-day hypoxia treated), mouse heart or HL-1 cardiac muscle cells were subjected to EMSA using wild type or mutant Probe #2. (C) Nuclear extracts were subjected to EMSA with wild type or truncated Probe #2. (D) Heart nuclear extracts were subjected to EMSA using ³²P-labeled Probe #2 in the presence of excess cold competitor (unlabelled Probe #2). (E) HL-1 cells were transfected with the luciferase construct controlled by the 250 bp proximal region of the wild type (WT) or mutant (Mut) Gata4 promoter. Values represent means ± SEM of the ratio of 250 bp Gata4 promoter-controlled firefly luciferase activity to thymidine kinase (TK) promoter-controlled Renilla luciferase activity (n = 6). *significantly different from the wild type Gata4 promoter. Some images were grouped from different parts of the same gel and such arrangements are indicated by dividing lines.

Fig. 4. Identification of CCAAT box binding protein

(A) EMSA was performed with Probe #2 and nuclear extracts from rat RV (7-day hypoxia treated) or HL-1 cells. Supershift experiments were performed with antibodies (ab) against C/EBPβ and CBF-B. (B) Nuclear extracts from rat RV (7-day hypoxia treated) or HL-1 cells were mixed with biotinylated Probe #2 and streptavidin-agarose. Samples were centrifuged, washed and boiled. Supernatant of the final spin was subjected to SDS-PAGE and immunoblotted with CBF-B antibody. Lanes denoted minus pull down show control experiments without biotinylated Probe #2. (C) HL-1 cells were transfected with siRNA for CBF-B, nuclear extracts were prepared and subjected to Western blotting. Values in bar graphs represent means ± SEM (n = 6).

Fig. 5. Regulation of CBF/NF-Y by annexin A1

(A) Rat RV nuclear extracts (NE) or buffer alone control were immunoprecipitated with CBF-B antibody or normal goat IgG control, and immuno-blotted with mouse annexin A1 antibody. (B) Recombinant human annexin A1 was added to binding reaction mixtures for EMSA with Probe #2 and RV nuclear extracts from rats subjected to 14 days of hypoxia. Values in the bar graph represent means ± SEM (n = 6). (C) Rats were subjected to hypoxia. Annexin A1 protein levels in RV and LV homogenates were monitored by Western blot. Values in bar graphs represent means ± SEM (n = 6). (D) Annexin A1 expression was monitored in LV and RV homogenates from normal rats (n = 6). (E) CBF-B protein expression was monitored in LV and RV homogenates from normal rats (n = 6). *significant difference.

Fig. 6. Role of metal-catalyzed protein carbonylation in Gata4 gene expression and the development of RV hypertrophy

(A) Rats were subjected to hypoxia, and RV and LV homogenates were prepared. Carbonylated annexin A1 was monitored by labeling carbonylated proteins with 2,4-dinitrophenylhydrazine (DNPH), immunoprecipitated with the antibody for DNPH-derivatized proteins, and Western blotting with annexin A1 antibody. Values in bar graphs represent means ± SEM of carbonylated annexin A1 and the ratio of carbonylated annexin A1 to annexin A1 protein level (n = 6). *significantly different from control. (B) Rats were injected with deferoxamine (DFO; 20 mg/kg) or saline daily during the 4 day exposure to hypoxia. Gata4 mRNA and 28s rRNA levels were monitored by RT-PCR. Values in the bar graph represent means ± SEM (n = 5). (C) Masses of RV, LV and the septum (S) were measured and RV/(LV+S) values were calculated as an estimate of RV hypertrophy (n = 3). *significantly different from each other.