Cardiac p300 is involved in myocyte growth with decompensated heart failure

Abstract

A variety of stresses on the heart initiate a number of subcellular signaling pathways, which finally reach the nuclei of cardiac myocytes and cause myocyte hypertrophy with heart failure. However, common nuclear pathways that lead to this state are unknown. A zinc finger protein, GATA-4, is one of the transcription factors that mediate changes in gene expression during myocardial-cell hypertrophy. p300 not only acts as a transcriptional coactivator of GATA-4, but also possesses an intrinsic histone acetyltransferase activity. In primary cardiac myocytes derived from neonatal rats, we show that stimulation with phenylephrine increased an acetylated form of GATA-4 and its DNA-binding activity, as well as expression of p300. A dominant-negative mutant of p300 suppressed phenylephrine-induced nuclear acetylation, activation of GATA-4-dependent endothelin-1 promoters, and hypertrophic responses, such as increase in cell size and sarcomere organization. In sharp contrast to the activation of cardiac MEK-1, which phosphorylates GATA-4 and causes compensated hypertrophy in vivo, p300-mediated acetylation of mouse cardiac nuclear proteins, including GATA-4, results in marked eccentric dilatation and systolic dysfunction. These findings suggest that p300-mediated nuclear acetylation plays a critical role in the development of myocyte hypertrophy and represents a pathway that leads to decompensated heart failure.

FIG. 1.

In vitro acetylation of GATA-4 in COS7 cells. COS7 cells were transfected with 2 μg of pcDNAG4 and 9 μg of pCMVwtp300 and were pulse-labeled with [¹⁴C]acetic acid sodium salt for 3 h. The nuclear extracts were immunoprecipitated with anti-GATA-4 antibody or with normal goat IgG, resolved by SDS-polyacrylamide gel electrophoresis, fixed, and autoradiographed using a bioimaging analyzer.

FIG. 2.

p300 acetylates lysine residues of GATA-4, enhances its DNA-binding activity, and is involved in GATA-4-dependent ET-1 transcription. (A) COS7 cells were transfected with 2 μg of pcDNAG4 or with (+) 9 μg of pCMVwtp300 (p300) and/or 1 μg of pwtE1A (E1A) as indicated. The total amount of DNA was kept constant by cotransfecting pCMVβ-gal. Nuclear extracts (300 μg of protein) from these cells were immunoprecipitated with anti-GATA-4 antibody, followed by sequential Western blotting with anti-acetylated lysine antibody and with anti-GATA-4 antibody. (B) The nuclear extracts used for panel A before immunoprecipitation were subjected to Western blotting using the anti-GATA-4 antibody, anti-p300 antibody, anti-E1A antibody, or anti-β-αctin antibody. (C) The same nuclear extracts were probed with a radiolabeled double-stranded oligonucleotide containing the GATA-4 site in the ET-1 promoter. Ab, antibody; small arrow, supershifted band of GATA-4. (D) COS7 cells were transfected with 2.0 μg of pETCAT, 0.1 μg of pRSVluc, 0.5 μg of pcDNAG4 or pCMVβ-gal, 2.5 μg of pCMVwtp300 or pCMVβ-gal, and 0.3 μg of pwtE1A or pCMVβ-gal. The results are expressed as n-fold activation of the normalized CAT activity (CAT/luc) relative to that resulting from transfection with 3.3 μg of pCMVβ-gal without pcDNAG4, pCMVwtp300, or pwtE1A. The data shown are the means and standard errors of the mean from three independent experiments.

FIG. 3.

PE induces p300 expression, acetylation, and DNA binding of GATA-4 in cardiac myocytes. (A) Primary cardiac myocytes from neonatal rats were stimulated with saline (SS) or PE (10⁻⁵ M) for 48 h. Nuclear extracts from these cells were subjected to Western blotting with anti-GATA-4 antibody, anti-p300 antibody, anti-PCAF antibody, or anti-β-actin antibody. (B) The same nuclear extracts (100 μg of protein) were immunoprecipitated with anti-GATA-4 antibody and sequentially subjected to Western blotting with anti-acetylated lysine antibody and with anti-GATA-4 antibody. (C and D) The same nuclear extracts were probed with a radiolabeled double-stranded oligonucleotide containing the ET-1 GATA site (C) and with one containing the Sp-1 site (D). −, absent.

FIG. 4.

p300(1514-1922) inhibits p300-induced acetylation and DNA binding of GATA-4. (A) COS7 cells were transfected with (+) 2 μg of pcDNAG4, 9 μg of pCMVwtp300, and 1 μg of pCMV1514-1922p300 as indicated. The total amount of DNA was kept constant by cotransfecting pCMVβ-gal. Nuclear extracts (300 μg of protein) from these cells were immunoprecipitated with anti-GATA-4 antibody or normal goat IgG, followed by sequential Western blotting with anti-acetylated lysine antibody and with anti-GATA-4 antibody. (B) The nuclear extracts used for panel B before immunoprecipitation were subjected to Western blotting using the anti-GATA-4 antibody, anti-p300 antibody, or anti-β-actin antibody. (C) The same nuclear extracts were probed with a radiolabeled double-stranded oligonucleotide containing the GATA-4 site in the ET-1 promoter. Small arrow, supershifted band of GATA-4.

FIG. 4.

p300(1514-1922) inhibits p300-induced acetylation and DNA binding of GATA-4. (A) COS7 cells were transfected with (+) 2 μg of pcDNAG4, 9 μg of pCMVwtp300, and 1 μg of pCMV1514-1922p300 as indicated. The total amount of DNA was kept constant by cotransfecting pCMVβ-gal. Nuclear extracts (300 μg of protein) from these cells were immunoprecipitated with anti-GATA-4 antibody or normal goat IgG, followed by sequential Western blotting with anti-acetylated lysine antibody and with anti-GATA-4 antibody. (B) The nuclear extracts used for panel B before immunoprecipitation were subjected to Western blotting using the anti-GATA-4 antibody, anti-p300 antibody, or anti-β-actin antibody. (C) The same nuclear extracts were probed with a radiolabeled double-stranded oligonucleotide containing the GATA-4 site in the ET-1 promoter. Small arrow, supershifted band of GATA-4.

FIG. 5.

p300(1514-1922) inhibits PE-induced nuclear hyperacetylation and GATA-4-dependent and PE-induced ET-1 transcription in cardiac myocytes. (A) Cardiac myocytes were transfected with 0.7 μg of pCMV1514-1922p300 or pCMVβ-gal (β-gal). Then, these cells were stimulated with saline or PE (10⁻⁵ M) for 48 h and subjected to immunocytochemical staining with antibody against acetylated lysine. +, present. (B) Nuclear extracts from these cells were subjected to Western blotting using the anti-p300 and anti-β-actin antibodies. (C) Cardiac myocytes were cotransfected with 1 μg of pETCAT, 0.05 μg of pRSVluc, 0.25 μg of pcDNAG4 or pCMVβ-gal, and 1.25 μg of pCMVwtp300, pCMV1514-1922p300, or pCMVβ-gal. The results are expressed as n-fold activation by GATA-4 of the normalized CAT activity (CAT/luc). The data shown are the means and standard errors of the mean of two independent experiments, each carried out in duplicate. (D) Cardiac myocytes were cotransfected with 2 μg of pETCAT, 0.1 μg of pRSVluc, and 0.4 μg of pCMVwtp300 or 0.4 or 1.2 μg of pCMV1514-1922p300. The total DNA content was equalized in each sample with pCMVβ-gal. The results are expressed as n-fold activation by PE of the normalized CAT activity (CAT/luc). The data shown are the means and standard errors of the mean of two independent experiments, each carried out in duplicate.

FIG. 5.

p300(1514-1922) inhibits PE-induced nuclear hyperacetylation and GATA-4-dependent and PE-induced ET-1 transcription in cardiac myocytes. (A) Cardiac myocytes were transfected with 0.7 μg of pCMV1514-1922p300 or pCMVβ-gal (β-gal). Then, these cells were stimulated with saline or PE (10⁻⁵ M) for 48 h and subjected to immunocytochemical staining with antibody against acetylated lysine. +, present. (B) Nuclear extracts from these cells were subjected to Western blotting using the anti-p300 and anti-β-actin antibodies. (C) Cardiac myocytes were cotransfected with 1 μg of pETCAT, 0.05 μg of pRSVluc, 0.25 μg of pcDNAG4 or pCMVβ-gal, and 1.25 μg of pCMVwtp300, pCMV1514-1922p300, or pCMVβ-gal. The results are expressed as n-fold activation by GATA-4 of the normalized CAT activity (CAT/luc). The data shown are the means and standard errors of the mean of two independent experiments, each carried out in duplicate. (D) Cardiac myocytes were cotransfected with 2 μg of pETCAT, 0.1 μg of pRSVluc, and 0.4 μg of pCMVwtp300 or 0.4 or 1.2 μg of pCMV1514-1922p300. The total DNA content was equalized in each sample with pCMVβ-gal. The results are expressed as n-fold activation by PE of the normalized CAT activity (CAT/luc). The data shown are the means and standard errors of the mean of two independent experiments, each carried out in duplicate.

FIG. 6.

p300(1514-1922) blocks hypertrophic responses in cardiac myocytes. (A) Cardiac myocytes were transfected with a total of 0.7 μg of pCMV1514-1922p300 or pCMVβ-gal, stimulated with saline (SS) or PE (10⁻⁵ M) for 48 h, and subjected to immunofluorescent staining with antibody to β-MHC. −, absent; +, present. (B) Measurement of cell diameter (in micrometers). The values are means and standard errors of the mean. The data are from 50 cells in each group.

FIG. 6.

p300(1514-1922) blocks hypertrophic responses in cardiac myocytes. (A) Cardiac myocytes were transfected with a total of 0.7 μg of pCMV1514-1922p300 or pCMVβ-gal, stimulated with saline (SS) or PE (10⁻⁵ M) for 48 h, and subjected to immunofluorescent staining with antibody to β-MHC. −, absent; +, present. (B) Measurement of cell diameter (in micrometers). The values are means and standard errors of the mean. The data are from 50 cells in each group.

FIG. 7.

Cardiac overexpression of p300 results in acetylation and increased DNA-binding activity of GATA-4. (A) Nuclear extracts (10 μg of protein) from WT or p300 TG mouse hearts were subjected to Western blotting with anti-p300 antibody, anti-GATA-4 antibody, or anti-β-actin antibody. (B) Nuclear extracts (400 μg of protein) from WT or TG mouse hearts were immunoprecipitated with anti-GATA-4 antibody and control goat IgG and sequentially subjected to Western blotting with anti-acetylated lysine antibody and anti-GATA-4 antibody. (C and D) Nuclear extracts from WT and TG mouse hearts were probed with a radiolabeled double-stranded oligonucleotide containing the GATA-4 site in the ET-1 promoter (C) and with one containing the Sp-1 site (D). Small arrow, supershifted band of GATA-4. (E) Analysis of ET-1 mRNA levels in WT and TG mouse hearts was performed by RT-PCR. (F) Northern blotting of total RNA from WT and TG mouse hearts for β-MHC, ANF, and GAPDH.

FIG. 8.

Histological sections of hearts from WT and p300 TG mice. (A) Gross view of histological sections of WT and TG mice at 24 weeks of age. Both sections were cut at the midsagittal level and parallel to the base. (B) Heart weight/body weight ratio (1,000) of WT and TG mice at 24 weeks of age (n = 5 for each group). (C) Histological sections at a magnification of ×200. (D) Cell diameter was measured as described in Materials and Methods. The values are means and standard errors of the mean. The data are from 50 cells in each group.

FIG. 8.

Histological sections of hearts from WT and p300 TG mice. (A) Gross view of histological sections of WT and TG mice at 24 weeks of age. Both sections were cut at the midsagittal level and parallel to the base. (B) Heart weight/body weight ratio (1,000) of WT and TG mice at 24 weeks of age (n = 5 for each group). (C) Histological sections at a magnification of ×200. (D) Cell diameter was measured as described in Materials and Methods. The values are means and standard errors of the mean. The data are from 50 cells in each group.

FIG. 9.

Echocardiographic parameters of WT and p300 TG mice. TG mice or WT littermates at the age of 24 weeks were subjected to transthoracic echocardiography. SEPth, septal wall thickness; PWth, left-ventricular posterior-wall thickness; HR, heart rate; FS, fractional shortening, which was calculated as [(LVDD − LVESD)/LVDD] × 100.