Retinoic acid and sodium butyrate suppress the cardiac expression of hypertrophic markers and proinflammatory mediators in Npr1 gene-disrupted haplotype mice

Abstract

The objective of the present study was to examine the genetically determined differences in the natriuretic peptide receptor-A (NPRA) gene (Npr1) copies affecting the expression of cardiac hypertrophic markers, proinflammatory mediators, and matrix metalloproteinases (MMPs) in a gene-dose-dependent manner. We determined whether stimulation of Npr1 by all-trans retinoic acid (RA) and histone deacetylase (HDAC) inhibitor sodium butyric acid (SB) suppress the expression of cardiac disease markers. In the present study, we utilized Npr1 gene-disrupted heterozygous (Npr1(+/-), 1-copy), wild-type (Npr1(+/+), 2-copy), gene-duplicated (Npr1(++/+), 3-copy) mice, which were treated intraperitoneally with RA, SB, and a combination of RA/SB, a hybrid drug (HB) for 2 wk. Untreated 1-copy mice showed significantly increased heart weight-body weight (HW/BW) ratio, blood pressure, hypertrophic markers, including beta-myosin heavy chain (β-MHC) and proto-oncogenes (c-fos and c-jun), proinflammatory mediator nuclear factor kappa B (NF-κB), and MMPs (MMP-2, MMP-9) compared with 2-copy and 3-copy mice. The heterozygous (haplotype) 1-copy mice treated with RA, SB, or HB, exhibited significant reduction in the expression of β-MHC, c-fos, c-jun, NF-κB, MMP-2, and MMP-9. In drug-treated animals, the activity and expression levels of HDAC were significantly reduced and histone acetyltransferase activity and expression levels were increased. The drug treatments significantly increased the fractional shortening and reduced the systolic and diastolic parameters of the Npr1(+/-) mice hearts. Together, the present results demonstrate that a decreased Npr1 copy number enhanced the expression of hypertrophic markers, proinflammatory mediators, and MMPs, whereas an increased Npr1 repressed the cardiac disease markers in a gene-dose-dependent manner.

Keywords: fibrosis; gene-disruption; histone acetyltransferase; histone deacetylase; hypertrophy; natriuretic peptide receptor-A.

Fig. 1.

Analysis of interstitial collagen in control and drug-treated Npr1 gene-targeted mice hearts. Representative heart tissue sections (A) and the fibrosis analysis (B) of collagen from control, retinoic acid (RA), sodium butyrate (SB), and hybrid (HB) drug-treated mice stained with Masson's trichrome. The color blue is indicative of fibrosis (×40 magnification). Values are expressed as means ± SE (n = 6 animals in each group). Statistical significance expressed as $P < 0.05, $$P < 0.01 compared with 2-copy control; *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle-treated controls.

Fig. 2.

Expression analysis of hypertrophic and fibrotic marker genes in the hearts of control and drug-treated Npr1 gene-targeted mice. mRNA expression of β-myosin heavy chain (β-MHC; A), α-skeletal actin (α-SKA; B), c-fos (C), c-jun (D), MMP-2 (E), MMP-9 (F), TIMP-1 (G), and TIMP-2 (H) in the heart tissue of control, retinoic acid (RA), sodium butyrate (SB) and hybrid (HB) drug-treated mice, normalized to GAPDH expression. Values are expressed as means ± SE (n = 6 animals in each group). Statistical significance expressed as $P < 0.05, $$P < 0.01 compared with 2-copy control; *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle-treated controls within the group.

Fig. 3.

Analysis of hypertrophic and proto-oncogenes protein levels in the heart of control, drug-treated Npr1 gene-targeted mice: A–F: representative Western blots showing the expression of β-myosin heavy chain (β-MHC), α-skeletal actin (α-SKA), c-fos, and c-jun in the heart tissue of control, retinoic acid (RA), sodium butyrate (SB), and hybrid (HB) drug-treated mice, respectively. G–J: densitometry analysis of proteins normalized to β-actin expression. Values are expressed as means ± SE (n = 6 animals in each group). Statistical significance expressed as $P < 0.05, $$P < 0.01 compared with 2-copy control; * P < 0.05, **P < 0.01 compared with vehicle-treated controls within the group.

Fig. 4.

Analysis of matrix metallo proteinase (MMP) and tissue inhibitors of MMPs (TIMP) expression levels in the heart tissues of controls and drug-treated Npr1 gene-targeted mice. A–F: representative Western blots showing the expression of MMP-2, MMP-9, TGF-β1, TIMP-1, and TIMP-2 proteins in the heart tissue of control, retinoic acid (RA), sodium butyrate (SB), and hybrid (HB) drug-treated mice, respectively. G–K: densitometry analysis of proteins normalized to β-actin expression. Values are expressed as means ± SE (n = 6 animals in each group). Statistical significance expressed as $P < 0.05, $$P < 0.01 compared with 2-copy control; *P < 0.05, **P < 0.01 compared with vehicle-treated controls within the group.

Fig. 5.

Analysis of p65, IκB phosphorylation in the heart tissues of Npr1 gene-disrupted and Npr1 gene-duplicated control and drug-treated mice. A–C: representative Western blots showing the expression of p-NF-κB (p65) and p-IκB proteins in the heart tissue of control, retinoic acid (RA), sodium butyrate (SB), and hybrid (HB) drug-treated mice, respectively. D and E: densitometry analysis of proteins normalized to β-actin expression. F: immunofluorescence of NF-κB in vehicle-treated and HB-treated Npr1 gene-targeted mice hearts. Red fluorescence indicates NF-κB expression, and blue fluorescence shows the nucleus stained with DAPI. G: quantitative fluorescence intensity analysis of NF-κB expression quantified by intracellular fluorescence intensity. Values are expressed as means ± SE (n = 8 animals in each group). Statistical significance expressed as $P < 0.05, $$P < 0.01 compared with 2-copy control; *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle-treated controls within the group. Scale bar = 50 μm.

Fig. 6.

Assay of histone deacetylase (HDAC) and histone acetyltransferase (HAT) activity in the heart tissues of Npr1 gene-targeted mice. A: quantitative analysis of HDAC activity in the heart tissues of Npr1 gene-targeted mice. B: representative Western blot showing expression of HDAC1 and HDAC2 in the heart tissue of control (C) and hybrid (HB) drug-treated mice. Densitometry analysis was done by AlphaInnotech phosphoimager software. C: HAT activity in heart tissues of Npr1 gene-targeted mice treated with HB. Values are expressed as means ± SE (n = 8 animals in each group). Statistical significance expressed as $P < 0.05, $$P < 0.01 compared with 2-copy control; *P < 0.05, **P < 0.01 compared with vehicle-treated controls within the group.

Fig. 7.

Cardiac protein expression of NPRA in control and drug-treated Npr1 gene-targeted mice. A: representative Western blot showing expression of NPRA in the heart tissue of control (C) and hybrid (HB) drug-treated mice. Densitometry analysis was done by AlphaInnotech phosphoimager software. B: plasma cGMP levels in control and drug-treated mice. Values are expressed as means ± SE of 3 independent experiments. *P < 0.05; **P < 0.01 (vehicle-treated vs. drug-treated same group); $$P < 0.01 (1-copy or 3-copy vs. 2-copy); n = 8 mice per group.

Fig. 8.

Schematic representation of the proposed mechanisms by which RA and SB treatments inhibit the development of cardiac remodeling in Npr1^+/− mice. Disruption of Npr1 gene leads to an unbalanced activation of NF-κB and AP-1 cascades that trigger the expression of MMPs, profibrotic cytokines, hypertrophic markers, NF-κB, AP-1, and HDACs, thereby activate specific molecular and structural changes leading to fibrosis and hypertrophic remodeling in 1-copy (Npr1^+/−) mice hearts. Treatment with retinoic acid and sodium butyrate increases the production of cGMP thus inhibits the expression of MMPs, hypertrophic markers, NF-κB, AP-1, and HDACs, which protect against fibrosis and hypertrophic remodeling in Npr1^+/− mice hearts. NF-κB, nuclear factor-kappa B; AP-1, activating protein-1; HDACs, histone deacetylases; HAT, histone acetyltransferase; MMPs, matrix metalloproteinases.