Genetic Disruption of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Triggers Differential Cardiac Fibrosis and Disorders in Male and Female Mutant Mice: Role of TGF-β1/SMAD Signaling Pathway

Abstract

The global targeted disruption of the natriuretic peptide receptor-A (NPRA) gene (Npr1) in mice provokes hypertension and cardiovascular dysfunction. The objective of this study was to determine the mechanisms regulating the development of cardiac fibrosis and dysfunction in Npr1 mutant mice. Npr1 knockout (Npr1^-/-, 0-copy), heterozygous (Npr1^+/-, 1-copy), and wild-type (Npr1^+/+, 2-copy) mice were treated with the transforming growth factor (TGF)-β1 receptor (TGF-β1R) antagonist GW788388 (2 µg/g body weight/day; ip) for 28 days. Hearts were isolated and used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analyses. The Npr1^-/- (0-copy) mice showed a 6-fold induction of cardiac fibrosis and dysfunction with markedly induced expressions of collagen-1α (3.8-fold), monocyte chemoattractant protein (3.7-fold), connective tissue growth factor (CTGF, 5.3-fold), α-smooth muscle actin (α-SMA, 6.1-fold), TGF-βRI (4.3-fold), TGF-βRII (4.7-fold), and phosphorylated small mothers against decapentaplegic (pSMAD) proteins, including pSMAD-2 (3.2-fold) and pSMAD-3 (3.7-fold), compared with wild-type mice. The expressions of phosphorylated extracellular-regulated kinase ERK1/2 (pERK1/2), matrix metalloproteinases-2, -9, (MMP-2, -9), and proliferating cell nuclear antigen (PCNA) were also significantly upregulated in Npr1 0-copy mice. The treatment of mutant mice with GW788388 significantly blocked the expression of fibrotic markers, SMAD proteins, MMPs, and PCNA compared with the vehicle-treated control mice. The treatment with GW788388 significantly prevented cardiac dysfunctions in a sex-dependent manner in Npr1 0-copy and 1-copy mutant mice. The results suggest that the development of cardiac fibrosis and dysfunction in mutant mice is predominantly regulated through the TGF-β1-mediated SMAD-dependent pathway.

Keywords: GW788388; SMAD; TGF-β1; cardiac fibrosis; guanylyl cyclase receptor; natriuretic peptides.

Figure 1

The expression analysis of the mRNA of fibrotic marker genes in the heart tissues of control and GW788388 drug-treated male Npr1 gene-disrupted mice: The expression levels of mRNA of Col-1-α, Col-3, α-SMA, MCP-1, CTGF, PAI-1, TGF-β1RI, and TGF-β1RII in the heart tissues of control and drug-treated mice are shown in panels (A–H). The expression of mRNA was normalized to GAPDH expression. Values are expressed as means ± S.E. (n = 6 animals in each group). Statistical significance is expressed as ### p < 0.001, vehicle-treated 0-copy and 1-copy vs. 2-copy; * p < 0.05, ** p < 0.01, *** p < 0.001, vehicle-treated 0-copy, 1-copy and 2 copy vs. drug-treated same gene copy number. NS: not significant.

Figure 2

Analysis of the protein levels of different SMAD and key fibrotic markers in the heart tissues of male Npr1 gene knockout mice: representative Western blots showing the levels of pSMAD-1, pSMAD-2, pSMAD-3, SMAD-4, SMAD-6, α-SMA, pTAK1, and pERK 1/2 proteins in the heart tissues of Npr1 mice, respectively (panels (A–H)). The densitometry analysis of proteins was normalized to total SMAD proteins and/or β-actin protein levels. Values are expressed as means ± S.E. (n = 6 animals in each group). Statistical significance is expressed as *** p < 0.001, Npr1^+/+ (2-copy) vs. Npr1^−/− (0-copy). In panels (A–F), Western blot was performed for the cytosolic extract and in panels (G,H), whereas Western blot was performed for both cytosolic and nuclear extracts.

Figure 3

Western blotting analysis of extracellular matrix and fibrotic proteins in the heart tissues of control and drug-treated male Npr1 gene-disrupted mice: representative Western blots showing the levels of MMP-2, MMP-9, PCNA, CTGF, and α-SMA proteins (panels (A–E)). The densitometry analysis of proteins was normalized to β-actin protein levels (panels (F–J). Values are expressed as means ± S.E. (n = 6 animals in each group). Statistical significance is expressed as ### p < 0.001, vehicle-treated 0-copy and 1-copy vs. 2-copy; * p < 0.05, ** p < 0.01, vehicle-treated 0-copy,1-copy and 2 copy vs. drug-treated same gene copy number.

Figure 4

Western blot analysis of TGF-β1 receptors and SMAD proteins in the heart tissues of control and drug-treated male Npr1 gene-ablated mice: representative Western blots showing the protein expression of TGF-β1RI and TGF-β1RII (panels (A,B)). The densitometry analysis of proteins was normalized to β-actin expression (panels (C,D)). Representative Western blots showing the protein levels of pSMAD-2 and pSMAD-3 (panels (E,F)). Densitometry analysis of pSMAD-2 and pSMAD-3 proteins was normalized to β-actin (panels (G,H)). Values are expressed as means ± S.E. (n = 6 animals in each group). Statistical significance is expressed as ### p < 0.001, vehicle-treated 0-copy and 1-copy vs. 2-copy; * p < 0.05, ** p < 0.01, vehicle-treated 0-copy,1-copy and 2 copy vs. drug-treated same gene copy number.

Figure 5

Immunohistochemical analysis of the TGF-β1RI, TGF-β1RII, and pSMAD-2 proteins in heart tissue sections of control and drug-treated male Npr1 gene knockout mice: representative immunohistochemistry of TGF-β1RI and TGF-β1RII (panels (A,B)). Quantitative analysis of protein immunoreactivity in heart tissues (panels (C,D)). Representative immunohistochemistry of pSMAD-2 protein immunoreactivity in heart tissues (panel (E)). Quantitative analysis of densitometry of pSAMD-2 (panel (F)). Values are expressed as means ± S.E. (n = 6 animals in each group). Statistical significance is expressed as ### p < 0.001, vehicle-treated 0-copy and 1-copy vs. 2-copy; * p < 0.05, ** p < 0.01, vehicle-treated 0-copy,1-copy and 2 copy vs. drug-treated same gene copy number.

Figure 6

Sex-based analysis of the systolic blood pressure and cardiac hypertrophy in Npr1 0-copy, 1-copy, and wild-type 2-copy male and female control and GW788388-treated mice: the SBP, HW/BW, LV/BW, and TL/HW ratios are shown in panels (A–D). Values are expressed as means ± S.E. (n = 6 animals in each group). Statistical significance is expressed as # p < 0.05, ## p < 0.01, ### p < 0.001, vehicle-treated 0-copy and 1-copy vs. 2-copy; ** p < 0.01, vehicle-treated 0-copy, 1-copy and 2 copy vs. drug-treated same gene copy number. NS: not significant.

Figure 7

Analysis of interstitial and perivascular fibrosis in the control and drug-treated male Npr1 gene-ablated mouse heart: Representative heart tissue sections for interstitial fibrosis from control and drug-treated male mice were stained with picrosirius red staining (panels (A,B)). The red color is indicative of fibrosis. Similarly, the representative heart tissue sections for perivascular fibrosis are presented from control and drug-treated mice (panels (C,D)). Values are expressed as means ± S.E. (n = 6 animals in each group). Statistical significance is expressed as ### p < 0.001, vehicle-treated 0-copy and 1-copy vs. 2-copy; *** p < 0.001, vehicle-treated 0-copy,1-copy and 2 copy vs. drug-treated same gene copy number. NS: not significant.

Figure 8

Representative images of the echocardiogram in the left ventricle of control and GW788388 drug-treated male Npr1 gene-disrupted mice: representative M-mode images of the left ventricle for each group were calculated from the average of 6 sessions/day for five consecutive days. The representative images are shown for 0-copy, 1-copy, and 2-copy mice with and without GW788388 treatments. Longer blue lines indicate LVED-d and shorter blue lines indicate LVED-s in the echocardiogram images. Each image is the representative of a minimum of three cardiac cycles per animal (n = 6 mice per groups).

Figure 9

Sex-based echocardiographic analyses of cardiac structures and functions in male and female Npr1 mutant and control mice: left ventricular end systolic dimension (LVED-s); left ventricular end diastolic dimension (LVED-d); interventricular septal wall thickness (IVST); posterior wall thickness (PWT); fractional shortening (FS); and heart rate (HR) are presented from male and female mice (panels (A–F)). Values are expressed as means ± S.E. (n = 6 animals in each group). Statistical significance is expressed as # p < 0.05, ## p < 0.01, ### p < 0.001, vehicle-treated 0-copy and 1-copy vs. 2-copy; * p < 0.05, ** p < 0.01, *** p < 0.001, vehicle-treated 0-copy, 1-copy and 2-copy vs. drug-treated same gene copy number. NS: not significant.

Figure 10

Schematic diagram representation of the proposed mechanisms whereby treatment with the TGF-β1R antagonist GW788388 inhibits the development of cardiac fibrosis and remodeling in Npr1 gene-ablated mutant mice: disruption of Npr1 gene leads to an unbalanced activation of TGF-β1 signaling cascade that triggers the expression of fibrotic markers, thereby activating the specific molecular and structural changes leading to fibrosis and hypertrophic remodeling in a mutant mouse heart. Treatment with the TGF-β1 receptor antagonist GW788388 ameliorates by blocking the activation and nuclear translocation of SMAD-mediated signaling networks, which protects against fibrosis and hypertrophic remodeling in 0-copy and 1-copy mutant mice hearts.