Ventricular expression of natriuretic peptides in Npr1(-/-) mice with cardiac hypertrophy and fibrosis

Abstract

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that regulate blood pressure and volume, and exert their biological actions via the natriuretic peptide receptor-A gene (Npr1). Mice lacking Npr1 (Npr(-/-)) have marked cardiac hypertrophy and fibrosis disproportionate to their increased blood pressure. This study examined the relationships between ANP and BNP gene expression, immunoreactivity and fibrosis in cardiac tissue, circulating ANP levels, and ANP and BNP mRNA during embryogenesis in Npr1(-/-) mice. Disruption of the Npr1 signaling pathway resulted in augmented ANP and BNP gene and ANP protein expression in the cardiac ventricles, most pronounced for ANP mRNA in females [414 +/- 57 in Npr1(-/-) ng/mg and 124 +/- 25 ng/mg in wild-type (WT) by Taqman assay, P < 0.001]. This increased expression was highly correlated to the degree of cardiac hypertrophy and was localized to the left ventricle (LV) inner free wall and to areas of ventricular fibrosis. In contrast, plasma ANP was significantly greater than WT in male but not female Npr1(-/-) mice. Increased ANP and BNP gene expression was observed in Npr1(-/-) embryos from 16 days of gestation. Our study suggests that cardiac ventricular expression of ANP and BNP is more closely associated with local hypertrophy and fibrosis than either systemic blood pressure or circulating ANP levels.

Fig. 1

Representative autoradiographs of longitudinal sections through entire mouse hearts from wild-type (WT) male (A) and female (B) mice and natiuretic peptide receptor-A knockout (Npr1^–/–) male (C) and female (D) mice hybridized with atrial natriuretic peptide (ANP) and ANP control probe (E). Positive ANP expression is observed as darkened regions in the left atria (la), left ventricle (lv), right atria (ra), and right ventricle (rv).

Fig. 2

Representative autoradiographs of longitudinal sections through entire male and female WT (A and B) and male and female Npr1^–/– (C and D) adult mouse hearts hybridized with brain natriuretic peptide (BNP) and BNP control probe (E). Positive BNP expression is observed as darkened regions (see Fig. 1 for abbreviations).

Fig. 3

A: Masson trichrome staining (top) and ANP immunohistochemistry (bottom) of female Npr1^–/– and WT hearts. Blue color with Masson trichrome staining indicates collagen deposition, which is characteristic of fibrosis. Brown staining indicates ANP immunoreactivity (IR) and can be seen colocalized with perivascular fibrosis. Contents of the blood vessels also stain brown due to IR of blood clots within the lumen. White arrows show regions of left ventricular fibrosis and the colocalization of ANP-IR to these regions. Black arrows show ANP-IR at nonfibrotic regions of the inner free wall of the left ventricle. Scale bars in left and middle represent 500 μM, and bars in right represent 50 μM. B: Masson trichrome staining (top) and dark-field photomicrographs of representative longitudinal sections through the left ventricles of female Npr1^–/– and WT hearts hybridized with ANP and BNP. Blue color with Masson trichrome staining indicates collagen deposition, which is characteristic of fibrosis. Positive ANP and BNP gene expression is seen as bright silver grains above expressing cells. White arrows illustrate regions of ANP and BNP expression colocalized to areas of left ventricular fibrosis. Scale bars represent 200 μM. a, Atria.

Fig. 4

Representative autoradiographs of longitudinal sections through 11-day-old (11d) and 16-day-old (16d) Npr1^–/– embryos (A and C) and WT embryos (B and D) hybridized with ANP and ANP control probe (E). Positive ANP expression is observed as darkened regions in the atrium (a), ventricle (v), lung (l), skeletal muscle (sm), bladder (b), and vertebrae (vt).

Fig. 5

Representative autoradiographs of longitudinal sections through 11- and 16-day-old Npr1^–/– embryos (A and C) and WT embryos (B and D) hybridized with BNP and BNP control probe (E). Positive BNP expression is observed as darkened regions (see Fig. 4 for abbreviations).