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. 2000 Apr 11;97(8):4239-44.
doi: 10.1073/pnas.070371497.

Cardiac fibrosis in mice lacking brain natriuretic peptide

N Tamura  1 , Y Ogawa, H Chusho, K Nakamura, K Nakao, M Suda, M Kasahara, R Hashimoto, G Katsuura, M Mukoyama, H Itoh, Y Saito, I Tanaka, H Otani, M Katsuki
Affiliations

Cardiac fibrosis in mice lacking brain natriuretic peptide

N Tamura et al. Proc Natl Acad Sci U S A. .

Abstract

Cardiac fibrosis, defined as a proliferation of interstitial fibroblasts and biosynthesis of extracellular matrix components in the ventricles of the heart, is a consequence of remodeling processes initiated by pathologic events associated with a variety of cardiovascular disorders, which leads to abnormal myocardial stiffness and, ultimately, ventricular dysfunction. Brain natriuretic peptide (BNP) is a cardiac hormone produced primarily by ventricular myocytes, and its plasma concentrations are markedly elevated in patients with congestive heart failure and acute myocardial infarction. However, its precise functional significance has been undefined. In this paper, we report the generation of mice with targeted disruption of BNP (Nppb(-/-) mice). We observed multifocal fibrotic lesions in the ventricles from Nppb(-/-) mice. No signs of systemic hypertension and ventricular hypertrophy are noted in Nppb(-/-) mice. In response to ventricular pressure overload, focal fibrotic lesions are increased in size and number in Nppb(-/-) mice, whereas no focal fibrotic changes are found in wild-type littermates (Nppb(+/+) mice). This study establishes BNP as a cardiomyocyte-derived antifibrotic factor in vivo and provides evidence for its role as a local regulator of ventricular remodeling.

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Figures

Figure 1
Figure 1
Targeted disruption of Nppb. (A) Restriction maps of the wild-type 129/Sv mouse Nppb allele, targeting vector, and predicted disrupted allele. Filled bars indicate exons (I–III). The genomic fragments used as 5′ and 3′ external probes are shown as hatched boxes. B, BamHI; Se, SpeI; Sl, SalI; N, NotI; tk, herpes simplex virus thymidine kinase gene; neo, neomycin-resistance gene. (B) Southern blot analysis of genomic DNAs from F2 offspring generated by heterozygous intercrosses of F1 mice upon digestion with BamHI. (C) Northern blot analysis of Nppb mRNA in atria and ventricles from Nppb+/+, Nppb+/−, and Nppb−/− mice. Total RNAs (1 and 10 μg) from atria and ventricles, respectively, were analyzed. (D) Cardiac BNP concentrations in Nppb+/+, Nppb+/−, and Nppb−/− mice (n = 3). Open and hatched bars represent Nppb+/+ and Nppb+/− mice, respectively. n.d., not detectable in Nppb−/− mice. ∗, P < 0.05 vs. Nppb+/+ mice.
Figure 2
Figure 2
Light and electron microscopic examinations of hearts from Nppb+/+ and Nppb−/− mice. (A and B) Masson's trichrome staining of transverse sections of ventricles from Nppb+/+ (A) and Nppb−/− (B) mice at the level of papillary muscles. Focal fibrotic lesions are indicated by arrowheads. (C and D) Higher magnification (×25) of subendocardial regions of ventricles from Nppb+/+ (C) and Nppb−/− (D) mice. (E and F) Electron microscopy of left ventricular free wall from Nppb−/− mice. Supercontracted sarcomeres and disorganized myofibrils are indicated by filled and open arrowheads, respectively. Z, Z-bands in sarcomeres. (G and H) Electron microscopy of atria from Nppb+/+ (G) and Nppb−/− (H) mice. Atrial granules are indicated by arrowheads. (Scale bars represent 1 μm in E–H.)
Figure 3
Figure 3
Gene expression in ventricles from Nppb+/+ and Nppb−/− mice. (A) Northern blot analysis of Acta1, Atp2a2, Nppa, Edn1, and Tgfb1 mRNAs in ventricles from Nppb+/+ mice (n = 10) and Nppb−/− mice with or without cardiac fibrosis (n = 5 for each) at 20 wk of age. Hybridization signal for Gapd mRNA was used as an internal control. Total RNAs (20 μg) were analyzed, and representative cases are presented. (B–F) Shown are RT-PCR and Southern blot analysis of Ace mRNA (B and C) and Northern blot analysis of Tgfb3 and Cola1 mRNAs (D–F) in ventricles from Nppb+/+ and Nppb−/− mice at 10 wk of age (n = 5 for each) and ventricles from Nppb+/+ mice (n = 15) and Nppb−/− mice with or without cardiac fibrosis (n = 8 for each) at 20 wk of age. Representative cases (B and D) and quantification of Ace (C), Tgfb3 (E), and Cola1 (F) mRNA levels are shown. Open, hatched, and filled bars represent mRNA levels for Nppb+/+ mice, Nppb−/− mice without cardiac fibrosis, and Nppb−/− mice with cardiac fibrosis, respectively. The mean mRNA levels in Nppb+/+ mice (open bars) were defined as 1.0 arbitrary unit. ∗, P < 0.05; ∗∗, P < 0.01 vs. Nppb+/+ mice.
Figure 4
Figure 4
Cardiac fibrosis and cardiac gene expression in Nppb+/+ and Nppb−/− mice that received abdominal aortic constriction. (A and B) Masson's trichrome staining of transverse sections of ventricles from Nppb+/+ (A) and Nppb−/− (B) mice at the level of papillary muscles 7 days after aortic constriction. (C) Fibrosis-to-total area ratios of ventricular transverse sections from Nppb+/+ and Nppb−/− mice that received sham operation (open bars) or aortic constriction (filled bars) (n = 7). ∗, P < 0.05 vs. sham-operated group by one-way ANOVA among four groups. (D) Northern blot analysis of Nppb, Nppa, Acta1, Atp2a2, Tgfb1, Tgfb3, and Cola1 mRNAs. Total RNAs (10 μg) were used in each lane. S, sham-operated; C, constricted. (E) RT-PCR and Southern blot analysis of Ace mRNA are shown. (F–H) Quantification of Tgfb3 (F), Cola1 (G), and Ace (H) mRNA levels in ventricles from sham-operated (open bars) and constricted (closed bars) mice of each genotype (n = 7). The mean mRNA level for each gene in sham-operated Nppb+/+ mice was defined as 1.0 arbitrary unit. ∗, P < 0.05; ∗∗, P < 0.01 vs. sham-operated mice by one-way ANOVA among four groups (F and G). †, P < 0.05 vs. Nppb+/+ mice by two-way ANOVA (H).
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