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

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)-beta 1 receptor (TGF-beta 1R) antagonist GW788388 (2 mu g/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-1 alpha (3.8-fold), monocyte chemoattractant protein (3.7-fold), connective tissue growth factor (CTGF, 5.3-fold), alpha-smooth muscle actin (alpha-SMA, 6.1-fold), TGF-beta RI (4.3-fold), TGF-beta RII (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-beta 1-mediated SMAD-dependent pathway.