Endothelial nitric oxide synthase promotes neonatal cardiomyocyte proliferation by inhibiting tissue inhibitor of metalloproteinase-3 expression

OBJECTIVE: We have recently demonstrated that endothelial nitric oxide synthase (eNOS) promotes cardiomyocyte proliferation. However, mechanisms by which eNOS regulates cardiomyocyte proliferation are not fully understood. The goal of the present study was to investigate the role of tissue inhibitor of metalloproteinase-3 (TIMP-3) in eNOS-mediated cardiomyocyte proliferation. METHODS AND RESULTS: Experiments were conducted in cultured neonatal mouse cardiomyocytes. TIMP-3 expression was significantly decreased in wild-type (WT) cardiomyocytes treated with an adenoviral eNOS (Ad-eNOS). Furthermore, TIMP-3 levels were significantly decreased in cardiomyocytes derived from eNOS transgenic mice. Conversely, TIMP-3 transcript levels were significantly elevated in eNOS(-/-) cardiomyocytes. The inhibitory effect of NO on TIMP-3 expression was dependent on S-nitrosylation of c-jun, a subunit of AP-1. Cell proliferation was increased in TIMP-3(-/-) cardiomyocytes while recombinant TIMP-3 decreased proliferation in both TIMP-3(-/-) and WT cardiomyocytes. Furthermore, the decline in proliferation observed in eNOS(-/-) cardiomyocytes was abrogated when TIMP-3 expression was blocked by an anti-TIMP-3 antibody. In vivo cardiomyocyte proliferation was assessed by Ki67 immunostaining on postnatal day 1 hearts. Ki67-positive cardiomyocytes were decreased in eNOS(-/-), but increased in eNOS-Tg and TIMP-3(-/-) hearts compared to WT controls. CONCLUSIONS: Our study suggests that eNOS promotes neonatal cardiomyocyte proliferation by inhibiting TIMP-3 expression.