Osteopontin (OPN) is a secreted protein involved in cardiac remodeling and hypertrophy induced by the expression of angiotensin 2 (ANG II). Transgenic mice lacking OPN in the heart prevent cardiac hypertrophy and fibrosis mediated by ANG II. ANG II is an activator of the Na+/H+ exchanger isoform 1 (NHE1), a plasma membrane protein that regulates intracellular pH and also implicated in cardiac hypertrophy. Interestingly, transgenic mice expressing active NHE1 developed spontaneous cardiac hypertrophy in association with elevated levels of osteopontin. Whether ANG II induces osteopontin expression via activation of NHE1 remains unknown.
ANG II (100 nM, 16h) induces cardiomyocyte hypertrophy associated with an increased OPN protein expression ( ANG II: 138% ±30%, p<0.05). ANG II stimulated cardiomyocyte hypertrophy and OPN protein expression regressed in the presence of the NHE1 inhibitor, EMD (10μM) and calcineurin inhibitor, FK506 (1μM). H9c2 cells infected with the active form of NHE1 showed that NHE1 activation is enough to induce the hypertrophic pathway NFAT3/GATA4 and OPN expression. These effects were prevent by FK506. Our results demonstrate that NHE1 activation, either using an active NHE1 or through induction with ANG II, induces NFAT3 translocation into the nucleus and also demonstrate a significant activation of the transcription factor GATA-4 (NHE1: 149% ±28%, p<0.05). The activation of GATA-4 and OPN protein expression induced by active NHE1 is inhibited by FK506 treatment.
These results suggest that NFAT3/GATA4 pathway is a critical component of the NHE1 hypertrophic pathways activated by ANG II in cardiomyoblast and plays a key role in the regulation of cardiomyocyte hypertrophy and OPN protein expression.