Nitric oxide inhibits endothelin-1-induced neonatal cardiomyocyte hypertrophy via a RhoA-ROCK-dependent pathway

Abstract 

Although nitric oxide (NO) has received extensive attention as an anti-hypertrophic agent the mechanisms underlying its regulation of endothelin-1 (ET-1) have not been fully elucidated. Since RhoA has been identified as an important mediator of cardiac hypertrophy and is inhibited by NO in vascular tissue, we sought to determine whether the anti-ET-1 effects of NO in cardiomyocytes were mediated via inhibition of the RhoA-ROCK cascade in the context of cardiac hypertrophy. Neonatal rat ventricular myocytes were cultured in the presence of ET-1 (10 nM) with or without pre-treatment with the NO donor S-nitroso-n-acetylpenicillamine (SNAP; 100 μM), 8-Br-cGMP (cGMP; 100 μM), the RhoA inhibitor C3 exoenzyme (C3; 30 ng/ml), or the ROCK inhibitor Y-27632 (10 μM). ET-1-induced cardiomyocyte hypertrophy was prevented by pre-treatment with SNAP, cGMP, C3, or Y-27632. The hypertrophic response to ET-1 was associated with significantly increased gene and protein expression of both NOS2 and NOS1 although NOS3 was unaffected. ET-1 treatment for 15 min increased membrane-bound RhoA 2.6-fold (p<0.05), which was prevented by both SNAP and cGMP (p<0.05). These effects were associated with a complete abrogation of ET-1-induced phosphorylation of the downstream target of RhoA, cofilin-2, that was mimicked by direct inhibition of RhoA and ROCK. In addition, confocal microscopy and Western blotting revealed that 24 h ET-1 treatment reduced the G- to F-actin ratio 67% (p<0.05) which was prevented by SNAP, cGMP, C3 and Y (p<0.05). Taken together, these results suggest that the anti-hypertrophic effects of NO are due, in part, to cGMP-dependent inhibition of the RhoA-ROCK-cofilin signalling pathway. These findings may be important in understanding the mechanisms of anti-ET-1 and anti-hypertrophic effects of NO as well as in the development of novel RhoA-targeted therapeutic interventions for treating cardiac hypertrophy.

Abbreviations: ET-1, Endothelin-1, NO, nitric oxide, ROCK, Rho-associated coiled coil containing kinase, MAPK, mitogen-activated protein kinase, NOS, nitric oxide synthase, PDE, phosphodiesterase, SNP, sodium nitroprusside, SNAP, S-nitroso-n-acetylpenicillamine, SRF, serum response factor, GDI, guanine dissociation inhibitor

Keywords: Endothelin, Cardiomyocyte hypertrophy, RhoA-ROCK, Nitric oxide, Cyclic GMP, G- and F-actin