Oxidant stress from nitric oxide synthase–3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load
Eiki Takimoto, Hunter C. Champion, Manxiang Li, Shuxun Ren, E. Rene Rodriguez, Barbara Tavazzi, Giuseppe Lazzarino, Nazareno Paolocci, Kathleen L. Gabrielson, Yibin Wang, David A. Kass
Eiki Takimoto, Hunter C. Champion, Manxiang Li, Shuxun Ren, E. Rene Rodriguez, Barbara Tavazzi, Giuseppe Lazzarino, Nazareno Paolocci, Kathleen L. Gabrielson, Yibin Wang, David A. Kass
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Article Cardiology

Oxidant stress from nitric oxide synthase–3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load

Abstract

Cardiac pressure load stimulates hypertrophy, often leading to chamber dilation and dysfunction. ROS contribute to this process. Here we show that uncoupling of nitric oxide synthase–3 (NOS3) plays a major role in pressure load–induced myocardial ROS and consequent chamber remodeling/hypertrophy. Chronic transverse aortic constriction (TAC; for 3 and 9 weeks) in control mice induced marked cardiac hypertrophy, dilation, and dysfunction. Mice lacking NOS3 displayed modest and concentric hypertrophy to TAC with preserved function. NOS3–/– TAC hearts developed less fibrosis, myocyte hypertrophy, and fetal gene re-expression (B-natriuretic peptide and α–skeletal actin). ROS, nitrotyrosine, and gelatinase (MMP-2 and MMP-9) zymogen activity markedly increased in control TAC, but not in NOS3–/– TAC, hearts. TAC induced NOS3 uncoupling in the heart, reflected by reduced NOS3 dimer and tetrahydrobiopterin (BH4), increased NOS3-dependent generation of ROS, and lowered Ca2+-dependent NOS activity. Cotreatment with BH4 prevented NOS3 uncoupling and inhibited ROS, resulting in concentric nondilated hypertrophy. Mice given the antioxidant tetrahydroneopterin as a control did not display changes in TAC response. Thus, pressure overload triggers NOS3 uncoupling as a prominent source of myocardial ROS that contribute to dilatory remodeling and cardiac dysfunction. Reversal of this process by BH4 suggests a potential treatment to ameliorate the pathophysiology of chronic pressure-induced hypertrophy.

Authors

Eiki Takimoto, Hunter C. Champion, Manxiang Li, Shuxun Ren, E. Rene Rodriguez, Barbara Tavazzi, Giuseppe Lazzarino, Nazareno Paolocci, Kathleen L. Gabrielson, Yibin Wang, David A. Kass

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BH4, but not H4N, prevented NOS3 uncoupling, ROS generation, and cardiac...
BH4, but not H4N, prevented NOS3 uncoupling, ROS generation, and cardiac remodeling induced by 3-week TAC. (A) Formalin-fixed (10%) hearts (upper panel) and histology (PAS methenamine; lower panel) showing concentric hypertrophy with BH4 cotreatment versus dilative hypertrophy with H4N accompanied by increased interstitial fibrosis. Scale bars: 10 mm (upper panel); 100 μm (lower panel). (B and C) Representative M-mode echocardiography (B) and PV loops (C) reveal corresponding functional improvement in BH4-treated, but not H4N-treated, hearts. (D) NOS3 dimer was preserved in BH4-treated, but not H4N-treated, hearts. (E) NOS Ca2+-dependent activity was restored by BH4 but not H4N treatment. *P < 0.05 versus sham. (F) Luminol chemiluminescence shows a decline in O2– generation in WT TAC hearts treated with BH4 but minimal effect with H4N treatment. *P < 0.05 versus sham. Lower graph shows the percent of luminol signal blunted by coincubation with L-NAME, confirming reduced NOS-derived O2– in BH4-treated hearts. **P < 0.05 versus BH4-treated. (G) Confocal images of DHE-stained (red) and DCF-stained (green) myocardium from WT TAC hearts treated with either BH4 or H4N. Scale bar: 50 μm. (H) Gelatin zymography for hearts with BH4 or H4N treatment and quantification results. The increased gel lysis in WT TAC hearts was reduced by BH4, but not H4N, therapy. *P < 0.05 versus sham.