Cardiomyocyte-restricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates beta-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction.

Abstract

In the heart, nitric oxide synthases (NOS) modulate cardiac contraction in an isoform-specific manner, which is critically dependent on their cellular and subcellular localization. Defective NO production by NOS3 (endothelial NOS [eNOS]) in the failing heart may precipitate cardiac failure, which could be reversed by overexpression of NOS3 in the myocardium. We studied the influence of NOS3 in relation to its subcellular localization on the function of cardiomyocytes isolated from transgenic mice overexpressing NOS3 under the alpha-myosin heavy chain promoter (NOS3-TG). Immunoblot analysis demonstrated moderate (5-fold) NOS3 overexpression in cardiomyocytes from NOS3-TG heterozygotes. Caveolar localization of transgenic eNOS was demonstrated by immunofluorescence, coimmunoprecipitation with caveolin-3, sucrose gradient fractionation, and immunogold staining revealed by electron microscopy. Compared with wild-type littermate, contractility of NOS3-TG cardiomyocytes analyzed by videomicroscopy revealed a lower incidence of spontaneous arrhythmic contractions (n=32, P<0.001); an attenuation of the beta-adrenergic positive inotropic response (isoproterenol, 10(-7) mol/L: 62.1+/-7.8% versus 90.8+/-8.0% of maximum Ca2+ response; n=10 to 17; P<0.05); a potentiation of the muscarinic negative chronotropic response (carbamylcholine, 3.10(-8) mol/L: -63.9+/-14% versus -27.7+/-5.6% of basal rate; n=8 to 10; P<0.05), confirmed by telemetry in vivo; and an attenuation of the accentuated antagonism of beta-adrenergically stimulated contraction (-14.6+/-1.5% versus -3.5+/-1.5; n=7 to 11; P<0.05). Cardiomyocyte NOS inhibition reversed all 4 effects (P<0.05). Moderate overexpression of NOS3, targeted to caveolae in murine cardiomyocytes, potentiates the postsynaptic muscarinic response and attenuates the effect of high concentrations of catecholamines. Cardiomyocyte NOS3 may represent a promising therapeutic target to restore the sympathovagal balance and protect the heart against arrhythmia.