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

Background-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. Methods and Results-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). Conclusions-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.