Coronary Hemodynamics in Endothelial NO Synthase Knockout Mice

Abstract

Abstract-For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOSϪ/Ϫ). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOSϪ/Ϫ mice. Aortic endothelial cells derived from eNOSϪ/Ϫ mice displayed only background levels of NO x formation compared with wild-type (WT) cells (88 versus 1990 pmol). eNOSϪ/Ϫ mice were hypertensive (mean arterial pressure, 135Ϯ15 versus 107Ϯ8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with N -nitro-L-arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, Ϫ35%) in WT hearts was followed by sustained vasodilation (ϩ190%). L-NAME significantly reduced vasodilation in WT hearts (ϩ125%) but did not alter the initial vasoconstriction. In eNOSϪ/Ϫ hearts, the initial vasoconstriction was augmented (Ϫ70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (Ϫ49% decrease of basal flow). This effect was even more pronounced in eNOSϪ/Ϫ hearts (Ϫ71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOSϪ/Ϫ mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) Key Words: heart Ⅲ gene targeting Ⅲ reactive hyperemia Ⅲ coronary flow Ⅲ blood pressure E ndothelial NO synthase, also called type III NO synthase, is the major NOS isoenzyme that is widely expressed in endothelial cells throughout the vascular bed. It is generally accepted that endothelium-derived NO is an important factor in the control of basal vascular tone.1 NO is also involved in receptor-mediated vasodilation in response to various agonists such as ACh, ATP, thrombin, bradykinin, and others. Through experiments using NOS inhibitors 2,3 but also by use of genetically modified animals, 4,5 it has been demonstrated that functional inactivation of eNOS activity results in hypertension. In addition to the control of vascular tone, NO inhibits platelet aggregation and leukocyte adhesion to the vessel wall as well as proliferation and migration of smooth muscle cells. Thus, eNOS is considered to play an important role in maintaining the antiatherogenic surface of the vessel wall. 6In the heart, eNOS is expressed primarily in the coronary and endocardial endothelia. In addition, eNOS has been localized to cardiac myocytes and the specialized cells of s...

Voluntary physical activity alterations in endothelial nitric oxide synthase knockout mice

“…Thus, in general, energy metabolism in the heart was preserved in both sedentary and active mice under conditions of eNOS knockout, so that the low physical activity of eNOSϪ/Ϫ animals did not result from altered cardiac energetics. In addition, eNOSϪ/Ϫ mice demonstrate no changes in basal coronary flow (15), and in basal conditions they have normal cardiac hemodynamics (56) as well as inotropic and lusitropic responses (11). Under conditions of ␤-adrenergic stimulation, eNOSϪ/Ϫ hearts even demonstrate augmented inotropy (2,35).…”

Deficiency in endothelial nitric oxide synthase impairs myocardial angiogenesis

“…It is possible that proangiogenic factors are upregulated and compensated for the loss of eNOS function and promote postnatal development of myocardial capillaries in those surviving eNOS Ϫ/Ϫ mice. Lack of eNOS may induce postnatally an increase in some angiogenic factors, such as angiotensin (22) and prostaglandins (13). Whether these factors are involved in the myocardial capillary development in adult eNOS Ϫ/Ϫ mice requires further investigation.…”

Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart

“…Importantly, Chaitagneau et al 18 demonstrated that endothelium-dependent relaxations to ACh in aortae and left descending coronary arteries of C57BL/ 6J mice were completely abolished by inhibition of NOS or by gene-targeted deletion of eNOS (eNOSde®cient mice), indicating that in these vessels NO is the main contributor to the response to ACh. However, in contrast, in the integrated coronary bed Godecke et al 7,19 demonstrated that, whilst responses to bradykinin are predominantly NOmediated,19±20 responses to ACh have a signi®cant prostacyclin-dependent component. EDHF did not appear to contribute to coronary relaxation in response to these endothelium-dependent vasodilators.…”

“…During a 20-min equilibration period, coronary¯ow was adjusted to achieve a coronary perfusion pressure (CPP) of 60 AE 5 mmHg as used previously. 7 A CPP of 60 mmHg was found to produce optimal vasodilator responses and preliminary experiments demonstrated that left ventricular (LV) function was normal at this level. CPP was measured via a pressure transducer and data recorded on a MacLab/8 s system (ADI, Australia).…”

“…Vascular responses in aortic rings were studied for comparison. Only one previous study has examined coronary vascular responses in the whole mouse heart 7 although vascular responsiveness to endothelium-dependent and -independent agonists has been studied in mouse aortic rings. 3,4,8,9 None of these studies, however, compared vascular responsiveness between different mouse strains.…”