Abstract-Human atrial fibrillation (AF) has been associated with increased atrial oxidative stress. In animal models, inhibition of reactive oxygen species prevents atrial remodeling induced by rapid pacing, suggesting that oxidative stress may play an important role in the pathophysiology of AF. NAD(P)H oxidase is a major source of superoxide in the cardiovascular system; however, whether this enzyme contributes to atrial oxidative stress in AF remains to be elucidated. We investigated the sources of superoxide production (using inhibitors and substrates of a range of oxidases, RT-PCR, immunofluorescence, and immunoblotting) in tissue homogenates and isolated atrial myocytes from the right atrial appendage (RAA) of patients undergoing cardiac surgery (nϭ54 in sinus rhythm [SR] and 15 in AF). A membrane-bound gp91phox containing NAD(P)H oxidase in atrial myocytes was the main source of atrial superoxide production in SR and in AF. NADPH-stimulated superoxide release from RAA homogenates was significantly increased in patients with AF in the absence of changes in mRNA expression of the p22phox and gp91phox subunits of the NAD(P)H oxidase. In contrast with findings in SR patients, NO synthases (NOSs) contributed significantly to atrial superoxide production in fibrillating atria, suggesting that increased oxidative stress in AF may lead to NOS "uncoupling." These findings indicate that a myocardial NAD(P)H oxidase and, to a lesser extent, dysfunctional NOS contribute significantly to superoxide production in the fibrillating human atrial myocardium and may play an important role in the atrial oxidative injury and electrophysiological remodeling observed in patients with AF.

Background-The neuronal isoform of nitric oxide synthase (nNOS) has been implicated in the regulation of basal and ␤-adrenergic inotropy in normal and chronically infarcted hearts. Furthermore, myocardial nNOS expression and activity increase in failing hearts, raising the possibility that nNOS may influence left ventricular (LV) remodeling progression and functional deterioration after myocardial infarction (MI) Methods and Results-We compared LV remodeling at 1, 4, and 8 weeks after MI in nNOS-knockout mice (nNOS -/-) and their wild-type (WT) littermates matched for infarct size by using a highly accurate 3-dimensional echocardiographic technique. Basal LV hemodynamics and the inotropic response to dobutamine infusion (4 and 16 ng · g Ϫ1 · min Ϫ1 ) were also evaluated 8 weeks after MI. Sham-operated nNOS -/-mice showed enhanced basal LV contractility (PϽ0.03 versus WT, as evaluated by preload-recruitable stroke work) but an attenuated inotropic response to dobutamine infusion (PϽ0.01 versus WT). Both basal and ␤-adrenergic LV relaxations were significantly impaired in nNOS -/-mice. Survival after MI did not differ between groups. However, nNOS -/-mice developed a faster and more severe LV dilation compared with WT mice (PϽ0.05 for both end-systolic and end-diastolic volume indices). WT mice maintained a positive inotropic response to dobutamine 8 weeks after MI. In contrast, infarcted nNOS -/-mice responded to dobutamine with a dramatic fall in LV contractility (PϽ0.01 for preload-recruitable stroke work). Conclusions-nNOS plays a crucial role in preventing adverse LV remodeling and maintaining myocardial ␤-adrenergic reserve after MI. Taken together, our findings suggest that upregulation of myocardial nNOS in infarcted hearts may be an important adaptive mechanism. (Circulation. 2005;112:3729-3737.)

Abstract-Stimulation of nitric oxide (NO) release from the coronary endothelium facilitates myocardial relaxation via a cGMP-dependent reduction in myofilament Ca 2ϩ sensitivity. Recent evidence suggests that NO released by a neuronal NO synthase (nNOS) in the myocardium can also hasten left ventricular relaxation; however, the mechanism underlying these findings is uncertain. Here we show that both relaxation (TR 50 ) and the rate of [Ca 2ϩ ] i transient decay (tau) are significantly prolonged in field-stimulated or voltage-clamped left ventricular myocytes from nNOS Ϫ/Ϫ mice and in wild-type myocytes (nNOS ϩ/ϩ ) after acute nNOS inhibition. Disabling the sarcoplasmic reticulum abolished the differences in TR 50 and tau, suggesting that impaired sarcoplasmic reticulum Ca 2ϩ reuptake may account for the slower relaxation in nNOS Ϫ/Ϫ mice. In line with these findings, disruption of nNOS (but not of endothelial NOS) decreased phospholamban phosphorylation (P-Ser 16 PLN), whereas nNOS inhibition had no effect on TR 50 or tau in PLN Ϫ/Ϫ myocytes. Inhibition of cGMP signaling had no effect on relaxation in either group whereas protein kinase A inhibition abolished the difference in relaxation and PLN phosphorylation by decreasing P-Ser 16 PLN and prolonging TR 50 in nNOS ϩ/ϩ myocytes. Conversely, inhibition of type 1 or 2A protein phosphatases shortened TR 50 and increased P-Ser 16 PLN in nNOS Ϫ/Ϫ but not in nNOS ϩ/ϩ myocytes, in agreement with data showing increased protein phosphatase activity in nNOS Ϫ/Ϫ hearts. Taken together, our findings identify a novel mechanism by which myocardial nNOS promotes left ventricular relaxation by regulating the protein kinase A-mediated phosphorylation of PLN and the rate of sarcoplasmic reticulum Ca 2ϩ reuptake via a cGMP-independent effect on protein phosphatase activity. Key Words: neuronal NOS Ⅲ nitric oxide Ⅲ relaxation Ⅲ phospholamban Ⅲ phosphatases T he facilitatory effects of nitric oxide (NO) on myocardial relaxation and left ventricular (LV) diastolic distensibility are well documented. In animal models and in humans, stimulation of NO release from the coronary endothelium hastens relaxation and enhances LV compliance. 1 The paracrine effects of endothelial-derived NO can be reproduced by applying cGMP analogs to isolated LV myocytes 2 and have been attributed to a reduction in myofilament Ca 2ϩ sensitivity secondary to troponin I phosphorylation by the cGMPdependent protein kinase (PK)G. 3 Through this mechanism, endogenous NO production may facilitate the Frank-Starling response 4 and maintain the LV preload reserve in failing hearts. 5 In contrast, the involvement of myocardial NO production in the regulation of relaxation has remained a matter of debate.An autocrine effect of NO on myocardial relaxation was first suggested in 1999, when inhibition of a neuronal-like NOS (nNOS) localized to the sarcoplasmic reticulum (SR) of LV myocytes was shown to increase the thapsigarginsensitive Ca 2ϩ uptake from cardiac SR vesicles. 6 These data implied that constitutiv...