Abstract-A neuronal isoform of nitric oxide synthase (nNOS) has recently been located to the cardiac sarcoplasmic reticulum (SR). Subcellular localization of a constitutive NOS in the proximity of an activating source of Ca 2ϩ suggests that cardiac nNOS-derived NO may regulate contraction by exerting a highly specific and localized action on ion channels/transporters involved in Ca 2ϩ cycling. To test this hypothesis, we have investigated myocardial Ca 2ϩ handling and contractility in nNOS knockout mice (nNOS Ϫ/Ϫ ) and in control mice (C) after acute nNOS inhibition with 100 mol/L L-VNIO. nNOS gene disruption or L-VNIO increased basal contraction both in left ventricular (LV)

Background-Evidence indicates that myocardial NO production can modulate contractility, but the source of NO remains uncertain. Here, we investigated the role of a type 1 NO synthase isoform (NOS1), which has been recently localized to the cardiac sarcoplasmic reticulum, in the regulation of basal and ␤-adrenergic myocardial contraction. Methods and Results-Contraction was assessed in left ventricular myocytes isolated from mice with NOS1 gene disruption (NOS1 Ϫ/Ϫ mice) and their littermate controls (NOS1 ϩ/ϩ mice) at 3 stimulation frequencies (1, 3, and 6 Hz) in basal conditions and during ␤-adrenergic stimulation with isoproterenol (2 nmol/L). In addition, we examined the effects of acute specific inhibition of NOS1 with vinyl-L-N-5-(1-imino-3-butenyl)-L-ornithine (L-VNIO, 500 mol/L). NOS1 Ϫ/Ϫ myocytes exhibited greater contraction at all frequencies (percent cell shortening at 6 Hz, 10.7Ϯ0.92% in NOS1 Ϫ/Ϫ myocytes versus 7.21Ϯ0.8% in NOS1 ϩ/ϩ myocytes; PϽ0.05) with a flat frequency-contraction relationship. Time to 50% relaxation was increased in NOS1 Ϫ/Ϫ myocytes at all frequencies (at 6 Hz, 26.53Ϯ1.4 ms in NOS1

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...