Sub-cellular targeting of constitutive NOS in health and disease

Journal of Molecular and Cellular Cardiology 52 (2012) 341–350

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Review article

Sub-cellular targeting of constitutive NOS in health and disease Yin Hua Zhang a, Barbara Casadei b,⁎ a b

Department of Physiology, Seoul National University, College of Medicine, Seoul, Republic of Korea Department of Cardiovascular Medicine, University of Oxford, Oxford, UK

a r t i c l e

i n f o

Article history: Received 18 April 2011 Received in revised form 7 September 2011 Accepted 8 September 2011 Available online 16 September 2011 Keywords: nNOS eNOS Compartmentalization Signaling Myocardial function

a b s t r a c t Constitutive nitric oxide synthases (NOSs) are ubiquitous enzymes that play a pivotal role in the regulation of myocardial function in health and disease. The discovery of both a neuronal NOS (nNOS) and an endothelial NOS (eNOS) isoform in the myocardium and the availability of genetically modified mice with selective eNOS or nNOS gene deletion have been of crucial importance for understanding the role of constitutive nitric oxide (NO) production in the myocardium. eNOS and nNOS are homologous in structure and utilize the same cofactors and substrates; however, they differ in their subcellular localization, regulation, and downstream signaling, all of which may account for their distinct effects on excitation–contraction coupling. In particular, eNOS-derived NO has been reported to increase left ventricular (LV) compliance, attenuate beta-adrenergic inotropy and enhance parasympathetic/muscarinic responses, and mediate the negative inotropic response to β3 adrenoreceptor stimulation via cGMP-dependent signaling. Conversely, nNOS-derived NO regulates basal myocardial inotropy and relaxation by inhibiting the sarcolemmal Ca2+ current (ICa) and promoting protein kinase A-dependent phospholamban (PLN) phosphorylation, independent of cGMP. By inhibiting the activity of myocardial oxidase systems, nNOS regulates the redox state of the myocardium and contributes to maintain eNOS “coupled” activity. After myocardial infarction, up-regulation of myocardial nNOS attenuates adverse remodeling and prevents arrhythmias whereas uncoupled eNOS activity in murine models of left ventricular pressure overload accelerates the progress towards heart failure. Here we review the evidence in support of the idea that NOS subcellular localization, mode of activation, and downstream signaling account for the diverse and highly specialized actions of NO in the heart. This article is part of a Special Issue entitled “Local Signaling in Myocytes”. © 2011 Elsevier Ltd. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential regulation of myocardial function by eNOS and nNOS . . . . . . . 2.1. Lessons from mice with selective knockout of the eNOS or nNOS gene 2.2. Lessons from myocardial-specific overexpression of eNOS or nNOS . . 3. Molecular mechanisms downstream of NO . . . . . . . . . . . . . . . . 3.1. cGMP-dependent signaling . . . . . . . . . . . . . . . . . . . . 3.2. S-nitrosylation of E–C coupling proteins and NOS-mediated regulation 3.3. nNOS regulation of myocardial redox signaling . . . . . . . . . . . 3.4. NOS uncoupling and myocardial function . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ⁎ Corresponding author at: Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK. Tel.: + 44 1865 234664; fax: + 44 1865 234667. E-mail address: [email protected] (B. Casadei). 0022-2828/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2011.09.006

Initially identified as endothelial derived relaxing factor by R.F. Furchgott et al. in 1980 [1], nitric oxide (NO) is a ubiquitous molecule that is involved in the regulation of almost all aspects of cellular

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function, including metabolism, cell growth, proliferation, apoptosis and cell death. In the cardiovascular system, NO plays a pivotal role in the regulation of contraction and Ca 2+ handling of cardiac myocytes and vascular smooth muscle cells by targeting an array of downstream effectors. NO is generated by a group of enzymes, named NO synthases (NOSs), which catalyze the conversion of L-arginine to L-citrulline in a reaction that requires O2 and cofactors. The neuronal isoform of NOS (nNOS or NOS1), which was first identified in neurons, has also been found to be constitutively expressed in cardiac myocytes [2] and vascular smooth muscle cells [3]. The endothelial isoform of NOS (eNOS or NOS3) is expressed both in endothelial cells and in cardiac myocytes [4]. Both eNOS and nNOS are dimers; each monomer contains an amino-terminal oxygenase domain (N-terminal) and a carboxy-terminal reductase domain (C-terminal). The oxygenase domain binds the substrate L-arginine and contains the co-factor tetrahydrobiopterin (BH4) and a cytochrome P-450-type heme active site. The reductase domain contains binding sites for flavins (FMN, FAD) and NADPH (the electron donor). The two monomers are dimerized and NOSs produce NO following Ca 2+ binding to calmodulin, which facilitates the electron transfer from the carboxyl-terminal reductase domain to its heme-containing amino-terminal domain, (Fig. 1). Since the initial observations in the late 1980s suggesting that NO might regulate myocardial function and Ca 2+ handling (reviewed in [4,5]), the effects of NO on myocardial contraction have been intensively studied in a variety of in vitro cardiac preparation (i.e., isolated cardiac myocytes, trabeculae, isolated hearts) and in vivo both in animal models and in humans. Findings from these investigations have suggested that constitutive NO production may be implicated in the rapid (Frank–Starling) [6,7] and sustained (Anrep effect) [8] inotropic response to mechanical loading, and in the force–frequency response, as observed by some investigators [9,10] but not by others [11,12]. Similarly, eNOS-derived NO has been thought to play a role in harnessing beta-adrenergic inotropy and in mediating the negative inotropic action of muscarinic receptor agonists in the presence of beta-adrenergic stimulation (i.e., the so-called “accentuated antagonism” [4,5]). It should be noted that constitutive NO production from within the heart can regulate myocardial function both via a paracrine mechanism, (i.e., via the NO released by eNOS in the coronary endothelium or in the endocardium) and in an intra/autocrine manner (i.e., via the NO produced from within the cardiomyocytes). For many years, eNOS was thought to be the only NOS isoform constitutively present in cardiomyocytes. The identification of a neuronal NOS (nNOS) in cardiac myocytes in 1999 [2] started a new era of discovery bringing some clarity in a field of research that is marred

by inconsistency. Once released, NO is short-lived (seconds) and has limited diffusion distance within a cardiomyocyte (due to the scavenging effect of myoglobin and superoxide anions); thus, the close proximity of eNOS and nNOS to their respective pool of effector proteins may be a pre-requisite for their specificity of action within the myocardium. Indeed, whereas eNOS is mostly localized at the sarcolemmal membrane where it is bound to caveolin 3 [13], nNOS appears to be predominantly (but not exclusively [14]), localized to the sarcoplasmic reticulum (SR) in close proximity to the RyR and xanthine oxidoreductase (XOR) [15,16] (Fig. 2). However, eNOS and nNOS can translocate to different subcellular compartments in response to external stimuli or in the presence of disease. For example, nNOS translocates to the sarcolemmal membrane in response to ischemia/reperfusion [17] and left ventricular (LV) injury [18,19] whereas eNOS traffics from the cytoplasmic face of caveolae to intracellular domains, such as the Golgi complex or the nucleus [20,21], and can also bind to the RyR [22]. Over the last three decades our understanding of the role of myocardial constitutive NOS has increased significantly; however, the molecular mechanisms involved in NO signaling remain incompletely understood. The aim of this review is to provide an update on the signaling pathways involved in the regulation of cardiac function by NO. 2. Differential regulation of myocardial function by eNOS and nNOS 2.1. Lessons from mice with selective knockout of the eNOS or nNOS gene One of the best studied paracrine action of NO released from the coronary endothelium is that on LV compliance. In humans, stimulation of eNOS activity by intra-coronary infusion of substance P has been shown to cause a small reduction in LV peak systolic pressure and systolic performance and a significant increase in LV end-diastolic distensibility [23]. These paracrine effects, which can be mimicked by NO donors and cGMP analogs, are thought to be mediated by phosphorylation of troponin I by protein kinase G (PKG) leading to a reduction in myofilament Ca 2+ sensitivity [24,25]. An increase in LV compliance (rather than a direct effect of NO on myocardial inotropy) is likely to be the mechanism by which endothelial NO production contributes to the Frank–Starling response [7]. However, contrary to expectations, LV diastolic function is normal in eNOS −/− mice under basal conditions (despite the mild hypertension-induced LV hypertrophy) and even faster than in wild type mice in the presence of beta-adrenergic stimulation [26]. Whether eNOS gene deletion affects myofilament Ca 2+ sensitivity or the Frank–Starling response remains to be investigated. Together, these findings suggest that eNOS-derived NO is unlikely to have a tonic effect on myocardial relaxation and LV compliance;

L-Citrulline + NO NADPH Arg

BH4 COOH

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FAD

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Fe NADP+

Ca2+

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Fig. 1. Schematic diagram of NOS structure. NOSs contain an oxygenase domain (− COOH terminal) and reductase domain (− NH2 terminal). These two domains are separated by calmodulin (CaM). CaM binding to Ca2+ dimerizes the two domains and activates the enzyme. Electrons (e−) transfer from FMN and FAD to the oxygenase domain so that NOS catalyzes the oxidation of L-arginine to L-citrulline and NO.

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Fig. 2. A. Immunogold labeling of SR vesicles with specific primary and secondary antibodies conjugated to 12 nm of colloidal gold shows that nNOS is located to the SR. B(a): A 150-kDa monomer band of XOR is identified in the purified SR fractions where nNOS and SERCA are also present. B(b): co-immunoprecipitation suggests that XOR associates with nNOS (bands 1, 4, and 7) but not with eNOS (bands 3, 6, or 9). Little XOR protein is detected in the total heart protein extract (bands 2, 5, and 8). TP, total heart protein; M, microsomal fraction; RC, rat cerebellum. Modified from refs. [2] and [16].

however, upon stimulation of eNOS (e.g., in response to an increase in LV preload) release of NO from the coronary endothelium [6] may facilitate LV diastolic function. Early work suggested that myocardial eNOS-derived NO might attenuate the inotropic and chronotropic effect of beta-adrenergic stimulation (both directly and by enhancing the effects of muscarinic receptor stimulation) through a PKG-dependent reduction in ICa [27]. Although the muscarinic regulation of ICa has been shown to be absent in LV myocytes from eNOS−/− mice, the increase in ICa in response to isoproterenol did not differ between eNOS−/− and wild type mice [28]. Similarly, a systematic study of the effect of eNOS gene deletion on beta-adrenergic responses in the murine myocardium confirmed the endothelial/endocardial origin of the NO involved in decreasing the LV inotropic response to isoproterenol and highlighted the importance of using littermate controls when assessing the effects of eNOS gene deletion [29]. Neither beta-adrenergic cell shortening nor the negative inotropic effect of muscarinic receptor stimulation in LV myocytes was affected by eNOS gene deletion when myocytes from wild type littermates were used as controls [29] (Fig. 3). In contrast, enhanced betaadrenergic inotropy has been reported in intact hearts of eNOS −/− mice or in vivo [26,30,31]. These and other findings [28,32,33] suggest that myocardial eNOS-derived NO is unlikely to have an important role in the physiological regulation of basal and beta-adrenergic contraction in mice; however, when myocardial NOS3/cGMP signaling is amplified by eNOS overexpression [34,35] or inhibition of cGMP hydrolysis (e.g., following PDE5 inhibition) [36], an eNOS-dependent inhibition of beta-adrenergic contraction has been observed, in the absence of changes in the amplitude of the intracellular Ca2+ transient, leading the authors to conclude that eNOS-derived NO may modulate beta-adrenergic responses through a PKG-dependent reduction in myofilament Ca2+ sensitivity [36]. Under these conditions, changes in other Ca2+ fluxes might counterbalance the reported inhibitory effects of PKG on Ca2+ entry via the sarcolemmal ICa [37]; for instance, stimulation of eNOS activity by stretch has also been reported to increase RyR open probability in isolated LV myocytes [8]. By contrast, nNOS gene deletion or inhibition has been associated with an increase in myocyte shortening and LV systolic function in vivo in some studies [11,38,39] but not in others [10,40,41]. Again, these differences might depend on the choice of controls, the mouse strain, and the experimental model and conditions. For instance Barouch et al. [40] showed a trend towards an increase in LV systolic

function in vivo when using C57BL/6 mice as controls whereas Wang et al. [10] showed no differences in the force development of right ventricular trabeculae from nNOS −/− mice stimulated at 4 Hz and 6 Hz (vs. C57BL/6 mice, both studied at 37 °C) but a significant lower cell shortening and amplitude of [Ca 2+]i transients in nNOS−/− LV myocytes field-stimulated at 0.5 Hz at room temperature. In our hands, the increase in cell shortening (3 Hz, 35 °C) resulting from nNOS disruption was associated with an increase in ICa density, SR Ca2+ content and [Ca 2+]i transient amplitude [38], as also observed by Burger et al. [42]. Similarly, nNOS gene deletion has been associated with a suppressed force–frequency response by some investigators [9,10] (but not by others [11]), which has been attributed to inability of these mice to increase their SR Ca 2+ load in response to increasing stimulation frequencies. nNOS has also been reported to co-localize with and be negatively regulated by the sarcolemmal Ca2+ pump [14,43,44] and with the RyR in the SR [41,45]. Such proximity between a Ca2+ regulated enzyme and a Ca 2+ release channel would be expected to have functional consequences; however, as for contraction and ICa, regulation of RyR function by nNOS-derived NO does not appear to be straightforward with one early report indicating that disruption of nNOS leads to an increase in RyR open probability and leak of Ca2+ during diastole [45] and a more recent publication reporting a reduction in RyR open probability and diastolic Ca 2+ leak in LV myocytes from nNOS knockout mice [41], both of which have been attributed to a reduction in RyR S-nitrosylation. Although differences in the balance between RyR oxidation and S-nitrosylation have been advocated to explain these disparities (as debated in [46,47]), the effect of myocardial constitutive NO production on the RyR open probability remains to be completely elucidated. A similar lack of consistency accompanies the results of studies investigating the effect of nNOS gene deletion on beta adrenergic inotropy; for instance, we found an increase in the cell shortening response to 2 nmol/L of isoproterenol in nNOS −/− mice [11] but no difference in the response to higher concentrations of isoproterenol [48]. In vivo, a blunted LV inotropic response to beta-adrenergic receptor agonists has been reported by most investigators [39,40,49,50]. The recent evidence indicating that nNOS inhibition causes an endothelium-independent reduction in coronary blood flow in humans [51] suggests that a mismatch between metabolic demand (that appears to be increased in the nNOS −/− myocardium [15]) and myocardial perfusion may depress the inotropic response to beta-adrenergic stimulation in nNOS−/− mice in vivo and

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myocardial function under physiological conditions are predominantly mediated by NO produced from the stimulated vascular endothelium. Conversely, myocardial nNOS derived NO appears to exert a tonic control on myocardial relaxation by regulating the rate of SR Ca 2+ uptake. However, a number of aspects remain unclear; it is tempting to suggest that the inconsistency among results from different laboratories may be biologically significant and reflect epigenetic mechanisms that are yet to be discovered; in reality, while animal experimentation is largely being conducted outside the rules that govern clinical studies (i.e., without accurate power calculations, standardized experimental procedures defining, among other things, the choice of controls, and with the experimenters not blinded of the genotype or intervention), it is difficult to exclude play of chance as the “mechanism” behind the reported phenotype variability, particularly when the differences between wild type and genetically modified mice are subtle. 2.2. Lessons from myocardial-specific overexpression of eNOS or nNOS

Fig. 3. The genetic background of mice alters the β-adrenergic (isoproterenol, ISO) and cholinergic (carbachol, CCh) inotropic response in LV myocytes. A: Sarcomere shortening under basal conditions, in the presence of ISO (100 nmol/L), ISO + CCh (1 μmol/L) and after wash-out in LV myocytes (1 Hz) from eNOS−/− mice, their wild type littermates (eNOS+/+) and C57BL/6 J mice. B: Mean cell shortening in LV myocytes from eNOS−/− and eNOS+/+ does not differ at each stage of the experimental protocol; by contrast, LV myocytes from C57BL/6 mice showed a significant reduction in the inotropic response to ISO compared with eNOS+/+ myocytes. From ref. [29].

explain the discrepancy between the in vivo findings and those obtained in isolated LV myocytes. Amidst this confusion, one thing seems certain, nNOS gene deletion or inhibition prolongs relaxation both in ventricular myocytes and in vivo, regardless of the experimental conditions and choice of wild-type controls [9–11,39,52]. The mechanism responsible for this effect of nNOS-derived NO differs from that by which NO release from the coronary endothelium exerts its facilitatory role on relaxation and LV compliance; instead of reducing myofilament Ca 2+ sensitivity [24,25], nNOS-derived NO increases the rate of Ca 2+ reuptake into the SR by maintaining PLN in its phosphorylated state (Fig. 4) [10,52]. In summary, the generation of genetically modified mice harboring eNOS or nNOS gene deletion has contributed to clarify some aspects of the role of constitutively produced NO within the myocardium. In particular, it seems now likely the effects of eNOS-derived NO on

Several murine models of myocardial-specific transgenic over-expression of eNOS have been generated [53–55]. In these mice, eNOS has been found to be mostly membrane bound and associated with caveolin-3 (Fig. 5) but not with RyR [54]. Transgenic eNOS expression has been associated with greater myocardial eNOS activity, as evaluated in vitro by the arginine to citrulline assay; however, even under these conditions, myocardial eNOS does not seem to affect basal contractile and relaxation parameters when compared to wild type mice [54,55]. These findings are consistent with a negligible autocrine effect of eNOS-derived NO on basal myocardial function. In an earlier study, Brunner et al. [53] reported a reduction in LV pressure development in a model of myocardial eNOS over-expression, which was reversed by nonselective NOS inhibition with L-NAME. However, myocardial eNOS expression in this model was very high (i.e., myocardial NOS activity, which when measured in vitro with added co-factors and substrate reflects the amount of eNOS protein, was 0.7 ±0.3 pmol·mg− 1·min− 1 in control mice and 61.8 ± 11.8 pmol·mg− 1·min− 1 in the transgenic mice). These conditions can result in a stochiometric imbalance between eNOS and its cofactor, tetrahydrobiopterin (BH4), or its substrate, L-arginine, leading to “uncoupling” of NOS activity (a condition whereby NOSs synthesize the reactive oxygen species, superoxide, rather than NO). Although differences in myocardial ROS production did not reach statistical significance in the small sample of mice investigated in this paper, the SOD-inhibitable ROS production was much greater in the eNOS transgenic mice. As L-NAME can inhibit both NO and ROS production from NOS, the myocardial phenotype observed in this model might reflect the effect of eNOS uncoupling and consequent increase in myocardial ROS production rather than that of an enhanced synthesis of eNOS-derived NO. In agreement with this interpretation, Brunner et al. did not observe any differences in the response to muscarinic or beta-adrenergic receptor stimulation in their mice, whereas more moderate cardiomyocyte-targeted overexpression of eNOS has been shown to attenuate the inotropic response to maximal beta-adrenergic stimulation and to increase the negative chronotropic and inotropic effects of muscarinic receptor stimulation [34,35,54]. The importance of increasing myocardial eNOS expression becomes clearer in animal models of human disease. For instance, myocardial eNOS overexpression was associated with a better preserved LV systolic and diastolic function after ischemia and reperfusion and with improved survival and attenuated LV hypertrophy and interstitial fibrosis after myocardial infarction [54,55]. As the eNOS protein level has been found to decrease in the remodeled and failing myocardium [19,56], these results imply that restoring or increasing myocardial eNOS expression may help in preventing adverse remodeling and LV dysfunction following myocardial infarction. However, this may not be always the case, as indicated by Takimoto et al. who showed that myocardial eNOS uncoupling exacerbated LV adverse remodeling and dysfunction in a murine model of severe LV pressure

Y.H. Zhang, B. Casadei / Journal of Molecular and Cellular Cardiology 52 (2012) 341–350

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Fig. 4. Phospholamban (PLN) phosphorylation is significantly reduced in nNOS LV myocytes compared to nNOS . Inhibition of PKA with PKI significantly reduces PLN phosphorylation (A) and prolongs relaxation only in nNOS+/+ LV myocytes (B), thereby abolishing the difference between genotypes. Inhibition of protein phosphatases (PP2A and PP1) with okadaic acid (2 uM) increases PLN phosphorylation only in nNOS–/– LV myocytes (C) and where it is associated with a significant reduction in the time to 50% relaxation (TR50, D). From ref. [48].

overload; under these conditions, eNOS gene deletion was found to be protective [57]. Whereas myocardial-specific overexpression of eNOS appears to blunt cardiac hypertrophic response [54], myocardial nNOS overexpression has been associated with increased LV wall thickness and myocyte width in two independent mouse models [58,59]. However, substantial differences between these models have been observed; for instance ICa density and inotropy was found to be increased by Loyer et al. [59] and decreased by Burkard et al. [58] (Fig. 6). Similarly Loyer et al. [59]reported a more rapid rate of decay of the [Ca2+]i transient and relaxation in their nNOS transgenic mouse in association with an increase in PLN phosphorylation whereas Burkard et al. [58] reported a slower Ca2+ reuptake in the presence of nNOS overexpression. Again, the reasons for this discrepancy are unclear. Although overexpression is often used in cell biology to better define the subcellular localization of a particular protein, there are many examples indicating that the results of these experiments may be misleading. For instance, in Burkard et al. [58] the native nNOS co-localized with SERCA whereas the transgenically over-expressed nNOS was mostly localized to the sarcolemmal membrane where it interacted with L-type Ca channel. In contrast, in Loyer et al. [59] the subcellular localization of nNOS was not altered by conditional overexpression. These data suggest that the specific subcellular localization of nNOS may be a key factor in determining the myocardial phenotype; for instance, a relative increase in sarcolemmal nNOS localization as observed by Burkard et al. [58] and in the presence of myocardial failure [18,19] may lead to a predominant inhibitory effect of NO on ICa and inotropy with little or no influence on PLN phosphorylation. In contrast, an increase in nNOS expression at the SR may maintain PLN phosphorylation and increase the speed of Ca2+ reuptake and relaxation. Although this hypothesis is supported, to some

extent, by experimental findings, a definitive demonstration is still lacking. More recent data have also demonstrated a mitochondrial localization of over-expressed nNOS as well as a translocation of native nNOS within the mitochondria and in the proximity of cytochrome C following ischemia–reperfusion [60]. The PDZ-binding domain of the nNOS α and μ isoforms and the nNOS adaptor protein, CAPON may be responsible for interaction with specific binding partners and for targeted subcellular localization [61]; however, whether nNOS is constitutively expressed in the mitochondria remains a matter of debate. Together, these data suggest that subcellular localization of nNOS is a highly dynamic and functionally relevant process of which, at present, we have only taken a handful of snapshots. 3. Molecular mechanisms downstream of NO 3.1. cGMP-dependent signaling As a signaling molecule that is released from spatially restricted NOSs, NO introduces post-translational modifications of numerous target proteins by using different chemical reactions. It was initially suggested that stimulation of soluble guanylate cyclase (sGC) by NO (and the consequent increase in cGMP synthesis) was the predominant mechanism sub-serving the function of NO in the cardiovascular system [62]. Whereas cGMP signaling has been involved in the regulation of protein phosphorylation (both via PKG and via the interaction with cGMP inhibited or stimulated phosphodiesterases of cAMP [63]), more recent evidence has suggested that S-nitrosylation of cysteine thiols by NO, leading to changes in the conformation and activity of a

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eNOS overexpression

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A

B eNOS-TG

WT

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50 µm

IP-CAV3 WT 50 µm

C

B

Fig. 5. Left: Cardiomyocyte-restricted eNOS overexpression (eNOS-TG) in the myocardium increases eNOS protein level and the formation of eNOS-caveolin 3 (CAV3) complexes (A and B), suggesting a predominant sarcolemmal localization of eNOS in this model. LV functional deterioration and adverse structural remodelling after myocardial infarction are prevented in this model (C). However, cardiac eNOS uncoupling (as observed following severe thoracic aorta constriction, TAC) exacerbates LV adverse remodeling and dysfunction (right, A); under these conditions eNOS gene deletion (NOS3–/–) is protective (B). Modified from refs. [54], [55] and [57].

number of enzymes, kinases and ion channels, may also be an important mechanism involved in NO signaling. Furthermore, myocardial nNOS has been found to inhibit mitochondrial respiration [15,60] and the activity of xanthine oxidases [15,16], suggesting that nNOS may also play an important part in the regulation of the myocardial redox state. Experiments using cGMP analogs or NO donors have shown that PKG can phosphorylate and modulate the activity of a number of proteins involved in E–C coupling; however, to which extent PKG regulates basal myocardial function downstream of constitutive NO production remains a matter of debate. In the presence of beta-adrenergic stimulation, blocking cGMP hydrolysis by inhibiting PDE5A has been associated with a PKG-dependent suppression of contractility that is not present in NOS3 knockout mice or after NOS inhibition, suggesting that, under these circumstances, PKG may account, at least in part, for the anti-adrenergic effects of NO [36]. Both inhibition of ICa and troponin I phosphorylation have been proposed as mechanism responsible for the PKG-mediated inhibition of beta-adrenergic inotropy [37,64]. A recent report has suggested that PKG may also limit cytosolic cGMP accumulation by NO donors by phosphorylating and activating PDE5 [65] while facilitating cGMP production downstream of natriuretic peptide receptors to increase the production of cGMP in the subsarcolemmal space. These findings suggest that PKG may contribute to subcellular cGMP compartmentalization. cGMP synthesis downstream of NO may also regulate cAMP/PKA signaling directly, by activating adenylate cyclase [66], and indirectly by stimulating or inhibiting the activity of PDEs of cAMP [67]. The general consensus is that NOSs and PDEs are compartmentalized within cardiac myocytes [40,68] and that by virtue of this spatial co-localization, NO-dependent regulation of cGMP and cAMP

can vary between subcellular compartments. Indeed, by using FRET imaging in live neonatal rat myocytes expressing cAMP and cGMP biosensors, Stangherlin et al. [69] have recently demonstrated that cGMP produced downstream of NO donors and soluble guanylate cyclase leads to a significant increase in the cAMP pool that activates PKA-RI (i.e., the isoform of PKA that is preferentially activated downstream of prostaglandin receptors) and to a reduction in the cAMP pool that activates PKA-RII (i.e., the isoform of PKA that is preferentially activated downstream of beta-adrenergic receptors). These opposing effects are determined by the cGMP-mediated regulation of PDE2 and PDE3, the activity of which is also confined to spatially distinct subcellular domains. Atrial natriuretic peptide (ANP), via cGMP-mediated activation of PDE2, significantly reduces cAMP levels in the PKA-RII compartment, suggesting that cGMP-mediated activation of PDE2 may contribute to the negative inotropy associated with ANP. Again, the relevance of these elegant studies to endogenous NO release from constitutive NOS in freshly isolated, adult cardiomyocytes remains to be investigated; indeed, whereas cGMP-dependent signaling pathways appear to be involved in the NO regulation of beta-adrenergic inotropy and myocardial relaxation by endothelial NO production, the tonic effects of myocardial nNOS-derived NO on basal cardiac function appear to be cGMP-independent. At least in the mouse, inhibition of soluble guanylate cyclase or PKG has no effect on the speed of myocardial relaxation and [Ca 2+]i decay in wild type or nNOS −/− LV myocytes [48]. By the same token, the slower rate of relaxation and Ca 2+ reuptake observed in nNOS −/− mice is due to a cGMP-independent reduction in PLN phosphorylation, secondary to an increase in serine–threonine protein phosphatase activity (Fig. 4) [48]. Carboxyl-methylation of catalytic subunits of PP2A is known to lead to the translocation and activation of this phosphatase

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C

C

Fig. 6. Left: nNOS over-expression in this model resulted in similar co-localization of nNOS in the sarcoplasmic reticulum (SR), as shown by the results of the co-immunoprecipitation with the SR Ca2+ ATPase (SERCA) (A) but also with significant co-localization of the enzyme with the L-type Ca channel (Cav1.2) in the LV myocardium (B). Under these conditions, the current density of ICa was significantly reduced (C). By contrast (right), in another model of nNOS overexpression, subcellular nNOS localization was not dramatically altered, i.e., nNOS association with caveolin 3 and RyR did not differ between transgenic (DT) and control mice in the presence or absence of thoracic aorta constriction (TAC) (A and B) and ICa was significantly increased (C). From refs. [58] and [59].

[70,71]. Whether nNOS regulates myocardial PP2A activity through this mechanism remains to be tested. 3.2. S-nitrosylation of E–C coupling proteins and NOS-mediated regulation of myocardial function S-nitrosylation refers to the incorporation of the NO moiety to a cysteine sulfur atom to form a S\NO bond. Like phosphorylation, S-nitrosylation is a precisely targeted post-translational modification that has the potential of fine-tuning the activity of numerous proteins involved in myocardial function. At present, over 1000 proteins are identified as SNO-proteins, among which there are ion channels and transporters involved in excitation–contraction coupling and proteins involved in contraction, metabolism, and cellular signaling [72]. In a recent proteomic study, Kohr et al. identified 116 constitutive SNO-proteins using the SNO-resin assisted capture (SNO-RAC) technique in tandem with mass spectrometry in mouse heart homogenates [73]. It is now known that several Ca 2+ handling proteins, such as RyR, the L-type Ca2+ channel and SERCA, can be S-nitrosylated. The rapidly reversible nature of this post-translational modification and its balance with the oxidative

state of the same cysteine residues may, in part, account for the reported variable effects of S-nitrosylation of protein involved in E–C coupling; for instance, S-nitrosylation of the L-type Ca channel has been associated with both an increase [74] or a reduction in ICa [75]. Hypo-nitrosylation of the RyR has been associated with both an increase [76] or a reduction [41] in RyR open probability whereas hyper-nitrosylation of RyR increases diastolic Ca2+ leak and arrhythmogenesis in the mdx mouse model of Duchenne muscular distrophy [77,78]. Recently, sarcomeric proteins in animal and human hearts have also been found to be nitrosylated by nitrosoglutathione [79]; although the functional effect of this post-translational modification has yet to be explored, increased tropomyosin S-nitrosylation has been shown to correlate with the severity of myocardial dysfunction [79]. The relative contribution of eNOS or nNOS-derived NO to the Snitrosylation of myocardial proteins in health and disease remains to be established. It has been suggested that both eNOS and nNOS may contribute to S-nitrosylation of the α1.2 subunit of the cardiac L-type Ca 2+ channel, since gene deletion of either NOS significantly reduced the channel's S-nitrosylation and increases ICa in female mice following ischemic/reperfusion [17].

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NO-dependent S-nitrosylation or S-glutathiolation of key cysteine residues of SERCA stimulates its activity and increases the rate of Ca2+ reuptake into the SR [80]. Proximity of nNOS to Ca 2+-handling proteins in the SR suggest that this NOS isoform may be more likely to be involved in the S-nitrosylation of SERCA than eNOS. In any case, specificity of S-nitrosylation seems to be dependent on the availability of accessible thiols on the target protein and the proximity of the source of NO. The dynamic nature and rapid reversibility of these modifications poses a particular challenge to the study of their functional correlates in live cells under physiological conditions.

4. Conclusion In summary, after more than 30 years of intensive scrutiny, we are only just starting to understand the role played by constitutive NO production in the regulation of cardiovascular function. The full complexity of the mechanisms involved in NO signaling is still far from being understood. Harnessing the power of this small molecule in its many facets and applying this knowledge to the clinical arena may prove elusive for many years to come. Disclosures

3.3. nNOS regulation of myocardial redox signaling Myocardial nNOS co-immunoprecipitates with XOR and inhibits superoxide production from this oxidase system (Fig. 2) [15,16]. Other studies have suggested that nNOS may also tonically inhibit NADPH oxidase activity [81]. In nNOS−/− mice NADPH oxidase or XOR inhibition significantly decreases LV myocyte cell shortening and abolishes the differences between nNOS−/− and wild type littermates [81]. nNOS signaling may involve peroxynitrite (ONOO−) as suggested by Kohr et al. [82,83] who demonstrated that the ONOO− donor, SIN-1, activates PKA-dependent PLN-phosphorylation and increases the rate of Ca2+-reuptake and relaxation in LV myocytes. However, the effect of SIN-1/ONOO− was independent of protein phosphatase activity and cAMP. Evidence indicates that the NO-redox balance of the myocardium may affect both PKG and PKA phosphorylation [84-86] as shown by the fact that exogenous hydrogen peroxide activated PKA RI by inducing the formation of an interprotein disulfide bond between its two regulatory subunits [84]. In the rat cerebral cortex, oxidative stress has been shown to decrease PP2A activity by inducing intramolecular disulfide cross-linking of the catalytic subunit of the phosphatase [87,88]. Whether enhanced myocardial oxidase activity associated with nNOS gene deletion may account for a reduction in PP2A activity and an increase in PKA-dependent signaling at the sarcolemmal membrane remains to be established; our data indicate that inhibition of myocardial oxidases or PKA significantly reduces contraction in nNOS−/− LV myocyte and abolishes the difference in cell shortening between nNOS−/− and nNOS+/+ myocytes [81].

3.4. NOS uncoupling and myocardial function A number of common cardiovascular disease states (e.g., hypertension, diabetes and atherosclerosis) have been associated with “uncoupling” of eNOS activity; more recently this phenomenon has also been described in the atrial or ventricular myocardium in the presence of pressure overload, ischemia or atrial fibrillation [57,89–91]. A number of mechanisms may account for NOS uncoupling. Oxidation of the co-factor, tetrahydrobiopterin (BH4) has so far been the most commonly reported mechanism responsible for eNOS uncoupling in the myocardium [57,90–92]. Reduced availability of L-arginine [93] and de-phosphorylation of eNOS at Thr495 (in mice and human, Thr497 in bovine) [94] have also been associated with NOS uncoupling. Recently, S-glutathionylation of two cysteine residues in eNOS has been shown to reversibly uncouple the synthase [95] by a unique mechanism involving a leak of superoxide from the reductase domain of eNOS. To date, our understanding of the importance of NOS uncoupling in the myocardium is still in its infancy, however, it has been established that myocardial eNOS uncoupling secondary to BH4 deficiency increases myocardial ROS production and accelerates the evolution towards heart failure in response to aortic coarctation [57]. More recently, NOS uncoupling secondary to BH4 deficiency has been associated with diastolic dysfunction and reduced PLN phosphorylation in animal models of hypertension [91] and in human and experimental atrial fibrillation [92].

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