Targeting NOS as a therapeutic approach for heart failure.

JPT-06645; No of Pages 10 Pharmacology & Therapeutics xxx (2014) xxx–xxx

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Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: G. Billman

Targeting NOS as a therapeutic approach for heart failure Lifei Tang, Honglan Wang, Mark T. Ziolo ⁎ Department of Physiology & Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, USA

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Keywords: cGMP S-nitrosylation Contraction Arrhythmia Hypertrophy Nitroso-redox balance

a b s t r a c t Nitric oxide is a key signaling molecule in the heart and is produced endogenously by three isoforms of nitric oxide synthase, neuronal NOS (NOS1), endothelial NOS (NOS3), and inducible NOS (NOS2). Nitric oxide signals via cGMP-dependent or independent pathways to modulate downstream proteins via specific post translational modifications (i.e. cGMP-dependent protein kinase phosphorylation, S-nitrosylation, etc.). Dysfunction of NOS (i.e. altered expression, location, coupling, activity, etc.) exists in various cardiac disease conditions, such as heart failure, contributing to the contractile dysfunction, adverse remodeling, and hypertrophy. This review will focus on the signaling pathways of each NOS isoform during health and disease, and discuss current and potential therapeutic approaches targeting nitric oxide signaling to treat heart disease. © 2013 Published by Elsevier Inc.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . 2. Heart failure. . . . . . . . . . . . . . . . . . . . . . 3. Therapeutic Targeting of NOS dysfunction in heart disease . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Nitric oxide (NO) is a soluble and highly diffusible gas that functions as a signaling molecule in the human body. Besides its well-recognized roles in endothelium-derived smooth muscle relaxation (Palmer et al., 1987), synaptic transmission and immunological responses (Mayer & Hemmens, 1997), NO is also a key regulator of heart function via Abbreviations: AP, action potential; β-AR, β-adrenergic; BH4, tetrahydrobiopterin; cGMP, cyclic GMP; DAD, delayed afterdepolarization; EAD, early afterdepolarization; ECC, excitation contraction coupling; FFR, force-frequency response; HF, heart failure; ICa, L-type Ca2+ current; LTCC, L-type Ca2+ channel; NF-κB, nuclear factor kappa B; NFAT, nuclear factor of activated T cells; NO, nitric oxide; NOS, nitric oxide synthase; NOS1, neuronal nitric oxide synthase; NOS2, inducible nitric oxide synthase; NOS3, endothelial − nitric oxide synthase; O− 2 , superoxide anion radical; ONOO , peroxynitrite; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PLB, phospholamban; ROS, reactive oxygen species; RyR, ryanodine receptor; SERCA, SR Ca2+ ATPase; SOD, superoxide dismutase; SR, sarcoplasmic reticulum; TAC, transverse aortic constriction; XOR, xanthine oxidoreductase; WT, wildtype. ⁎ Corresponding author at: Department of Physiology & Cell Biology, The Ohio State University, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA. Tel.: 614 688 7905; fax: 614 688 7999. E-mail address: [email protected] (M.T. Ziolo).

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modulation of excitation-contraction coupling (ECC) (Ziolo et al., 2008) and myocardial growth (Booz, 2005).

1.1. NO signaling NO signals via either the cyclic GMP (cGMP)-dependent or the cGMP-independent pathways (Ziolo, 2008). In the cGMP-dependent signaling pathway, NO activates soluble guanylate cyclase to increase cGMP levels, which modulates cGMP-regulated phosphodiesterases (PDE; cGMP-stimulated: PDE2; cGMP-inhibited: PDE3) or the activation of cGMP-dependent protein kinase G (PKG) to modulate a variety of protein targets (Kojda et al., 1996; Layland et al., 2002; Yang et al., 2007; Stangherlin et al., 2011). In the cGMP independent pathway, NO directly regulates protein function via a post-translational modification of S-nitrosylation, which is the addition of a nitrosyl group to a free thiol on a cysteine residue of the target protein (Stamler et al., 2001). However, other reactive oxygen species (e.g., superoxide anion radical — O− 2 ) can act as a chemical trap to buffer and quench NO levels to impair its signaling (Paolocci et al., 2001). On the other hand, these reactions can also result in the formation of new signaling molecules. For

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Please cite this article as: Tang, L., et al., Targeting NOS as a therapeutic approach for heart failure, Pharmacology & Therapeutics (2014), http:// dx.doi.org/10.1016/j.pharmthera.2013.12.013

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L. Tang et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

example, NO will react with O− 2 with an extremely high rate constant to favor the formation of peroxynitrite (ONOO−) (Huie & Padmaja, 1993). Within cardiac myocytes, low levels of ONOO− are reported to mediate beneficial protein glutathionylation and nitrosylation, while high levels of ONOO− can lead to irreversible and harmful nitration/oxidation of proteins (Kohr et al., 2008a, 2012). Thus, while NO is a simple molecule, its signaling is very complex due to its multiple pathways (cGMP-dependent and independent) and interaction with other radicals to form additional signaling molecules. The complexity of NO is further evident when we also consider the enzymes that produce NO. 1.2. NO synthases NO is endogenously produced by enzymes termed Nitric Oxide Synthase (NOS). In mammals, three isoforms of NOS are encoded by different genes (Knowles & Moncada, 1994). Within ventricular myocytes, neuronal NOS (NOS1, nNOS) and endothelial NOS (NOS3, eNOS) are constitutively expressed (Balligand et al., 1995; Xu et al., 1999), while inducible NOS (NOS2, iNOS) is expressed due to environmental cues (i.e. cytokine production during immune responses) (Balligand et al., 1994). Production of NO via NOS1 and NOS3 is calcium-dependent (Busse & Mulsch, 1990; Fleming & Busse, 1999), while NOS2 is a calcium-independent enzyme (Cho et al., 1992). Furthermore, within cardiac myocytes, NOS2 produces much higher levels of NO compared to NOS1 and NOS3 (Ziolo et al., 2001a). NOS functions as a dimer composed of two identical monomers. The C-terminus (reductase domain) of the monomer consists of nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) binding sites, while the N-terminus (oxygenase domain) consists of tetrahydrobiopterin (BH4), oxygen (O2), and L-arginine binding sites along with a heme group for NOS dimerization (Stuehr et al., 2001; Forstermann & Munzel, 2006). There is also a calmodulin binding domain located between the reductase and oxygenase domains. Flavin serves to transfer an electron from NADPH in the reductase domain of one monomer to the oxygenase domain of the other monomer resulting in the production of NO via oxidation of L-arginine to L-citrulline (Stuehr et al., 1991; Andrew & Mayer, 1999). A state of oxidative stress (i.e., increased levels of reactive oxygen species — ROS) can interrupt NO production by NOS leading to the generation of O− 2 instead of NO, termed NOS uncoupling (Vasquez-Vivar et al., 1998; Xia et al., 1998). BH4 is known to bind to NOS at the interface of two monomers to stabilize the dimer structure. Depletion or oxidation (to BH2) of BH4 causes the electrons to flow from the reductase domain to oxygen at the oxidase domain within the monomer, producing O− 2 (Crabtree et al., 2009). Additionally, glutathionylation of NOS3 at Cysteine689 and Cysteine908 blocks the reduction of NADPH to NADP, leading to O− 2 generation at the reductase domain (Chen et al., 2010). ONOO− toxicity is also reported to uncouple NOS3 via oxidation of the central zinc bound in the heme domain (Zou et al., 2002). Thus, NOS serves as the primary source of NO within ventricular myocytes. However, under disease conditions, NOS can also result in − O− 2 production and increased ONOO levels. 1.3. NOS1 signaling NOS1 was first identified in the brain (Knowles et al., 1989), and was termed neuronal (or brain) NOS. However, expression of this isozyme has been found in numerous cell types (including ventricular myocytes) and has since been renamed NOS1. In the myocyte, NOS1 is known to be predominantly localized to the sarcoplasmic reticulum (SR) (Xu et al., 1999; Barouch et al., 2002), but it is also found at the mitochondria (Burkard et al., 2010) and sarcolemmal membrane (Williams et al., 2006). At the SR, NOS1 has been shown to co-immunoprecipitate with the SR Ca2+ release channel, ryanodine receptor (RyR), and xanthine oxidoreductase (XOR) (Xu et al., 1999; Barouch et al., 2002; Khan

et al., 2004). NOS1 signals through the cGMP-independent signaling pathway (i.e, S-nitrosylation via generation of low ONOO−) (H. Wang et al., 2008a), and acts as a major regulator of ECC. It appears that the chief source of NO that modulates myocyte Ca2+ handling is produced by NOS1. Contractile studies performed on myocytes with genetic deletion of NOS1 (NOS1−/−) demonstrated that these myocytes had depressed basal contraction along with prolonged relaxation (H. Wang et al., 2008a). We believe that these observed functional effects in NOS1−/− myocytes were due to loss of NOS1 signaling (and not some compensatory adaptation) since these results could be recapitulated with acute NOS1 inhibition (S-methyl-Lthiocitrulline — SMLT) in wildtype (WT) myocytes (Barouch et al., 2002; Khan et al., 2003; H. Wang et al., 2008a; Zhang et al., 2008). NOS1 signaling is also imperative for the positive force-frequency response (FFR) and the β-adrenegic (β-AR)-induced positive inotropy (Barouch et al., 2002; Khan et al., 2003; H. Wang et al., 2008a). Indeed, myocytes have a blunted FFR and reduced contractile response to βAR stimulation after chronic deletion (NOS1 knockout mice) or acute pharmacologic inhibition of NOS1. The major factor responsible for the positive FFR and inotropy with β-AR stimulation is an increase in the Ca2+ transient amplitude, which is not observed with NOS1 knockout or inhibition. A major determinant of Ca2+ transient amplitude is the SR Ca2+ load (Shannon et al., 2000). We and others have reported that there is a significant reduction in the SR Ca2+ load with NOS1 knockout or inhibition (Khan et al., 2003; H. Wang et al., 2008a). The SR Ca2+ load is mainly determined by SR Ca2+ uptake by the SR Ca2+-ATPase (SERCA)/phospholamban (PLB) complex (Bers, 2002). PLB is a phosphoprotein that acts to inhibit SERCA to limit Ca2+ uptake. However, upon PLB phosphorylation, this inhibition of SERCA is relieved to increase SR Ca2+ uptake. This phosphorylation has two major effects: 1) increase SR Ca2+ load, and 2) accelerate the rate of [Ca2+]i decline. We previously published that the Ca2+ transient decline of myocytes is predominantly regulated by PLB phosphorylation at serine16 (the cAMP dependent protein kinase (PKA) site) (Roof et al., 2011). In NOS1−/− myocytes, basal PLB serine16 phosphorylation is decreased (H. Wang et al., 2008a; Zhang et al., 2008). Thus, the decrease in PLB phosphorylation is largely responsible for the slowed Ca2+ transient decline and reduced SR Ca2+ load (Roof et al., 2012). We believe the decreased phosphorylation is due to a decrease in basal PKA activity. Specifically, we found that NOS1 signals via low ONOO− formation, and that exogenous ONOO− can directly (in the absence of cAMP) activate PKA (Kohr et al., 2010b), probably via S-nitrosylation (Burgoyne & Eaton, 2009). Consistent with these findings, myocyte-specific NOS1 over-expression mice showed increased basal PLB Serine16 phosphorylation (Loyer et al., 2008). However, direct effects of NOS1 signaling on the regulation of PKA activity remains to be determined. There is also evidence that NOS1−/− myocytes have increased phosphatase (both PP1 and PP2a) activity (Zhang et al., 2008). This is most likely due to the increased oxidative stress that is present in these myocytes (Khan et al., 2004; Kinugawa et al., 2005; Zhang et al., 2009; Traynham et al., 2012), as O− 2 can activate both PP1 and PP2a (Sommer et al., 2002; Liu & Hofmann, 2004). In fact, in a model with upregulated NOS1 expression (exercise), we have found that there is increased PLB Serine16 phosphorylation due to increased PKA activity and decreased PP activity (Roof et al., 2013). Interestingly, there was no difference between WT and NOS1−/− myocytes in PLB Serine16 phosphorylation and Ca2+ transient decline during β-AR stimulation (H. Wang et al., 2008a; Roof et al., 2012), suggesting that the cAMPinduced activation of PKA is normal in NOS1−/− myocytes. Thus, it appears that NOS1 signaling mediates PLB phosphorylation at serine16 via regulation of PKA and PP activity under basal conditions, leading to positive inotropy of the heart. Although PKA activation during β-AR stimulation is intact, the functional response to β-AR stimulation is blunted in NOS1−/− myocytes (Barouch et al., 2002; H. Wang et al., 2008a). These data indicate that NOS1 signaling has another target, the ryanodine receptor (RyR). RyR



is an essential protein for contraction, regulating the release of Ca2+ from the SR (Bers, 2002). We have previously shown that NOS1 signaling activates RyR via S-nitrosylation (Wang et al., 2010). Provocatively, RyR protein levels are increased in NOS1−/− myocytes (Sears et al., 2003; Gonzalez et al., 2007; Wang et al., 2010), which we believe to be a compensatory mechanism for the decreased SR Ca2+ release and Ca2+ cycling in these cells. We believe the decrease in RyR activity due to a lack of S-nitrosylation limits Ca2+ release from the SR to blunt basal and β-AR stimulated contraction (although further studies are still needed to validate the latter). XOR, which is involved in purine degradation and is one of the major sources of O− 2 (Berry & Hare, 2004), also co-localizes with NOS1 and RyR at the SR (Khan et al., 2004). O− 2 production by XOR is inhibited by NO (Ichimori et al., 1999), and the loss of XOR inhibition in the NOS1 knockout is the foremost reason for the oxidative stress in these myocytes (Khan et al., 2004). Hence, in addition to S-nitrosylation, NOS1 signaling also helps maintain normal RyR activity by preventing RyR oxidation. Under certain conditions (disease, FFR, β-AR stimulation, etc.), RyR in NOS1−/− myocytes becomes oxidized resulting in enhanced activity (Gonzalez et al., 2007, 2010; Dulce et al., 2013). This enhanced activity results in diastolic SR Ca2+ leak to empty the SR and blunt contraction. This increased leak also contributes to the generation of spontaneous Ca2+ waves and triggered arrhythmias. Thus, NOS1 signaling promotes protein S-nitrosylation and limits oxidation to directly regulate RyR function for proper contraction. In addition to producing NO, it has been suggested that NOS1 also produces nitroxyl (HNO), the 1-electron reduction product of nitric oxide (Schmidt et al., 1996; Ishimura et al., 2005). Studies have shown that exogenous HNO has similar contractile effects and protein end targets (RyR and SERCA/PLB) as NOS1 signaling (Tocchetti et al., 2007; Kohr et al., 2010a; Sivakumaran et al., 2013), making this a very realistic possibility. NOS1 signaling occurs at the subcellular SR domain to directly (S-nitrosylation) and indirectly (phosphorylation via possibly regulating PKA and PP activity) regulate protein function and contraction. Loss of this compartmentalized signaling results in decreased contraction and prolonged relaxation through alterations in PLB phosphorylation, RyR activity, and increased XOR function. Interestingly, these phenomenon are broadly observed in heart failure as well (Sag et al., 2013) Therefore, dysfunction of NOS1 is very likely involved in the process of heart failure, and studies on NOS1 deficiency models can help to reveal, in part, the molecular mechanisms of heart failure. Likewise, HNO donors have shown beneficial effects in animal models of heart failure, and appears to be a promising approach to treat human patients (Sabbah et al., 2013). 1.4. NOS3 signaling NOS3 was first identified in the coronary endothelium and was termed endothelial NOS (Palmer et al., 1988). However, expression of this isozyme has been found in numerous cell types (including ventricular myocytes) and has since been renamed NOS3. In the myocyte, NOS3 is known to be localized to caveolae by binding with Caveolin-3 (Cav-3) (Feron et al., 1998; Barouch et al., 2002). In addition to increased [Ca2+]i, NOS3 is also activated via phosphorylation at Serine1179 by Akt (protein kinase B) (Fulton et al., 1999), which is important for cardioprotection in end-stage heart failure (Fukushima et al., 2010). Although NO is a highly diffusible gas, NOS signaling is temporally and spatially localized (i.e., compartmentalized signaling) (Barouch et al., 2002; Ziolo & Bers, 2003). Since NOS1 and NOS3 are localized to different domains (caveolae vs SR) within the ventricular myocyte, each has unique signaling pathways, protein end targets, and functional effects on the heart. Unlike NOS1, NOS3 signaling does not regulate basal myocyte contraction but only β-AR stimulated contraction (Barouch et al., 2002; Wang et al., 2008b). In the caveolar microdomain,

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NOS3 closely localizes with L-type Ca2+ channels (LTCC) and βadrenergic receptors (Balijepalli et al., 2006). Superoxide dismutase (SOD), which degrades O− 2 , is also localized with NOS3 (Brahmajothi & Campbell, 1999). The buffering of O− 2 by SOD drives NOS3 to signal via the cGMP/sGC/PKG pathway to phosphorylate various protein targets (i.e. LTCC). In contrast to NOS1−/− myocytes, NOS3−/− myocytes showed an increased functional response to β-AR receptor stimuli (Gyurko et al., 2000; Barouch et al., 2002; Champion et al., 2004; H. Wang et al., 2008b), whereas mice with NOS3 overexpression showed a decreased functional response to β-AR stimulated contraction (Brunner et al., 2001; Massion et al., 2004). Due to the same subcellular localization, NOS3 modulates LTCC. Specifically, acute NOS3 inhibition in WT myocytes or NOS3 knockout (NOS3−/−) myocytes had a greater β-AR stimulated Ca2+ current (ICa) (H. Wang et al., 2008b). In addition, our data demonstrated that PKG is involved in this pathway and likely phosphorylates LTCC at Serine496 on the beta2a subunit (Yang et al., 2007). Furthermore, we determined that the effect of NOS3 on LTCC is restricted to the caveolar microdomain via phosphodiesterasetype 5 (PDE5) (Ziolo et al., 2003; Wang et al., 2009). PDE5 is specific for cGMP and thus will prevent cGMP from freely diffusing. There also appears to be gender differences with NOS3 signaling. Sun et al., 2006 observed greater effects of NOS3 on LTCC in female myocytes compared to male myocytes (Sun et al., 2006). We believe it is this NOS3/PDE5/PKG/LTCC pathway that blunts the contractile response to β-AR stimulation. In addition to limiting β-AR stimulated Ca2+ influx, NOS3 signaling also shortens the action potential waveform. Specifically, NOS3−/− mice exhibit prolonged action potential (AP) duration with β-AR stimulation (H. Wang et al., 2008b). A key determinant of AP duration is the various K+ channels (Nerbonne & Kass, 2005). However, we observed no differences in K+ channels (transient outward (Ito), sustained (IKsus), or inward rectifier (IK1)) between WT and NOS3−/− myocytes (Wang et al., 2012). Thus, we deduce that the increased Ca2+ influx in NOS3−/− myocytes results in greater Na+/Ca2+ exchange (NCX) current that prolongs AP duration (Ferreiro et al., 2012). While NOS3 signaling does blunt the contractile effects of β-AR stimulation (Gyurko et al., 2000; Barouch et al., 2002; Champion et al., 2004; H. Wang et al., 2008b), this effect is minor and not always observed (Martin et al., 2006). Therefore, NO produced via NOS3 has a different role compared to NOS1. We propose that NOS3 signaling is protective against adrenergic toxicity. We have, in fact, observed greater β-AR induced spontaneous activity [early (EAD) and delayed (DAD) afterdepolarizations] in NOS3−/− myocytes (H. Wang et al., 2008b). It is known that EADs are caused by abnormal ICa activity (January & Riddle, 1989). In addition, the increased Ca2+ influx will lead to increased SR Ca2+ load, which we have observed in NOS3−/− myocytes (Wang et al., 2012). If there is SR Ca2+ overload, this will lead to spontaneous release and DADs (Tweedie et al., 2000). These effects were also reported in telemetric studies in NOS3−/− mice (Rakhit et al., 2001). Therefore, NOS3 inhibition of LTCC will prevent triggered arrhythmias by limiting afterdepolarizations. It is also known that prolonged AP duration is a contributing factor to reentrant arrhythmias (Pogwizd & Bers, 2004). Unfortunately, arrhythmias account for the majority of deaths in heart failure patients (Go et al., 2013). Therefore, dysfunction of NOS3 is likely involved in the arrhythmogenesis of heart failure. In addition to limiting the generation of arrhythmias, NOS3 signaling attenuates hypertrophy. A major pathway to induce pathological hypertrophy is the calcineurin-nuclear factor of activated T cells (NFAT) pathway. Activation of calcineurin by Ca2+ influx via LTCC leads to dephosphorylation and nuclear translocation of the cytoplasmic latent NFAT, resulting in expression of a series of hypertrophic genes (Houser & Molkentin, 2008). Thus, NOS3 and NO/cGMP/PKG inhibition of LTCC will attenuate calcineurin activation to prohibit NFAT translocation and hypertrophy (Fiedler et al., 2002). This can be observed in NOS3−/− mice which have hypertrophy (Wenzel et al., 2007). PDE5


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plays a significant role in this pathway especially with its upregulation in HF (Takimoto et al., 2005a; Vandenwijngaert et al., 2013). Recent work has also shown that NOS3 is localized to the nucleus where it can regulate transcription via nuclear β-AR (Vaniotis et al., 2013). While the role of nuclear NOS3 in hypertrophy has not been investigated, one could speculate that altered nuclear NOS3 signaling may contribute to the modulation of gene transcription during HF (Ziolo & Biesiadecki, 2013). In summary, NOS3-derived NO is a negative regulator of β-AR stimulated contraction via inhibition of LTCC. Furthermore, limiting Ca2+ influx has a protective role to help prevent arrhythmias and the progression of hypertrophy in heart failure. 2. Heart failure Heart failure (HF) is defined as the inability of the heart to pump enough blood to meet the metabolic demands of the body. Various experimental animal models are generated to produce heart failure, such as myocardial infarction, pressure overload, toxic drug treatment or mutations of sarcomeric or cytoskeletal proteins (Houser et al., 2012). Severe heart failure displays ventricular dilation and limited contractile reserve (Feldman et al., 2008). At the myocyte level, a final common pathway of HF, regardless of the causative event, is a lower SR Ca2+ load resulting in depressed cell contraction (Houser et al., 2000). An element of the lower SR Ca2+ load is a reduction in SR Ca2+ uptake. This is due to decreased SERCA expression and/or reduced PLB Serine16 phosphorylation. Another factor is an increase in diastolic SR Ca2+ leak due to increased RyR activity. The expression and activity of NCX is also increased in HF leading to a loss of Ca2+ from the myocyte. Associated with the Ca2+ mishandling, Na+ handling is also altered (increased NCX and Na+/H+ exchanger activity, late Na+ current, etc.) leading to Na+ overload to increase the propensity of arrhythmias. Much of the altered Ca2+ and Na+ handling in HF can be attributed to oxidative stress caused by increased ROS levels (Sag et al., 2013). O− 2 is the main type of ROS, with the main sources within cardiac myocytes being the mitochondria (W. Wang et al., 2008), NADPH oxidase (Li et al., 2002), and XOR (Ekelund et al., 1999). Additionally, NOS uncoupling (Takimoto et al., 2005b) and monoamine oxidase (Kaludercic et al., 2010) also contribute to the increased O− 2 production during HF. There is also a decrease in ROS degradation due to downregulation of SOD (Sam et al., 2005) and glutathione (Watanabe et al., 2013). The − higher O− 2 levels will result in increased levels of ONOO (by reacting with NO as described above), hydrogen peroxide (via SOD), and hydroxyl radical (via the Haber–Weiss reaction). An increase in these signaling molecules will lead to protein post-translational modifications (e.g., oxidation) resulting in contractile dysfunction. However, the focus of this review is NOS and the functional effects of ROS/oxidative stress has been reviewed elsewhere (Hafstad et al., 2013). 2.1. NOS signaling in heart failure While there is decreased [NO]i due to buffering with O− 2 in HF, there are also alterations in NOS activity resulting in lowered NO production. Thus, we will discuss how signaling via each NOS isoform is disrupted during HF. 2.2. NOS2 and heart failure NOS2 was first identified in activated (but not quiescent) macrophages and was termed inducible NOS (Lowenstein et al., 1992) and renamed to NOS2 to be consistent with the new nomenclature. During an inflammatory response, expression of NOS2 can also be induced within ventricular myocytes where it is a cytosolic protein (Schulz et al., 1992). Since the initial observation NOS2 in cardiac myocytes (Balligand et al., 1994), NOS2 is now widely considered to be involved

in various pathophysiological conditions of the myocardium, such as ischemia-reperfusion injury (Wildhirt et al., 1999), septicemia (Ullrich et al., 2000; Ziolo et al., 2001b; Ichinose et al., 2003), aging (Yang et al., 2004), infarction (Sam et al., 2001), and heart failure (Ziolo et al., 2004). NOS2 signals via the cGMP-dependent and independent pathways leading to targeting of different end proteins with distinctive post-translational modifications (phosphorylation, S-nitrosylation, nitration, and oxidation). In the diseases discussed above, expression of NOS2 has been found to contribute to the contractile dysfunction (Wildhirt et al., 1999; Ullrich et al., 2000; Sam et al., 2001; Ziolo et al., 2001b; Ichinose et al., 2003; Yang et al., 2004; Ziolo et al., 2004). Conversely, studies have also found a beneficial effect of NOS2 expression (Heymes et al., 1999; Paulus, 2000). This discrepancy may be due to which pathway is activated (cGMP-dependent or independent) and/or end targets, which may be dependent upon the ROS levels in the heart. Early on in the disease state and/or when NOS2 is first expressed, ROS levels in all likelihood will be low. This will allow NOS2 to signal via cGMP, which is protective. For example, NOS2 signaling has been found to decrease β-AR stimulated ICa via PKG phosphorylation to limit detrimental Ca2+ influx (as discussed above) (Ziolo et al., 2001a). However, since NOS2 activity is Ca2+/calmodulin independent, continuing NOS2 expression will deplete L-arginine concentrations resulting in the uncoupling of NOS2 − and the generation of O− 2 (Xia et al., 1996). In addition, in HF O2 production via mitochondria, NADPH oxidase, and XOR is increased. The resulting high levels of NO and O− 2 favors the formation of correspondingly high levels of ONOO−. In fact, it has been shown that high levels of ONOO− elicit contractile dysfunction with NOS2 expression (Ferdinandy et al., 2000). Thus, the role of NOS2 in the myocardium (beneficial or detrimental) may be time-dependent. However, studies using NOS2 knockout (NOS2−/−) mice clearly show that NOS2 expression is detrimental in HF. Specifically, with pressure overload (transverse aortic constriction — TAC) or myocardial infarction, NOS2−/− mice were better protected with diminished hypertrophy, remodeling, dilation, fibrosis, contractile dysfunction, apoptosis, and mortality (Sam et al., 2001; Zhang et al., 2007; Dias et al., 2010). The mechanism by which NOS2 results in contractile dysfunction in HF is by blunting basal contraction and the positive inotropic response during β-AR stimulation by reducing Ca2+ transient amplitude (Ziolo et al., 1998, 2004). This is accomplished by altering RyR activity and reducing PLB phosphorylation (Ziolo et al., 2001b; Yu et al., 2005; Kohr et al., 2008b, 2009). In addition to contributing to the contractile dysfunction, NOS2 signaling may contribute to pathological hypertrophy. NOS2 expression is achieved via the transcription factor nuclear factor kappaB (NF-κB) (Oddis & Finkel, 1996). NF-κB when complexed with the IκB inhibitor protein exists in an inactive form in the cytoplasm. Phosphorylation of IκB via IκB kinase (IKK) allows NF-κB translocation to the nucleus where it binds DNA and regulates gene transcription. IKK can be activated by a multitude of factors including O− 2 . NF-κB regulates the transcription of a wide array of genes; consequently NF-κB signaling can be protective and/or harmful to the heart in various cardiomyopathies (Van der Heiden et al., 2010). There have been several reports demonstrating that NF-κB knockout reduces pathological hypertrophy, including after myocardial infarction (Kawano et al., 2006). However, other studies found that NF-κB is essential for compensatory remodeling (Timmers et al., 2009). We believe that NOS2 signaling contributes to the damaging NF-κB pathway. Interestingly, specific inhibition of NOS2 results in decreased expression of NOS2 (Hu et al., 2006), suggestive of a feedback loop since ONOO− can also regulate NF-κB activity (Loukili et al., 2010). In situations (e.g., HF) of already activated NF-κB, NOS2 and high levels of ONOO− will cause greater NF-κB nuclear translocation and gene transcription. We believe that this NOS2/NF-κB axis drives the heart into hypertrophy and this pathway warrants much more study. Thus, in the setting of HF, specific inhibition of NOS2 (and not the other NOS isoforms) is an attractive therapeutic approach.



2.3. NOS1 and heart failure There is ample evidence that NOS1 is also involved in the contractile dysfunction in HF. Particularly, studies have shown that myocardial infarction in NOS1−/− mice display worse ventricular remodeling, contractile dysfunction, and mortality compared to WT mice (Dawson et al., 2005; Saraiva et al., 2005). Whereas, mice with cardiac myocyte-specific NOS1 overexpression developed less dilation, maintained contractile function and prevented the development of HF in a TAC model (Loyer et al., 2008). The protein end targets and components of the NOS1 signaling pathway exhibit altered function in HF (i.e., decreased PLB Serine16 phosphorylation (Huang et al., 1999; Schwinger et al., 1999), increased diastolic SR Ca2+ leak (Ai et al., 2005), and increased XOR activity (Gonzalez et al., 2010)). The increased diastolic SR Ca2+ leak is due to enhanced RyR activity and has been explained by increased RyR phosphorylation via CaMKII (Maier, 2005; Curran et al., 2010; Neef et al., 2013), or thiol oxidation of RyR (Mochizuki et al., 2007; Terentyev et al., 2008; Gonzalez et al., 2010). ROS production from XOR is increased, which contributes to the increased RyR oxidation (Gonzalez et al., 2010) and possibly CaMKII activation, which can occur in the absence of Ca2+/calmodulin (Erickson et al., 2008). In addition, NOS1 is found to be uncoupled under oxidative stress in vitro (Sun et al., 2008), but whether this process exists in HF and contributes to the oxidative stress is still not known. Interestingly, expression and activity of NOS1 were found to be increased in human HF and a rat myocardial infarction HF model. However, NOS1 translocates from the SR to caveolae in the sarcolemmal membrane by binding to caveolin-3 (Damy et al., 2003; Bendall et al., 2004; Damy et al., 2004). We believe that this is analogous to knockout of NOS1 arising to similar functional effects we and others observe in NOS1−/− mice. These data suggest that the loss of NOS1 signaling at the SR microdomain contributes to the dysregulation of Ca2+ handling in HF. NOS1 signaling has also been reported to be protective against arrhythmias. The translocation of NOS1 has been shown to decrease β-AR stimulated contraction via reducing LTCC by PKG phosphorylation (Bendall et al., 2004; Oceandy et al., 2007) acting similar as NOS3 in healthy hearts. This occurs since NOS3 expression is decreased in HF and NOS1 recoups the loss of NO in this microdomain. Consistent with that assertion, NOS1−/− mice have increased arrhythmias after myocardial infarction (Burger et al., 2009). Furthermore, CAPON, a NOS1 adaptor protein, is reported to be beneficial in preventing prolonged QT interval and sudden cardiac death (Arking et al., 2006; Kao et al., 2009), by mediating NOS1 and ICa reduction. With the decrease in NOS3 expression (Piech et al., 2002) and/or uncoupling of NOS3 (Takimoto et al., 2005b) in HF, we believe the translocation of NOS1 plays as a compensatory mechanism for the loss of NOS3 to prevent arrhythmias. This will also limit detrimental Ca2+ influx to prevent pathological hypertrophy. Thus, a beneficial therapeutic approach is to increase NOS1 or NO. In fact, we have shown that the NO donor SNAP was much more beneficial in increasing contraction in NOS1−/− myocytes compared to WT myocytes (Wang et al., 2010). However, care must be taken to assure that this occurs at the SR microdomain. NOS1 is also shown to form a protein complex with PMCA4b, sodium channel (Nav1.5), and α-syntrophin located at a different subcellular domain — the sarcolemma membrane (Williams et al., 2006). S-nitrosylation of Nav 1.5 by NOS1 increases late Na+ current and derangement of this complex can lead to increased late Na+ current causing long QT syndrome (Ueda et al., 2008).

2.4. NOS3 and heart failure NOS3 expression levels have been shown to be decreased in HF (Piech et al., 2002). There is also a reduction in Caveolin-3 expression (Feiner et al., 2011), that may further mediate the loss of NOS3 signaling in the correct subcellular location. The decrease in NOS3 activity

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decreases NO production, which is an enormous detriment to the failing heart. The negative effect of losing NOS3 signaling is prominently illustrated in NOS3−/− mice. After a myocardial infarction, NOS3−/− mice display worse ventricular remodeling, hypertrophy, and contractile dysfunction (Scherrer-Crosbie et al., 2001), whereas cardiomyocytetargeted NOS3 expression rescued the LV remodeling after pressure overload (Buys et al., 2007). As previously discussed, NOS3 acts as an antihypertrophic factor in a cGMP dependent manner by inhibition of the calcineurin-NFAT pathway and protects the heart from arrhythmias. Thus, in the setting of HF, increasing expression of NOS3 is an attractive therapeutic approach. However, caution must be taken with this approach since NOS3 can become uncoupled in HF to produce O− 2 and increase ROS levels in the heart (Takimoto et al., 2005b). This effect may be further exacerbated by a decrease in SOD expression with HF (Sam et al., 2005). Thus, a more advisable approach would be to target the beneficial downstream targets of NOS3 (e.g., PDE5 to increase cGMP levels). 3. Therapeutic Targeting of NOS dysfunction in heart disease The decreased NO bioavailability and oxidative stress in HF is directly the result of dysfunctional NOSs with the consequence of contractile dysfunction, hypertrophy, and adverse remodeling. Numerous therapeutic approaches have been tried to repair NO signaling in human HF, but have had little success. As mentioned, NOS2 inhibition would be beneficial for HF to lower ONOO− levels. However, trials have been terminated early due to negative effects in patients most likely due to inhibition of the other NOS isoforms (Bailey et al., 2007). Consequently, there is a great need to develop a specific NOS2 inhibitor that will not affect the other NOS isoforms. Another approach used was to broadly increase NO levels. For example, organic nitrates are clinically used for treatment of ischemic symptoms of angina and congestive heart failure via increasing blood NO concentration, but showed side effects of increasing superoxide production with long-term consumption (Dikalov et al., 1999; Szocs et al., 2007). This could result in high levels of ONOO− doing more harm to the patient. Another approach has been to pharmacologically supplement BH4 to recouple NOS3 to increase NO production. Unfortunately, BH4 in the setting of HF is easily oxidized to BH2 and has limited effects on NOS3 (Moens et al., 2008, 2011a, 2011b). There are also compounds (e.g., nebivolol) that increase NOS3 expression and prevent uncoupling. These compounds had positive effects in animal HF models (Wohlfart et al., 2008) and human cell lines (Cominacini et al., 2003; Ignarro, 2008), but were ineffective in human failing myocardium (Brixius et al., 2006). Other compounds, such as hydrogen sulfide can increase NOS3 activity via an Akt-dependent process (Predmore et al., 2011) and may be promising for the treatment of HF (Kondo et al., 2013). Additional approaches have been to target components downstream of NOS3 to increase cGMP levels. These tactics used direct, non-NO, activators of sGC (such as cinaciguat) or using specific PDE5 inhibitors (such as udenafil, vardenafil and sildenafil). These compounds have undergone clinical trials in systolic/diastolic heart failure patients with various outcome measurements (peak oxygen consumption, 6 minute walk, pulmonary capillary wedge pressure, left ventricular systolic/diastolic parameters). While, cinaciguat did not show any beneficial effects (Gheorghiade et al., 2012), chronic PDE5 inhibition appeared to improve functional capacity and clinical status (Behling et al., 2008; Guazzi et al., 2011). Additional trials are currently ongoing to test long lasting PDE5 inhibition (udenafil) or additional forms of HF (e.g., heart failure with preserved ejection fraction) (Kim et al., 2013; Redfield et al., 2013). Thus, a promising approach in using the NOS pathway to treat HF patients is manipulating downstream components of the signaling cascade (specifically by inhibiting PDE5 to increase cGMP levels).


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3.1. Nitroso-redox balance Instead of trying to increase NO levels, antioxidants to lower ROS levels and reduce the oxidative stress have been tested. However, general antioxidant strategies such as Vitamins E and C supplementation showed disappointing or even risky effects on cardiovascular events (Lonn et al., 2005). Specific inhibitors of proteins that produce O− 2 have also been tested. Statins are currently prescribed for the treatment and prevention of HF. While statins lower serum cholesterol levels, they have many additional affects such as the inhibition of NADPH oxidase (Wassmann et al., 2002). NADPH oxidase inhibitors have proved to be useful for the treatment of HF in animals (Qin et al., 2007). However, it appears that statins may not have beneficial effects in human patients with HF (Bonsu et al., 2013). Inhibitors of XOR (allopurinol or oxypurinol) have also been tested since there were beneficial results in canine and rat HF models (Saavedra et al., 2002; Minhas et al., 2006). Unfortunately, XOR inhibition had no beneficial effects in human failing patients (Hare et al., 2008; Nasr & Maurice, 2010). A possible reason why these antioxidant therapies (general scavengers, NAPDPH oxidase/XOR inhibitors etc.) did not work is that they only target one side of the nitroso-redox balance, which has a tight functional and often chemical coupling between O− 2 and NO. Therefore, it may not be adequate to treat only one part of the balance; but it may also be necessary to simultaneously restore the partner molecule (i.e., both NO (increase) and O − 2 (decrease) levels). We developed a novel cell permeable nitrone (EMEPO), which scavenges O − 2 and produces NO as a reaction byproduct to repair the nitroso-redox imbalance. Because NO is only released − upon O − 2 binding, EMEPO targets decreased O 2 and increased NO where it is needed within each cell. Acute EMEPO treatment showed beneficial effects on contractile function in isolated myocytes from a post-MI canine model (Traynham et al., 2012). In fact, this approach (using two pharmacological compounds — hydralazine and isosorbide dinitrate) to restore nitroso-redox balance showed beneficial effects (ameliorated contractile dysfunction and decreased mortality) in human HF patients (Taylor et al., 2004). However, the myocyte (or other cell types) may differentially uptake the two compounds and the nitroso-redox imbalance is some cells may remain. We believe a better approach is to use one compound to ascertain that the nitroso-redox balance is restored in each cell. Thus, fixing the nitroso-redox imbalance provides a promising strategy to correct NOS dysfunction in the treatment of various cardiomyopathies. Given the complexity of NOS dysfunction in heart disease (i.e., isoforms, compartmentalized function, translocation, uncoupling, and ROS), the use of general NOS inhibitors, NO donors, and ROS scavengers/inhibitors may not represent the best option. Further work is required to find “perfect” strategies to specifically target signaling pathway components and end targets to regain proper NO signaling.

4. Conclusions NO signaling plays a key role in the modulation of heart function and growth. In addition to its regulation of normal physiology, altered NOS function is a major contributor to the syndrome of HF. Changes of expression, location, and activation of each NOS isoforms results in deranged downstream protein function, causing irregular contractile function and adverse remodeling (Fig. 1). Various therapeutic strategies have been proposed to address the dysfunction of NOS signaling by restoring NO production or targeting downstream mechanisms; however, limited beneficial results have been obtained in human HF. A promising therapy may be the simultaneous restoration of both NO and O− 2 levels directly where needed and warrants further investigation.

Fig. 1. NOS isozymes and signaling in a healthy (top) and failing (bottom) cardiac myocyte. Top) NOS1 co-localizes with ryanodine receptors (RyR) and xanthine oxidoreductase (XO) on the sarcoplasmic reticulum (SR). It regulates Ca2+ handling directly (RyR S-nitrosylation and protection from RyR oxidation) and indirectly (PLB phosphorylation) via a cGMP independent pathway. NOS3 is located closely to L-type Ca2+ channels (LTCC) and superoxide dismutase (SOD) in caveolae. It protects myocytes from arrhythmias and hypertrophy via inhibition of LTCC and the calcineurin/NFAT pathway in a cGMP/PKG dependent pathway. NOS3 signaling is compartmentalized via phosphodiesterasetype 5 (PDE5). Bottom) NOS1 expression is increased but translocates to caveolae, leading to decreased localization at the SR and decreased NO but increased O− 2 levels. NOS3 expression is decreased, leading to arrhythmias via increased LTCC and hypertrophic remodeling via enhanced calcineurin-NFAT signaling. NOS2 is expressed and regulates contraction/hypertrophy via both cGMP dependent and independent pathways. In addition, both NOS2 and NOS3 can − be uncoupled to produce O− 2 and ONOO contributing to the increased oxidative stress in − HF. NO: nitric oxide, O− 2 : superoxide, ONOO : peroxynitrite, PLB: phospholamban, and SERCA: SR Ca2+-ATPase, solid line: positive regulation, and dashed line: negative regulation.

Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This research was supported by the National Institutes of Health (K02HL094692, MTZ). References Ai, X., Curran, J. W., Shannon, T. R., Bers, D.M., & Pogwizd, S. M. (2005). Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor


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Targeting NEK2 as a promising therapeutic approach for cancer treatment.

Targeting CBLB as a potential therapeutic approach for disseminated candidiasis.

Targeting the mevalonate cascade as a new therapeutic approach in heart disease, cancer and pulmonary disease.

Current therapeutic approach in heart failure with preserved ejection fraction.

Therapeutic effects of pentoxifylline on diabetic heart tissue via NOS.

Myocardial viability as integral part of the diagnostic and therapeutic approach to ischemic heart failure.

[Organ-protection therapy. A new therapeutic approach for acute heart failure?].

Targeting CD44 as a novel therapeutic approach for treating pancreatic cancer recurrence.

Targeting microRNAs in heart failure.

Angiotensin-converting enzyme 2 as a therapeutic target for heart failure.

G protein-coupled Receptor Kinase 2 as a Therapeutic Target for Heart Failure.

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Targeting hydrogen sulfide as a promising therapeutic strategy for atherosclerosis.

Targeting α-synuclein as a therapeutic strategy for Parkinson's disease.

Therapeutic targeting of the oncostatin M receptor-β prevents inflammatory heart failure.

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New diagnostic and therapeutic possibilities for diastolic heart failure.

Heart Failure: Targeting miRNA pathology in heart disease.

Targeting interleukin-1 in heart failure and inflammatory heart disease.

Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach.

[Video games as a therapeutic approach for patients: coming soon?].

Thoracic impedance as a therapeutic marker of acute decompensated heart failure.

Epigenetic modulation as a therapeutic approach for pulmonary arterial hypertension.

Reducing methylglyoxal as a therapeutic target for diabetic heart disease.