Nitric oxide signalling and neuronal nitric oxide synthase in the heart under stress

Introduction

Nitric oxide (NO) is an essential molecule that plays fundamental roles in maintaining cardiovascular functions in animals and humans^1–5. The NO that exerts biological functions in the myocardium can be acquired through exogenous sources or is produced from the endogenous endothelial and neuronal NO synthases (eNOS and nNOS, respectively, which are constitutively expressed in the myocytes) and from inducible NOS by inflammatory cytokines following infection^4–6 (Figure 1). In the last few decades, our understanding of the detailed mechanisms, the effects of NO on myocardial functions, and the roles for NOSs in diseased hearts has improved. Comprehensive approaches have been undertaken to achieve this outcome, including manipulation of the upstream and downstream effectors of NOSs (pharmacologically or genetically modified NOS regulation and viral infections of specific NOS genes), supplementation of NO mimetics (for example, exogenous NO donors or NO substrates), and systematic detection of plasma and tissue NO^4,5,7,8. Nevertheless, the practical implications of NO and its precursors or regulators in translational and therapeutic strategies in cardiovascular diseases are hampered because of the complex nature of NO and the array of downstream signalling cascades and effectors in the myocardium. In the initial part of this review, I will provide a systematic overview of the sources and post-transcriptional modification of NO in the myocardium; the latter part of the review will focus on recent advances of nNOS-targeting proteins and responses in the heart under stress. Ultimately, I will delineate the prospect for using the therapeutic platform of nNOS and NO to target human heart diseases.

Figure 1. Schematic diagram demonstrating the sources of nitric oxide (NO) in the heart and mechanisms mediating the effects of NO and its derivatives.

Both exogenous sources (nitrate-rich vegetables and food through the entero-salivary nitrate–nitrite–NO pathway and skeletal muscle nitrate → NO pathway) and endogenous sources (neuronal nitric oxide synthase [nNOS], endothelial NOS [eNOS], or inducible NOS [iNOS]) determine the bioavailable NO in the myocardium. NO regulates downstream targets through soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-dependent phosphorylation, S-nitrosylation, and transnitrosylation. Alternatively, NOS-derived radicals and NO-related derivatives (H₂O₂, O₂^–, and peroxynitrite [ONOO^–], etc.) affect downstream effectors through the oxidation and S-glutathionylation. As such, NO regulates membrane proteins, Ca²⁺-handling proteins, membrane proteins, and organelle effectors in the cardiac myocytes.

Exogenous sources of nitric oxide

It is generally acknowledged that NO is derived from the classic L-arginine–NOS–NO pathway. In fact, NO that exerts functions in the myocardium may also be acquired from the alternative source of NO, the nitrate–nitrite–NO pathway (Figure 1). Nitrate (NO₃⁻) in various types of green leafy vegetables and food^9–11 is taken up into the plasma to become a reliable reservoir and the stable precursor of NO (the half-life of nitrate in the plasma is 5–6 hours). Nitrate from this source is actively taken up by the salivary gland, is secreted in concentrated form in the saliva (about 10-fold that in the plasma), and is subsequently reduced to more active nitrite (NO₂⁻) in the oral cavity by nitrate reductases of commensal bacteria (entero-salivary NO pathway). Nitrite is reduced to NO in the acidic environment of the stomach and is absorbed into the blood in the upper gastrointestinal system (the half-life of nitrate in the plasma is about 30 minutes), while the rest mixes with the nitrite formed from nitrate derived from endogenous NOS-produced NO. Various enzymes and proteins are known to be involved in the NO metabolite cycle and nitrite’s reduction to NO, including xanthine oxidase^12,13, deoxyhaemoglobin and deoxymyoglobin^14–16, neuroglobin¹⁷, respiratory chain enzymes¹⁸, cytochrome P450¹⁹, aldehyde oxidase²⁰, carbonic anhydrase²¹, and NO synthase²². Overall, about 25% of nitrate undergoes re-uptake by the salivary gland and produces functional NO in the circulation; the rest of the nitrate is eventually excreted in the urine. The amount of NO from exogenous sources can be as high as the amount that is produced from NOSs in the tissues (with enough daily consumption of green leafy vegetables or food, nitrite intake varies from 0 to 20 mg/day²³), indicating the importance of this pathway in supplementing local NO in the tissue. Notably, unlike NO from the L-arginine–NOS–NO pathway, food-derived functional NO is oxygen independent^10,11. Accordingly, NO from this source becomes more important in ischaemic or hypoxic conditions, such as myocardial infarction, hypertrophy, and heart failure.

NO or nitrite can undergo an oxidative process via oxyhaemoglobin or oxymyoglobin to produce stable nitrate, which can be reduced back to nitrite and NO by molybdopterin-containing mammalian nitrate reductases, such as xanthine oxidoreductase or aldehyde oxidase^10,11. Therefore, there is a constant recycling of NO precursors, metabolites, and NO that maintains exogenous NO in the human body. The respective contributions of the endogenous versus exogenous NO to intracellular signalling and function in healthy and diseased hearts in vivo remain to be revealed.

A number of organs or tissues (for example, neurons, liver, heart, skeletal muscle, kidney, arteries such as the aorta, and endothelium) are the active sites for NO production from constitutive NOSs. Recently, it has been shown that skeletal muscle is a dynamic nitrate reservoir that increments plasma nitrate and nitrite because of the abundance of the tissue in the mammalian body²⁴. nNOS in the skeletal muscle contributes to the supply because it is the only isoform in the skeletal muscle²⁵. However, the proportions of NO from the specific sources that contribute to the bioavailable NO in the myocardium remain undetermined.

Endogenous sources of nitric oxide in the myocardium

Multifaceted mechanisms mediating the effects of neuronal nitric oxide synthase

It is generally accepted that S-nitrosylation (or S-nitrosation) and soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-dependent phosphorylation are the predominant mechanisms that mediate the effects of NO in biological systems (Figure 1). The former mechanism involves post-translational modification of a thiol group in proteins by NO (transferring NO to cysteine residues, -SNO), and the latter implicates PKG-dependent phosphorylation of serine residues of the target proteins. S-nitrosylation is explicitly initiated by NO, but dinitrogen trioxide (N₂O₃), the nitrosonium ion (NO⁺), peroxynitrite (ONOO⁻), and SNO proteins are also able to deliver NO to the cysteine residues of the target proteins^44,45. Protein-protein transfer of NO (trans-S-nitrosylation) is now known to represent one of the most important mechanisms of NO⁴⁶ (Figure 1). In this process, the SNO “donor” proteins are referred to as nitrosylases. Trans-S-nitrosylation possesses advantages for effective interactions between proteins⁴⁷. Furthermore, transnitrosylation is important when NO bioavailability is limited in an oxidative and/or nitrosative stress environment, such as during ischaemic reperfusion. S-nitrosylation can be terminated by the action of denitrosylases (for example, S-nitrosoglutathione reductase and thioredoxin), with NADH and NADPH serving as electron donors to regenerate glutathione and thioredoxin^48,49.

Various types of proteins are targeted by NO, which in turn triggers an array of signalling cascades depending on the properties of the target proteins, e.g. inhibition of protein phosphatase 2A/protein phosphatase 1 by NO leads to protein kinase A (PKA) and Ca²⁺-calmodulin-dependent kinase II-dependent phosphorylation of downstream effector proteins such as phospholamban (PLN)⁵⁰, whereas sGC activation by NO in the myocardium of hypertensive rats causes cGMP/PKG-dependent phosphorylation of cTnI and cMyBPC³⁴. Conversely, phosphodiesterase 5 (PDE5) activation by NO/sGC/cGMP/PKG limits cytosolic cGMP, a negative feedback mechanism of NO regulation of cGMP in cardiac myocytes⁵¹. In addition, by targeting cardiac oxidases, such as xanthine oxidoreductase⁵², NADPH oxidase^53,54, and mitochondrial reactive oxygen species (ROS) production⁵⁵, nNOS-derived NO controls intracellular oxidative status and ROS-dependent downstream effects in the myocardium. Cysteine residues are the targets of ROS to cause S-glutathionylation in the proteins^56,57; therefore, S-nitrosylation by NO may “block” critical cysteine residues from irreversible oxidation under the conditions, such as increased oxidative stress. Consequently, post-transcriptional modifications downstream of NO change the effector proteins, altering their localisation, binding partners, activity, and, ultimately, function.

nNOS has also been demonstrated to produce H₂O₂ in the endothelium of large arteries, such as the aorta, and H₂O₂ mediates endothelium-dependent vascular relaxation^58,59. Conversely, impairment of endothelial nNOS-derived H₂O₂ has been shown to worsen endothelial dysfunction in both atherosclerosis^60,61 and hypertension⁶², indicating a protective role of nNOS-derived H₂O₂ in the vasculature. Similarly, both eNOS-derived NO and nNOS-derived H₂O₂ contribute to acetylcholine stimulation of vasodilatation⁵⁹ by regulating similar downstream protein kinases and phosphatases^63–65. In contrast, uncoupling of eNOS and nNOS (secondary to the deficiency of L-arginine, tetrahydrobiopterin [BH4] oxidation, or S-glutathionylation^52,66–68) results in the production of superoxide (O₂⁻) instead of NO; under such conditions, eNOS and nNOS become the sources of oxidative stress for pathological progression in the myocardium.

Taken together, the mechanisms mediating the effect of NO are complex. S-nitrosylation, transnitrosylation, and sGC/cGMP/PKG-dependent phosphorylation provide major post-transcriptional modifications of NO. By producing H₂O₂, O₂⁻, and the NO derivatives, NOSs also function as the upstream regulators of redox-dependent signalling.

Effector targets of neuronal nitric oxide synthase maintaining cardiac contraction and relaxation during disease progression in the heart

Nitric oxide, nitric oxide synthase, and cardiovascular therapy

Because of the importance of NO and its significance in the cardiovascular system, approaches that manipulate the bioavailability of NO in the myocardium are essential therapeutic strategies for the better treatment of cardiovascular diseases (CVDs). In fact, nitroglycerin, an organic nitrate that releases NO, has been used clinically in the treatment of CVD for more than 150 years (despite the fact that the effective molecule for the response, NO, was identified in the late 1970s¹). Enhanced acknowledgement of the mechanistic insights into NO signalling, exogenous versus endogenous NO sources, the maintenance and the degradation of NO, and the properties of NOSs as well as modern technology enables novel approaches to increase NO bioavailability in target tissues for the desired responses. In principle, enhancement of NO and its signalling can be achieved through three routes: increase exogenous and endogenous sources to promote NO production, reduce NO metabolism/degradation, and stimulate downstream signalling of NO.

A number of strategies are used to promote NO formation. For example, inhaled NO is registered to be applied to newborn babies with persistent pulmonary artery hypertension^106,107 to support ventilation-perfusion match and to prevent systemic ischaemia. Nitrite can be similarly applied; in fact, the effectiveness of oral, inhaled, and intravenous nitrite on a number of CVDs (such as pulmonary artery hypertension [PAH], peripheral vascular diseases, myocardial infarction, and cerebral vasospasm after subarachnoid haemorrhage) are under study with promising prospects on some occasions^108–110. Delivering organic and inorganic nitrate and nitrite to amplify systematic or local NO through nitrate–nitrite–NO and the nitrate–nitrite–NO–fatty acid pathways are probably the most active area under investigation experimentally and in the clinic¹⁰. So far, a number of putative precursors of NO (nitroxyl [HNO], S-nitrosothiols, sodium nitrite, sodium nitrate, nitrated fatty acids, and nitrate from beetroot juice and green leaves, such as spinach, etc.) have been identified and are under development. Dietary consumption of NO precursors is a cheap, safe, and effective way of nitrate delivery; programming of a suitable diet regime for vulnerable populations will be important to reduce the cardiovascular risks as well as the economic burden on national healthcare systems. The relationship between the daily consumption of nitrate and cardiovascular events is noticeable. For example, high fruit and green vegetable intake in a Japanese population historically known to have low rates of CVD (daily consumption of nitrate >1,100 mg/60 kg) is associated with greater circulating nitrate and nitrite¹¹¹ compared to those in the US and Europe, where average daily nitrate consumption ranges from 40–100 mg and 30–180 mg, respectively, and the rates of CVD are high^112,113. Moreover, the consumption of “healthy” fats, as in the Mediterranean diet, in the form of unsaturated fatty acids such as oleic and linoleic acid (to form nitrated fatty acids), is beneficial in preventing the development of CVD and reduces the risk factors¹¹⁴. Notably, nitrite reduction to NO preferentially occurs in the presence of hypoxia and acidosis, during physical exercise, at the time when cardiac muscle needs NO the most.

Alternatively, supplementation of NO substrates, e.g. arginine, L-citrulline, and BH4 (a co-factor of NOS), and inhibition of arginase and asymmetric dimethylarginine (an endogenous NOS inhibitor) are the necessary strategies to increase NO through promoting NOS activity¹¹. Statins and nebivolol or carvedilol (new-generation beta1-adrenergic receptor blockers) exert anti-adrenergic responses via the stimulation of the beta3-adrenergic receptor and increasing NOS production of NO^115–118. Targeting NOS is advantageous in mediating the specific downstream signalling of NOSs in the compartments.

Attempts to prevent NO reduction and inhibition of NOS uncoupling are also important in maintaining or increasing cytosolic NO. Decreasing the formation of ROS using blockers of angiotensin-converting enzyme (ACE), angiotensin I type 1 receptor (AT1R), or NADPH oxidases (NOXs) or reducing ROS by using antioxidants and scavengers are the putative mechanisms to reduce NO “sink” and therefore maintain or increase NO level¹¹. However, the complex NOX isoforms and the redox–nitrosol network weaken the effectiveness of the developed drugs in clinical use. Indeed, our recent results indicate that NOX and ROS are upstream regulators of cardiac nNOS protein and activity downstream of Ang II and AT1R¹¹⁹. Furthermore, Ang II type 2 receptor (AT2R) mediates Ang II enhancement of nNOS protein expression via ROS activation of eNOS activity¹¹⁹. As such, it is wrong to simply assume that NO level can be increased by using ACE and AT1R inhibitors. The development of selective NOX inhibitors and specific ROS-manipulating drugs that do not affect NOS protein should be pursued, and the effect of NOX and ROS on nNOS protein expression in the myocardium should be taken into consideration.

Stimulation of the downstream signalling pathway of NO is an improved strategy to target the effector proteins directly, bypassing the complex scenario of NOSs and the redox–nitrosol network. Synthetic benzyl indazole compound YC-1, the oral sGC stimulators riociguat and vericiguat, and atrial, brain, and C-type natriuretic peptides are in use to increase cellular cGMP¹¹. Inhibition of a negative regulator of cGMP, PDE5 (e.g. using sildenafil), is another therapeutic approach to stimulate cGMP/PKG signaling^120–122. Stimulation of the PKG-dependent pathway has been shown to exert potent protective effects in a wide array of cardiovascular disease models, including hypertension, PAH, heart failure, haemolytic anaemia, and infarct-reperfusion injury^120–124. However, the application of the drugs in a large cohort of patients with CVD show minor responses to the treatment, suggesting further investigation on cGMP enhancers is still needed.

Primary S-nitrosothiols (RSNOs) are the endogenous NO carriers and donors and have emerged as platforms for the localised delivery of NO, which mediates S-nitrosation-dependent mechanisms¹²⁵. S-nitrosoglutathione (GSNO) is one of the main RSNOs and is a central intermediate in the formation and degradation of cellular S-nitrosothiols with potential therapeutic applications¹²⁵. So far, GSNO has not been implied in pharmaceutical composition owing to the fast decomposition in aqueous solutions. To sustain the bio-available NO donors and optimise their kinetics and to control the delivery of NO to targeted tissues or proteins, various biomaterial-based carriers (microparticles and nanoparticles) are under development¹²⁶.

Taken together, a number of validated ways have been developed to increase systemic and local NO levels and are promising in mediating the beneficial effects in CVD. However, in order to translate the research innovations into the application to a large population, more research is necessary, with special attention to the specificity and effectiveness, i.e. nitrate/nitrite regime in the diet and strategies of increasing nNOS (as well as eNOS) and improving NO-effector interactions in CVD settings. NO-dependent therapy holds promise as a therapeutic platform for CVD in humans.

Future perspectives

Our understandings of the NO and NOSs that regulate myocardial contraction, relaxation, and pathological signalling are advanced, but the dynamic paradigm in the myocardium under stress is not clearly presented. NO from both exogenous and endogenous sources supply the bioavailable NO in the myocardium, and the level and effectiveness of NO is determined by multiple regulation mechanisms including daily consumption of NO precursors, nitrate from skeletal muscles, NO production through the entero-salivary NO pathway (oral and gut microbiome function as essential regulators) and from NOSs as well as redox environment and the nature and the abundance of target proteins. In general, NO regulates downstream effector proteins through three mechanisms (sGC/cGMP/PKG-dependent phosphorylation, S-nitrosylation, and transnitrosylation) and the numbers and types of effectors regulated by NO are diverse. As such, modification of these effectors by NO subsequently triggers an array of signalling cascades that lead to different physiological and pathological consequences. By and large, NO and its downstream signalling pathway exert potent cardiovascular protection; however, translational research of NO and NOS that are applicable for CVD and therapeutic efficiency using an NO-dependent regime are still far from satisfactory¹¹. Further research needs to be carried out aiming to increase the specificity of NO-effector interaction at the site of relevant proteins. Better design of a dietary program and the promotion and targeting of nNOS and eNOS in specific locations with the effector target(s) of importance will maximise the effectiveness of NO application in cardiovascular physiology and pathology.