Treating oxidative stress in heart failure: past, present and future

Abstract

Advances in cardiovascular research have identified oxidative stress as an important pathophysiological pathway in the development and progression of heart failure. Oxidative stress is defined as the imbalance between the production of reactive oxygen species (ROS) and the endogenous antioxidant defence system. Under physiological conditions, small quantities of ROS are produced intracellularly, which function in cell signalling, and can be readily reduced by the antioxidant defence system. However, under pathophysiological conditions, the production of ROS exceeds the buffering capacity of the antioxidant defence system, resulting in cell damage and death. Over the last decades several studies have tried to target oxidative stress with the aim to improve outcome in patients with heart failure, with very limited success. The reasons as to why these studies failed to demonstrate any beneficial effects remain unclear. However, one plausible explanation might be that currently employed strategies, which target oxidative stress by exogenous inhibition of ROS production or supplementation of exogenous antioxidants, are not effective enough, while bolstering the endogenous antioxidant capacity might be a far more potent avenue for therapeutic intervention. In this review, we provide an overview of oxidative stress in the pathophysiology of heart failure and the strategies utilized to date to target this pathway. We provide novel insights into modulation of endogenous antioxidants, which may lead to novel therapeutic strategies to improve outcome in patients with heart failure.

Introduction

Oxidative stress is involved in the development and progression of clinical and experimental heart failure.1, 2, 3, 4 Oxidative stress is defined as a dysregulation between the production of reactive oxygen species (ROS) and the endogenous antioxidant defence mechanisms, the so called ‘redox state’. When present in low concentrations, ROS plays a critical function in cell homeostasis. However, excess ROS causes cellular dysfunction, protein and lipid peroxidation, DNA damage, and eventually leads to irreversible cell damage and death. This is also evident in the heart where high sensitive troponin assays have demonstrated an increase in troponin release during heart failure progression, suggesting a gradual loss in cardiomyocytes.5

In the heart, an overabundance of ROS can lead to the development and progression of maladaptive myocardial remodelling and heart failure (Figure 1). ROS directly impairs the electrophysiology and the contractile machinery of cardiomyocytes by modifying proteins central to excitation–contraction coupling, including L‐type calcium channels, sodium channels, potassium channels, and the sodium–calcium exchanger.6 ROS can also alter the activity of the sarcoplasmic reticulum Ca²⁺‐adenosine triphosphatase (SERCA) as well as reduce myofilament calcium sensitivity.6 Furthermore, ROS induces an energy deficit by affecting the function of proteins involved in energy metabolism.6 Finally, ROS has a pro‐fibrotic function, by inducing cardiac fibroblast proliferation and matrix metalloproteinases resulting in extracellular remodelling.6

Figure 1.

The effects of excessive oxidative stress on the myocardium. As a result of cardiac injury there is a severe accumulation of oxidative stress (reactive oxygen species, ROS), which has several detrimental effects on the myocardium. 1. Cardiomyocyte electrophysiology is severely affected by increased ROS. ROS reverses the function of the Na⁺/Ca²⁺ exchanger (NCX), leading to Ca²⁺ influx and Na⁺ efflux. ROS also increases the influx of Ca²⁺ via the L‐type calcium channels. Increased ROS also increases sarcK_ATP currents, leading to action potential duration shortening, while also reducing K_V currents and increasing late sodium currents leading to prolonged action potential durations. 2. Excessive ROS promotes ryanodine receptor 2 (RyR2) activity and inhibits sarcoplasmic reticulum Ca²⁺‐adenosine triphosphatase 2 (SERCA2) activity, resulting in calcium overload and reduced myofilament calcium sensitivity, eventually leading to contractile dysfunction. 3. The mitochondria react to ischaemic injury by producing increased levels of ROS, however the overabundance of ROS inversely results in further mitochondrial and energy metabolism dysfunction. 4. The increase in ROS is also responsible for increased fibrosis resulting from an increase in tissue inhibitors of metalloproteinases (TIMP) and reduction in matrix metalloproteinase (MMP) expression.

This review will summarize the current knowledge regarding oxidative stress production and the antioxidant defence mechanism in the heart, under physiological and pathophysiological conditions. Furthermore, we recapitulate the current knowledge, failures and successes, regarding the treatment of heart failure by targeting oxidative stress. Finally, we discuss the future potential of targeting endogenous oxidative stress defence mechanisms, to improve clinical outcome in patients with heart failure.

Reactive oxygen species in heart failure and antioxidant mechanisms: a brief summary

Reactive oxygen species production in the heart is primarily achieved by the mitochondria, NADPH oxidases, xanthine oxidase, and uncoupled nitric oxide synthase (NOS) (Figure 2). Under pathological conditions, the electron transport chain of the mitochondria induces the formation of large quantities of superoxide. This increase has been shown to contribute to cardiomyocyte damage and larger myocardial injury after an acute myocardial infarction.7, 8 ROS production is also enhanced due to an increased expression and activity of NADPH oxidase, resulting from several pathological stimuli, including mechanical stretch, angiotensin II, endothelin‐1, and tumour necrosis factor (TNF)‐α.9, 10, 11 Similarly, xanthine oxidase expression and activity is also increased in the failing heart, again leading to an increased production of ROS.11 Finally, as a result of cardiac injury, NOS becomes uncoupled and structurally unstable leading to an increased generation of ROS. In mice, increased generation of ROS leads to left ventricular (LV) dilatation, contractile dysfunction, and LV remodelling.12

Figure 2.

Oxidative stress production and scavenging in cardiomyocytes under physiological and pathophysiological conditions. (Top) Under physiological conditions oxidative stress in the form of reactive oxygen species (ROS) is produced in small quantities by the mitochondrial electron chain, NADPH oxidase (NOX), xanthine oxidase (XO), and nitric oxide synthase (NOS). Mitochondrial respiration converts oxygen to water, resulting in the production of small quantities of superoxide (O₂ ^‐) as a by‐product. The process starts with electrons derived from NADH₂ and FADH₂ moving along the respiratory transport chain through a series of cytochrome‐based complexes (I, III, and IV). These complexes eventually transport electrons to molecular oxygen. The high free energy of the electrons is gradually extracted and converted into adenosine triphosphate. NOX is a multimeric complex composed of a plasma membrane spanning cytochrome b₅₅₈ (NOX2) and cytosolic components (Rac1, p47^phox, p67^phox, p40^phox). Under physiological conditions this complex is in a resting state, producing minimal O₂ ^‐, by transferring an electron from NADPH to molecular oxygen. XO, which is a cytoplasmic enzyme that catalyzes the oxidation of hypoxanthine and xanthine to uric acid using molecular oxygen as an electron receptor, produces O₂ ^‐ and hydrogen peroxide (H₂O₂) in the process. NOS oxidizes the NOS cofactor BH₄ utilizing NADPH to generate nitric oxide and L‐citrulline from L‐arginine and oxygen. Superoxide dismutase (SOD) initiates the detoxification of ROS, by scavenging O₂ ^‐ and converting it to H₂O₂. Both catalase and glutathione peroxidase (GPx) further detoxify the H₂O₂ to water and oxygen. GPx utilizes two glutathione (GSH) molecules as electron donors in the reduction of H₂O₂ to water, producing oxidized glutathione (GSSG) in the process. Once GPx oxidizes GSH to GSSG, GSH reductase (GR) can reduce GSSG back to GSH at the expense of NADPH, forming the GSH redox cycle. The ratio of GSH to GSSG largely determines the intracellular redox potential. (Bottom) Under pathophysiological conditions, oxidative stress production is increased as a result of increased NOX and XO expression, coupled to blockage of the mitochondrial electron chain and uncoupling of NOS. Furthermore, the expression and activity (dotted lines) of SOD, catalase, and GPx are reduced. The levels of GSH are also reduced, while the levels of GSSG are increased. This severe increase in oxidative stress eventually leads to hypertrophy, fibrosis, apoptosis, and contractile dysfunction in the myocardium.

Besides the drastic increase in oxidative stress production, heart failure is also characterized by an exhaustion of the innate antioxidant defence mechanism. In cardiomyocytes, as in most cell types, the major endogenous components of the antioxidant defence mechanism responsible for the inactivation of ROS are superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), nicotinamide adenine dinucleotide (NAD⁺) and glutathione (GSH) (Figure 2). Several studies have observed a significant decrease in the activities of SOD, catalase, and GPx in animal models for heart failure.13, 14, 15 Furthermore, mice lacking SOD or GPx exposed to cardiac injury have demonstrated worse outcomes when compared to their wild‐type littermates.16, 17, 18, 19, 20 NAD⁺, together with its reduced dinucleotide NADH, are pivotal in driving oxidation–reduction reactions involved in energy production.21, 22 Besides its role in regulating cellular energy metabolism, NAD⁺ is a precursor for the phosphorylated dinucleotide pair NADP⁺/NADPH, which plays a major role in the detoxification of ROS.21, 22 In several murine models of heart failure, a reduction in myocardial NAD⁺ levels has been observed.23, 24, 25 Interestingly, a recent study demonstrated that nicotinamide mononucleotide adenylyl transferase (Nmnat), an enzyme responsible for the production of NAD⁺, is significantly repressed in both models of murine heart failure and in heart failure patients.26 This observation suggests that NAD⁺ reduction also occurs in the human setting. Furthermore GSH, like NAD⁺, is another major antioxidant of mammalian cells, by scavenging radicals and the elimination of lipid peroxidation products.27 Interestingly, a reduction in total GSH has been observed in animals post‐cardiac injury.28, 29 Furthermore, depletion of GSH was highly correlated with serum TNF‐α levels.29 In LV tissue of end‐stage dilated or ischaemic cardiomyopathy patients, total GSH was decreased by 54% when compared to controls.28 In another study, serum GSH levels highly correlated with the symptom severity in heart failure patients.30

Lessons from previous oxidative stress treatments in heart failure

Based on the observation that the redox state is in disarray during heart failure,31 several experimental and clinical studies targeted oxidative stress producers (i.e. NADPH oxidases, xanthine oxidase, and uncoupled NOS) or scavengers [i.e. SOD, catalase, exogenous antioxidant (vitamin E, or folic acid), and GPx] to treat heart failure (Table 1).11, 12, 18, 19, 26, 28, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54

Table 1.

Summary of pre‐clinical and clinical trials using anti‐oxidative stress treatments

Experimental animal studies	Target	Treatment	Model	Results	Ref.
Inhibition of oxidative stress producers	NADPH oxidase	Cytosolic NADPH oxidase component p47phox knock‐out	MI mice	Protected the heart from LV remodelling and dysfunction post‐MI	32
	Xantine oxidase	Oxypurinol administration	Spontaneous hypertensive/HF (SHHF) rat	Improved LV contractility and myocardial efficiency	33
		Allopurinol administration	Exercise‐induced HF in dogs	In pacing‐induced CHF, allopurinol improved LV systolic function	34
	NOS uncoupling	Sapropterin administration	Chronic transverse aortic constriction mice	Improved cardiac function	12
Improving endogenous antioxidant capacity	SOD	SOD overexpression	Ischaemia/reperfusion injury in mice	Reduced oxidative stress production, improved contractility, and reduced infarct size	35
	Catalase	Catalase overexpression	Myocyte‐specific overexpression of G(alpha)q mice (a model for dilated cardiomyopathy) crossbred with myocyte‐specific overexpression of catalase	Reduced myocyte hypertrophy, myocyte apoptosis, and fibrosis	36
	GPx	GPx overexpression	MI mice	Prevention of adverse LV remodelling	18
			Ischaemia/reperfusion injury in mice	Improved contractility and reduced infarct size
	GSH	N‐acetylcysteine administration	MI rats	Improved LV GSH levels, improved contractility, and reduced LV remodelling	28
			Hypertensive rat model (induced by NOS inhibitor N(G)‐nitro‐L‐arginine methyl ester and high‐salt diet)	Improved cardiac GSH levels, reduced LV remodelling and dysfunction, improved TNF‐α levels, and reduced cardiac fibrosis	29
	NAD+	Nicotamide riboside administration	Mouse model of dilated cardiomyopathy	Improved cardiac function and redox state	26
Supplementation of exogenous antioxidants	ROS	Vitamin E supplementation	Ascending aortic banding in guinea pigs (cardiac hypertrophy)	Improved myocardial redox state and cardiac function	37
			Diabetic rat model by injection of streptozotocin	Improved myocardial redox state and cardiac function	38
			Volume overload dog model	Reduced oxidative stress and improved myocardial contractility	39
		Folic acid supplementation	Mouse model of high‐fat diet‐induced obesity	Reduced cardiac dysfunction, oxidative stress, and myocardial fibrosis	40
Inhibition of oxidative stress producers	Xanthine oxidase	Oxypurinol administration	Chronic HF (n = 60)	Improved LV ejection fraction	41
			Symptomatic HF (n = 405)	No improved clinical outcome	42, 43
		Allopurinol administration	Idiopathic dilated cardiomyopathy (n = 9)	Improved myocardial efficiency	11
			Chronic HF (n = 50)	Reduced plasma BNP levels	44
			Primary percutaneous transluminal coronary angioplasty in patients with acute MI (n = 38)	Reduced oxidative stress and improved LV function	45
			Hyperuricaemic chronic HF (n = 19)	Improved peripheral vasodilator capacity and blood flow locally and systemically	46
			Chronic HF (n = 11)	Improved endothelial dysfunction	47
	NOS uncoupling	Sapropterin administration	Coronary artery disease (n = 49)	No effect on vascular function or redox state	48
Improving endogenous antioxidant capacity	GSH	N‐acetylcysteine administration	Acute MI (n = 30)	Improved cardiac function	49
		N‐acetylcysteine and streptokinase administration	Acute MI (n = 1, case study)	Improved cardiac function	50
		N‐acetylcysteine, nitroglycerin and streptokinase administration	Acute MI (n = 27)	Reduced oxidative stress and improved LV function	51
Supplementation of exogenous antioxidants	ROS	Vitamin E supplementation	Ischaemic heart disease (n = 2002)	Reduced rate of non‐fatal MI	52
		Combined vitamin A, C, E, and β‐carotene	Suspected acute MI (n = 125)	Reduced cardiac necrosis and oxidative stress	53
		Meta‐analysis of randomized controlled trials	Cardiovascular diseases (50 studies, n = 294 478), including coronary heart disease, acute MI, unstable angina, TIA, stroke, and angiographically proved coronary atherosclerosis	No beneficial effects of vitamin supplementation on preventing cardiovascular disease	54

BNP, B‐type natriuretic peptide; CHF, congestive heart failure; GPx, glutathione peroxidase; GSH, glutathione; HF, heart failure; LV, left ventricle; MI, myocardial infarction; NAD⁺, nicotinamide adenine dinucleotide; NOS, nitric oxide synthase; SOD, superoxide dismutase; ROS, reactive oxygen species; TIA, transient ischaemic attack; TNF, tumour necrosis factor.

Pre‐clinical animal models for anti‐oxidative stress therapies

Targeting oxidative stress in the pre‐clinical setting has been extensively studied and demonstrated highly promising results. These experimental studies have focused on three distinct approaches to target oxidative stress in heart failure: (i) inhibition of oxidative stress producers, (ii) improving endogenous antioxidant capacity, and (iii) improving antioxidant capacity by supplementation of exogenous antioxidants.

Initial experimental animal studies focused on inhibiting oxidative stress production by targeting NADPH oxidases, xanthine oxidase, or NOS uncoupling. NADPH oxidase inhibition in mice lacking the cytosolic NADPH oxidase component p47^phox, protected the heart from LV remodelling and dysfunction post‐myocardial infarction.32 Inhibition of xanthine oxidase, by means of oxypurinol (rats) or allopurinol (dogs), protected the heart from LV remodelling, improved LV contractile function and myocardial efficiency post‐cardiac injury.33, 34 The production of ROS by the uncoupling of NOS has also been studied as a possible target for heart failure. Mice, with a knock‐out for NOS3, exposed to transverse aortic constriction (TAC), demonstrated a reduction in fibrosis and myocyte hypertrophy.12 Similarly, inhibiting NOS by means of BH4 (sapropterin) treatment also protected mice from TAC‐induced cardiac injury.12 These observations suggested that directly inhibiting ROS producers, thereby reducing oxidative stress, can result in improved survival and cardiac function following cardiac injury.

Besides targeting oxidative stress production, early experimental studies demonstrated that increasing the endogenous antioxidant capacity leads to improved cardiac function in rodent models for heart failure. Specifically, these studies focused on the primary antioxidant enzymes (SOD, catalase, and GPx) and antioxidants (NAD⁺, GSH, vitamin E, and folic acid). Mice overexpressing SOD when exposed to ischaemia/reperfusion injury were found to have severely decreased levels of superoxide production, improved contractile function, and a decrease in infarct size.35 Similarly, mice with a cardiomyocyte specific overexpression of catalase in a transgenic model for dilated cardiomyopathy were found to have a significant reduction in adverse remodelling (i.e. myocyte hypertrophy, myocyte apoptosis, and interstitial fibrosis) and the progression of heart failure.36 Finally, mice with an overexpression of GPx exposed to ischaemia/reperfusion injury were also found to have improved cardiac tissue survival and function, resulting from an inhibition of LV remodelling.18, 19

Following these positive results obtained by increasing the endogenous expression of antioxidant enzymes, several experimental studies tried to improve the endogenous antioxidant capacity. Early studies focused on increasing the endogenous levels of GSH, by the administration of N‐acetylcysteine (NAC), a precursor of GSH. NAC is readily absorbed into cells, where it is converted into cysteine, the rate‐limiting amino acid in the synthesis of GSH. These studies demonstrated that NAC can improve GSH levels, reduce oxidative stress, and improve cardiac function in rat models of cardiac injury.28, 29 To further address the importance of the antioxidant capacity, several experimental studies demonstrated that supplementation of vitamin E and folic acid leads to improved cardiac function in models for heart failure.37, 38, 39, 40 Recently, a study has demonstrated that by increasing the levels of NAD⁺, by supplementation of nicotinamide riboside (NR), a precursor of NAD⁺, leads to improved cardiac function and redox state in a murine heart failure model.26 These findings suggest that by increasing the antioxidant capacity of the heart, cardiomyocyte survival is improved, and the myocardium is better able to cope with injury.

Clinical anti‐oxidative stress therapies

Due to these highly promising results in animal models, several studies have assessed the potential of anti‐oxidative stress therapies in the clinical setting. Similar to the animal studies, clinical trials have taken three approaches to targeting oxidative stress in heart failure patients: (i) inhibition of oxidative stress producers (xanthine oxidase and NOS uncoupling), (ii) improving endogenous antioxidant capacity (NAC), and (iii) improving antioxidant capacity by supplementation of exogenous antioxidants (vitamin A, vitamin C, vitamin E, and folic acid).

At present, the best studied therapy in patients with heart failure is the inhibition of xanthine oxidase by the administration of allopurinol or oxypurinol.11, 41, 42, 43, 44, 45, 46, 47 The initial clinical trials were small studies (n = 9–60) in patients with dilated cardiomyopathy and chronic heart failure. These trials all demonstrated that treatment with allopurinol or oxypurinol improved myocardial function, peripheral vasodilatation capacity, blood flow, endothelial dysfunction, reduced plasma B‐type natriuretic peptide levels, and increased LV ejection fraction.11, 41, 44, 45, 46, 47 However, in a larger randomized controlled trial in 405 patients with heart failure, oxypurinol did not improve clinical outcome.42, 43 The primary endpoint of the study was a combined clinical endpoint that classified the patient's clinical status as improved, worsened, or unchanged 24 weeks after the initiation of the study. Compared to the placebo group, patients demonstrated no improvement in clinical status following oxypurinol treatment.42, 43 Similarly, inhibition of NOS uncoupling, by means of sapropterin treatment, has also been studied in the clinical setting. Several small clinical trials have been performed with oral sapropterin administration in patients (n = 18–49) with systemic or pulmonary hypertension.48, 55, 56 However, these trials all failed to demonstrate significant differences in nitric oxide synthesis, oxidative stress, systemic haemodynamics, vascular redox state, or endothelial function.

Following these disappointing results by inhibiting oxidative stress production in patients, clinical studies went on to assess the potential of increasing the antioxidant capacity in heart failure patients. The majority of the trials performed to date have involved the supplementation of exogenous antioxidants (vitamin A, vitamin C, vitamin E, and folic acid). Initial studies found that the supplementation of exogenous antioxidants leads to a reduction in cardiovascular events, infarct sizes, and oxidative stress.52, 53 However, a recent meta‐analysis of 50 randomized controlled trials studying the effects of vitamin and antioxidant supplementation, including 294 478 participants, concluded that supplementation with exogenous vitamins and antioxidants was not associated with reductions in the risk of major cardiovascular diseases.54 More interestingly, several clinical trials have demonstrated that improving the antioxidant capacity, by bolstering endogenous GSH levels, does show some promise in heart failure patients. These trials demonstrated that supplementation of NAC in patients resulted in a reduction in oxidative stress, as measured by an increase in the GSH/oxidized GSH (GSSG) ratio, infarct size and an improved cardiac function in patients with heart failure and acute myocardial infarction.49, 50, 51, 57

Why have clinical anti‐oxidative stress therapies largely failed?

Taken together, these findings suggested that although targeting oxidative stress is theoretically logical, the majority of the strategies currently employed in the clinical setting have failed to improve patient prognosis. Furthermore, the exact reasons and mechanisms as to why these studies have failed to produce the expected beneficial effects, remain largely unknown. One reason could be that in the experimental setting, the majority of the studies utilized heart failure models to test the efficacy of anti‐oxidative stress treatments, while in the clinical setting anti‐oxidative stress treatments were primarily tested in patients with acute myocardial infarction and not heart failure. Although it is well documented that following myocardial infarction there is a surge in oxidative stress,58 it maybe that at this stage anti‐oxidative stress therapies are not capable of limiting the production of oxidative stress. Rather, these strategies may serve to improve outcome in heart failure patients, where there is less oxidative stress production. Another reason for this discrepancy could be that only specific patient populations benefit from anti‐oxidative stress treatments. Or else the inadequate understanding of the mechanistic mode of action of the administered antioxidant.

With regard to targeting oxidative stress production in human heart failure, it has been speculated that these therapies may be beneficial for a specific subset of patient. Oxypurinol has been shown to improve heart failure symptoms in a specific subset of patients with elevated uric acid, the product of xanthine oxidase.43, 59 Thus, identifying patients with increases in oxidative stress production resulting from xanthine oxidase activity may still benefit from this therapeutic approach. Several current anti‐oxidative stress therapies have also been found to result in some off‐target effects. Sapropterin administration was found to result in an unexpected increase in oxidized BH₄, BH₂, a competitive inhibitor of BH₄ that promotes NOS uncoupling.48, 60 Similarly, supplementation with α‐tocopherol (vitamin E) has been shown to drastically suppresses the levels of γ‐tocopherol (the more potent antioxidant, found primarily in the diet), thereby possibly reducing rather than increasing total antioxidant capacity.61

Although the findings of clinical trials aimed at reducing ROS production and increasing exogenous antioxidants have been disappointing, targeting oxidative stress, specifically the endogenous antioxidant capacity, in heart failure should not be entirely disregarded. Noteworthy has been the observation that increasing the antioxidant capacity, by bolstering endogenous GSH levels via NAC supplementation, results in improved patient outcome, without any adverse side effects.49, 50, 51, 57 Therefore, future oxidative stress therapies should focus on improving the endogenous antioxidant capacity, rather than inhibiting oxidative stress production or supplementation of exogenous antioxidants.

The future of oxidative stress as a therapeutic target in heart failure

The major endogenous antioxidants in mammalian cells are NAD⁺ and GSH, the latter produced via the γ‐glutamyl cycle. NAD⁺ has a multitude of cellular functions, and more recently it has become evident that it plays an important role in the detoxification of cellular ROS. Similarly, GSH protects cells against oxidative stress, and both NAD⁺ and GSH levels have been shown to be associated with heart failure in the experimental and clinical setting.23, 24, 25, 26, 27, 28, 29, 30 Furthermore, supplementation of NAD⁺ or GSH precursors has been found to improve cardiac function and redox state in models for heart failure.26, 28, 29 Thus, we propose two approaches for future anti‐oxidative stress therapies in heart failure patients: (i) increasing the endogenous antioxidant capacity, and (ii) increasing the expression/activity of antioxidant producing enzymes.

Improving endogenous antioxidant capacity in heart failure

Increasing the endogenous antioxidant capacity can be primarily achieved by supplementation of precursors of the major cellular antioxidants GSH and NAD⁺. The effectiveness and safety of this approach has previously been demonstrated with the supplementation of NAC, a precursor of GSH, to heart failure patients and acute myocardial infarction patients, resulting in improved patient outcome.49, 50, 51, 57 Thus, increasing the levels of endogenous antioxidants seems to be a promising target for not only treating oxidative stress in myocardial infarction, but also in heart failure patients.

Improving the endogenous levels of GSH can be achieved by supplementation with GSH precursors which can be utilized by the γ‐glutamyl cycle for de novo GSH synthesis (Figure 3). Besides NAC, γ‐glutamylcysteine, another GSH precursor, and 2‐oxothiazolidine‐4‐carboxylate (OTC), an analogue of 5‐oxproline, also seem to have the potential to increase endogenous GSH levels. Both γ‐glutamylcysteine and OTC increased GSH and reduced oxidative stress in experimental and clinical studies.62, 63, 64, 65, 66, 67, 68 Similar to NAC, supplementation of γ‐glutamylcysteine increased the levels of GSH in patients with cancer, with no adverse effects.62 OTC, which is converted to cysteine by 5‐oxoprolinase (OPLAH), increased GSH levels in the experimental setting.63, 64 Interestingly, early experimental studies have shown that OTC improved cardiac function following cardiac injury.65, 67 Furthermore, in several clinical trials OTC treatment had no adverse effects and increased GSH concentrations and decreased oxidative stress in patients with acute respiratory distress syndrome and HIV patients.67, 68

Figure 3.

Drug therapies targeting endogenous glutathione (GSH) synthesis. GSH is synthesized from cysteine (the rate‐limiting amino acid), glutamate, and glycine by the γ‐glutamyl cycle. GSH is then utilized by GSH peroxidase (GPx) to reduce oxidative stress, and in the process forming oxidized GSH (GSSG). GSSG is then reduced by action of GSH reductase (GR). Improving the γ‐glutamyl cycle's ability to produce GSH has been characterized as a treatment target in heart failure. N‐acetylcysteine (NAC), γ‐glutamylcysteine, and 2‐oxothiazolidine‐4‐carboxylate (OTC, also known as pro‐cysteine) are compounds which have demonstrated the capacity to increase the endogenous production of GSH. OTC is converted to cysteine, by action of 5‐oxoprolinase (OPLAH), to be used for de novo synthesis of GSH. Similarly, NAC is converted to cysteine intracellularly, and used for GSH synthesis. γ‐Glutamylcysteine is utilized by the γ‐glutamyl cycle to form GSH, by addition of glycine. GCL, glutamate cysteine ligase.

Besides improving the endogenous GSH levels in heart failure, another approach could be to increase the levels of endogenous NAD⁺. NAD⁺ can be derived from several precursors, including deamidated precursors, such as tryptophan, nicotinic acid, amidated vitamin B3 nicotinamide and NR.21 The most promising of these precursors to date is NR, which is converted to NAD⁺ by NR kinase (Nmrk) and nicotinamide mononucleotide adenylyl transferase (Nmnat). It has been recently demonstrated that supplementation of NR in murine models for dilated cardiomyopathy and pressure overload‐induced heart failure, can restore NAD⁺ levels and preserve cardiac function.26

Although only few studies have focused on increasing the endogenous antioxidant capacity in models for heart failure, the results to date may be promising. Furthermore, the observation that NAC supplementation in heart failure patients leads to increased GSH levels suggests that this might be a potential strategy for reducing the increase in oxidative stress resulting from cardiac injury. Future studies should thus focus on further characterizing the beneficial effects of the supplementation of GSH precursors (γ‐glutamylcysteine and OTC) and NAD⁺ precursors (NR) on heart failure patient outcome.

Targeting the endogenous production of antioxidants in heart failure

Besides improving endogenous antioxidant capacity by administration of GSH and NAD⁺ precursors, another avenue for reducing oxidative stress in heart failure is to improve the expression and/or activity of the γ‐glutamyl cycle and NAD⁺ producers.

Recent experimental studies have demonstrated that several components of the γ‐glutamyl cycle are strongly associated with the development and progression of heart failure (including γ‐glutamylcysteine synthetase, GPx, and OPLAH). Furthermore, modulation of these enzymes, by overexpression, has resulted in cardioprotection.18, 19, 69, 70 Of particular interest is OPLAH, a cytoplasmic enzyme of the GSH cycle whose only function is the conversion of 5‐oxoproline, a degradation product of GSH, into glutamate (Figure 4). Interestingly, studies have demonstrated that excessive 5‐oxoproline accumulation can lead to the induction of intracellular oxidative stress.70, 71, 72 Therefore, OPLAH plays a pivotal role not only in the γ‐glutamyl cycle, by producing glutamate for de novo GSH synthesis, but also as an antioxidant by scavenging 5‐oxoproline.

Figure 4.

Targeting 5‐oxoprolinase (OPLAH) to reduce oxidative stress in heart failure. Following cardiac injury, OPLAH expression is reduced, leading to the accumulation of 5‐oxoproline. 5‐Oxoproline then leads to drastic increase in oxidative stress (reactive oxygen species, ROS). To help reduce the insult of 5‐oxoproline to the injured myocardium, two strategies could be developed: (i) to pharmacologically improve the remaining OPLAH's ability to reduce 5‐oxoproline, or (ii) to increase OPLAH expression by means of gene therapy. ADP, adenosine diphosphate; ATP, adenosine triphosphate; GSH, glutathione.

OPLAH expression is suppressed in heart failure, both in the experimental and clinical setting.70, 73 In the murine setting, reduction in OPLAH increased plasma 5‐oxoproline levels in cardiac tissue and plasma, which coincided with an increase in oxidative stress.70 Interestingly, elevated levels of plasma 5‐oxoproline in chronic heart failure patients were associated with higher N‐terminal pro‐B‐type natriuretic peptide, incidence of atrial fibrillation, and all‐cause mortality.70 In a recent study, OPLAH overexpression in mice exposed to ischaemia/reperfusion injury or permanent myocardial ischaemia improved cardiac function, reduced infarct size and fibrosis, when compared to wild‐type littermates.70 Improved cardiac function in the OPLAH overexpression mice was coupled to reduced 5‐oxoproline levels and improved GSH/GSSG ratio post‐cardiac injury.70 Thus, stimulating the expression and/or activity of OPLAH could lead to novel therapeutic strategies for patients with heart failure.

Similar to the γ‐glutamyl cycle genes, there are several NAD⁺ producers, most noteworthy is Nampt. Nampt has been shown to be repressed in both model for heart failure and the clinical setting.26 Furthermore, overexpression of Nampt in mice was found to protect these animals against ischaemia/reperfusion injury and isoproterenol‐induced hypertrophy.23, 24 Therefore, increasing the expression and/or activity of Nampt might be a potential target for the treatment of heart failure.

To date there are no known pharmacological agents (i.e. drugs or small molecules) that have the capacity to induce OPLAH activity. There are however several Nampt activators, although limited information is currently available about these compounds in patients.74, 75 Future studies should therefore focus on (i) identifying novel pharmacological agents that specifically target OPLAH, and (ii) characterize Nampt activators in the clinical setting. Similarly, the development of an OPLAH or Nampt gene therapy, as recently described for SERCA2, could also serve as a viable therapeutic strategy.76 Furthermore, besides OPLAH and Nampt, other members of the γ‐glutamyl cycle and NAD⁺ producers should also be screened for their potential use as therapeutic targets in heart failure.

Conclusion

The role of oxidative stress in the onset and progression of heart failure has been extensively studied. Pre‐clinical studies showed promising results with various anti‐oxidative strategies, but these beneficial effects did not translate into positive results in clinical studies in patients with heart failure. This might be caused by inadequate patient inclusion criteria or off target effects of currently employed therapies. Therefore, we believe there is still room for novel antioxidant approaches in heart failure. In particular, targeting the endogenous antioxidant capacity might be interesting new targets in the treatment of heart failure. Of interest would be the development of medications capable of interacting with the components of the γ‐glutamyl cycle or NAD⁺ production, which may lead to novel treatment options for heart failure in the future.

Conflict of interest: none declared.