Effects of inorganic nitrate supplementation on cardiovascular function and exercise tolerance in heart failure

Abstract

Heart failure (HF) results in a myriad of central and peripheral abnormalities that impair the ability to sustain skeletal muscle contractions and, therefore, limit tolerance to exercise. Chief among these abnormalities is the lowered maximal oxygen uptake, which is brought about by reduced cardiac output and exacerbated by O₂ delivery-utilization mismatch within the active skeletal muscle. Impaired nitric oxide (NO) bioavailability is considered to play a vital role in the vascular dysfunction of both reduced and preserved ejection fraction HF (HFrEF and HFpEF, respectively), leading to the pursuit of therapies aimed at restoring NO levels in these patient populations. Considering the complementary role of the nitrate-nitrite-NO pathway in the regulation of enzymatic NO signaling, this review explores the potential utility of inorganic nitrate interventions to increase NO bioavailability in the HFrEF and HFpEF patient population. Although many preclinical investigations have suggested that enhanced reduction of nitrite to NO in low Po₂ and pH environments may make a nitrate-based therapy especially efficacious in patients with HF, inconsistent results have been found thus far in clinical settings. This brief review provides a summary of the effectiveness (or lack thereof) of inorganic nitrate interventions on exercise tolerance in patients with HFrEF and HFpEF. Focus is also given to practical considerations and current gaps in the literature to facilitate the development of effective nitrate-based interventions to improve exercise tolerance in patients with HF.

INTRODUCTION

Our view of the biological significance of nitric oxide (NO) has expanded greatly since the initial discovery as an endothelium-derived vasodilator to now being appreciated as a pluripotent signaling molecule involved in numerous cardiovascular, metabolic, renal, and immunological processes. Conversely, reduced NO bioavailability has been coupled to the pathophysiology of cardiometabolic and renal disorders. Generation of this small molecule is interestingly complex and is carried out by specific NO synthases (NOS) in a five-step oxidation process requiring l-arginine, molecular oxygen, and several crucial cofactors (Fig. 1) (1). In blood and tissues, NO is very short lived due to rapid oxidation to the more stable anions nitrate (NO₃⁻) and nitrite (NO₂⁻), which have been widely used as surrogate measures of NOS activity (2). Canonical NO signaling, such as in the vasculature, involves activation of soluble guanylyl cyclase (sGC) that increases the levels of the second messenger cyclic guanosine monophosphate (cGMP), activating cGMP-dependent protein kinases that induce relaxation of vascular smooth muscle. Modulation of protein functions also exists via cGMP-independent mechanisms that contribute to the regulatory effects of NO signaling (3). Considering the wide array of effects controlled by NO in health and disease, the purpose of this mini-review is to shed light on the potential therapeutic applications of the nitrate-nitrite-NO pathway for the treatment of heart failure (HF). Attention has been given to both reduced and preserved ejection fraction HF (HFrEF and HFpEF, respectively), with a special emphasis on inorganic nitrate supplementation and exercise tolerance.

Figure 1.

Top: schematic of the two parallel pathways for NO formation: the NOS-dependent and nitrate-nitrite-NO pathways. Middle: impact of inorganic nitrate and nitrite supplementation on many of the dysfunctional cardiorespiratory and skeletal muscle elements found in patients with HFpEF and HFrEF. Bottom: impact of inorganic nitrate and nitrite supplementation on exercise efficiency and tolerance in patients with HFpEF and HFrEF. See text for additional information. a-Vo₂ difference, arteriovenous O₂ content difference; NO, nitric oxide; NO₃⁻, nitrate; NO₂⁻, nitrite; NOS, nitric oxide synthase; PCWP, pulmonary capillary wedge pressure; V̇o_2peak, peak oxygen uptake; WR_max, maximal power output.

THE NITRATE-NITRITE-NITRIC OXIDE PATHWAY

Ten years after the discovery of NO as a signaling molecule, an alternative pathway for NO generation was found wherein nitrate and nitrite are serially reduced to NO and other bioactive nitrogen species (Fig. 1) (4, 5). As mentioned earlier, circulating nitrate originates from oxidation of NOS-generated NO but also to a large extent from dietary sources, such as green leafy vegetables and beetroot (6). Regardless of source, circulating nitrate is actively taken up by the salivary glands and secreted in the saliva (7). In the oral cavity, several bacterial species belonging to the commensal flora contain nitrate reductases that can catalyze the first reduction of salivary nitrate to nitrite (8, 9). In humans, but not to the same extent in rodents, the salivary levels of these anions normally exceed plasma levels by several orders of magnitude (9, 10). The crucial role of the oral microflora in nitrate bioactivation is evident from experiments where antibacterial mouthwash products abrogate the effects of both endogenous and dietary nitrate (11, 12). Under normal conditions, swallowed nitrite is rapidly protonated due to the acidic conditions in the stomach, instantly and nonenzymatically giving rise to NO and several other nitrogen intermediates with seemingly protective effects on the gastric mucosa (13). However, in the presence of dietary amines, formation of carcinogenic nitrosamines from nitrite can also occur, which has led to very strict regulations of nitrate levels in our food and drinking water (14). These measures are debatable since the relationship between dietary nitrate and cancer has not been demonstrated in humans (15, 16). After passage from the stomach, remaining nitrite is rapidly and very efficiently absorbed in the gut and enters the systemic circulation. There are several enzymatic and nonenzymatic pathways in blood and tissues that reduce nitrite to NO and other nitrogen intermediates, including those involving deoxyhemoglobin, xanthine oxidoreductase, and mitochondrial complexes (17). Interestingly, these pathways for NO generation from nitrite are potentiated during hypoxia and low pH, conditions where NOS activity is markedly reduced.

Accumulating data from experimental and human studies show that dietary stimulation of the nitrate-nitrite-NO pathway can provide beneficial cardiovascular (18) and metabolic (19) effects as well as renoprotection (20). These include lowering of blood pressure, increased aerobic efficiency during exercise, protection against ischemia-reperfusion injury and inflammation, and improved glucose control in models of diabetes. In view of these promising data, it is noteworthy that despite extensive NO research over the past 30 years, which indeed has accumulated considerable knowledge on the biological role of NO, there is unfortunately to date only a limited number of effective clinical applications. Inhaled NO gas is given to treat pulmonary hypertension in newborns (21), and exhaled NO is measured to monitor airway inflammation in asthma (22). Apart from nitroglycerin, known long before the discovery of NO in physiology, few novel NO-related drugs have emerged into clinical practice. Intravenous nitroprusside is recommended in the management of acute decompensated HF and typically requires close hemodynamic monitoring for marked hypotension (23). Phosphodiesterase inhibitors that block cGMP breakdown are used to treat pulmonary hypertension and erectile dysfunction (24). Novel sGC stimulators are prescribed for pulmonary hypertension, but promising studies may indicate a wider use of this class of drugs (25). In addition to therapies that target downstream signaling of NO, stimulation of the nitrate-nitrite-NO pathway (either by the diet or by administration of nitrate or nitrite) may represent an alternative way to enhance NO bioavailability, especially in HF where NOS function is impaired.

CONTEXT FOR THE DEVELOPMENT OF NITRATE-BASED HEART FAILURE THERAPIES

HF is a complex, multifactorial clinical syndrome with high morbidity and mortality. The disease is broadly defined by the inability to provide adequate cardiac output to meet peripheral metabolic demand at normal intracardiac filling pressures. HFpEF and HFrEF are the two main distinct phenotypes of HF identified among patients. Although patients with HFpEF demonstrate a similar poor prognosis as those with HFrEF, the pathophysiology of HFpEF is poorly understood, thus limiting the development of effective pharmacological therapies. Neurohumoral activation dominates HFrEF pathophysiology, whereas variable extracardiac mechanisms (e.g., arterial hypertension, metabolic risk, and renal insufficiency) seem to promote chronic systemic inflammation and result in the heterogeneous clinical presentation of HFpEF (26–28). Common to both HF classifications is the hallmark symptom of severe exercise intolerance, which constitutes a critical determinant of poor prognosis and reduced quality of life (27).

Recent advances indicate that noncardiac, peripheral skeletal muscle abnormalities contribute significantly to exercise intolerance in both HFpEF and HFrEF. Such muscle abnormalities include reductions in capillary density, vasodilatory capacity, oxygen transport (i.e., impaired perfusive and diffusive O₂ fluxes), percentage of type I muscle fibers, and mitochondrial content and function (27, 29–31). These HF-induced alterations culminate in muscle oxygen delivery-utilization (Q̇o₂/V̇o₂) mismatch during contractions predisposing to reduced exercise tolerance and maximal oxygen uptake (V̇o_2max) (27). Pursuant to the scope of this mini-review, several lines of evidence suggest that reduced NO bioavailability plays a crucial role in exercise intolerance in HFpEF and HFrEF (15, 27, 32).

Although the notion that nitrite reduction to NO is potentiated in relatively low Po₂ and pH environments is well enshrined in studies dating before the discovery of NO’s signaling properties, the holistic and applied effects of nitrate bioactivation have only become evident within the past two decades. For example, seminal work from Cosby et al. (33) revealed that relaxation of isolated vessels was potentiated largely at a Po₂ <30 mmHg when nitrite was combined with red blood cells. These data suggest that microvascular Po₂ (PO₂mv) values often observed in contracting muscle could facilitate the reduction of nitrite to NO in vivo and thus promote a local increase in blood flow. About a decade later, Ferguson et al. (34) demonstrated in rats that short periods (5 days) of nitrate supplementation enhanced blood flow during treadmill running in muscles composed predominantly of fast-twitch fibers, thereby showcasing the integrated aspects of low Po₂-induced nitrite reduction to NO during exercise. It was around this time that other investigations showed preferential effects of nitrate supplementation on fast-twitch muscles and athletic events where these types of muscle fibers are relied heavily upon (35).

The fiber-type specific effects of nitrate supplementation are likely facilitated by the large degree of blood flow heterogeneity across the spectrum of fast- and slow-twitch muscle phenotypes, such that at rest and during contractions, the PO₂mv of fast-twitch muscles is significantly lower than that of their slow-twitch compatriots (35–37). In HF, peripheral vascular derangements decrease PO₂mv predominantly in slow- but not fast-twitch muscles (38), bringing the contracting PO₂mv of the soleus (primarily slow-twitch) to ∼15 mmHg, which is well below the suggested Po₂ required to reduce nitrite to NO optimally in vivo (33). In this sense, HF may “level the playing field” for slow-twitch fibers to also benefit from nitrate supplementation, as it creates an environment ripe for nitrite bioactivation in muscles that may not usually reach the “optimal” PO₂mv range for those effects during submaximal contractions (26).

Considering the impact of inorganic nitrate supplementation in healthy humans and animal models and the muscle fiber-type specific effects mentioned above many have postulated that a nitrate-based therapy could improve contracting muscle Q̇o₂/V̇o₂ matching and ameliorate exercise intolerance in HF. These latter improvements could be achieved by enhanced local perfusion and oxygenation, increased muscle contractility and resistance to fatigue, reduced O₂ cost of muscle contractions, faster O₂ uptake kinetics, and/or lowered arterial blood pressure (26, 27, 32, 39, 40). In this context, inorganic nitrate and nitrite supplementation has recently emerged as a potential clinical intervention for both patients with HFpEF and HFrEF. Inorganic nitrate is further attractive because it can be supplemented orally via natural products (e.g., beetroot juice and leafy green vegetables) with no evidence of tachyphylaxis or significant adverse effects during chronic dosing, which is not the case with organic nitrates (15). The following sections will summarize the effects of inorganic nitrate and nitrite supplementation on peripheral responses to exercise and exercise tolerance in patients with HFpEF and HFrEF.

INORGANIC NITRATE AND NITRITE SUPPLEMENTATION IN HFPEF

Despite the potential for nitrate and nitrite reduction to NO in HF and the increased NO bioavailability afforded by nitrates, the pathway to an effective NO-based clinical intervention is not without complexity. For example, initial multicenter trials aiming at improving NO bioavailability and exercise tolerance in patients with HFpEF used organic nitrate (isosorbide mononitrate; NEAT-HFpEF trial) (41) and phosphodiesterase-5 inhibition (sildenafil; RELAX trial) (42). However, neither intervention improved exercise performance or the clinical status of patients with HFpEF. This prompted the evaluation of inorganic nitrate and nitrite as a potential therapy for HFpEF. While organic nitrates promote the tonic release of NO, stimulation of the nitrate-nitrite-NO pathway is potentiated by hypoxic and acidic conditions likely to be found in the contracting skeletal muscle of patients with HF, as noted previously (15, 38). As such, preferential vasodilation and increased perfusion to tissues of high metabolic demand form the basis for improved peripheral Q̇o₂/V̇o₂ matching and enhanced exercise tolerance with inorganic nitrates.

To date, only five studies have examined the effects of inorganic nitrate and nitrite on cardiorespiratory function and exercise tolerance in patients with HFpEF (Table 1). Most studies have used oral nitrate supplementation (via beetroot juice or potassium nitrate; KNO₃), whereas one multicenter trial used inhaled (nebulized) inorganic nitrite. These different oral nitrate sources (i.e., beetroot juice vs. KNO₃) produce similar increases in dose-normalized plasma nitrate and nitrite concentrations in healthy individuals (52). In terms of administration routes, inhaled and intravenous nitrite administration have resulted in similar pharmacokinetic and pharmacodynamic effects in patients with HFpEF (53, 54). Targeting the pulmonary alveolar-capillary interface with nebulized nitrite circumvents first-pass metabolism inherent to dietary nitrate supplementation, thus avoiding potential impairments in oral bacterial nitrate reductase activity. Nonetheless, the efficiency of pulmonary drug delivery depends on particle size and velocity of inspiratory flow produced by the nebulizer device (45, 55). Inorganic nitrate and nitrite supplementation are generally well tolerated among patients with HFpEF, with few adverse effects but mixed results with respect to exercise tolerance.

Table 1.

Effect of inorganic nitrate and nitrite supplementation on select physiological responses to exercise and exercise tolerance in patients with HFpEF and HFrEF

HF Phenotype and Study	Duration	Design	Participants/Groups	Dose and Administration Route	Exercise Protocol	Main Exercise Outcomes
Heart failure with preserved ejection fraction (HFpEF)
Eggebeen et al. (43)	Acute	Randomized, double-blind, placebo-controlled, crossover	18 patients with HFpEF (85% female) 69 ± 7 yr	Oral (beetroot juice, 6.1 mmol nitrate, single dose)	Upright submaximal cycling exercise (∼75% WRmax)	↔V̇o2peak, ↔tlim, ↔BP
	Chronic	Randomized, double-blind, placebo-controlled		Oral (beetroot juice, 6.1 mmol nitrate/day for 1 wk)		↔V̇o2peak, ↑tlim, ↔BP
Shaltout et al. (44)*	Chronic	Randomized, double-blind, placebo-controlled	Placebo and exercise training: 9 patients with HFpEF (89% female) 71 ± 8 yrBeetroot juice and exercise training: 10 HFpEF patients (80% female) 68 ± 6 yr	Oral (beetroot juice, 6.1 mmol nitrate 3× per wk for 4 wks)	Upright maximal and submaximal cycling exercise (∼75% WRmax)	↔V̇o2peak, ↔WRmax, ↔tlim, ↔BP
Borlaug et al. (45)†	Chronic	Multicenter, randomized, double-blind, placebo-controlled, crossover	105 patients with HFpEF (56% female) 68 yr	Inhaled (nebulized inorganic nitrite, 46 mg 3× per day for 1 wk + 80 mg 3× per day for 3 wks)	Upright maximal cycling exercise	↔V̇o2peak, ↔tlim
Zamani et al. (46)	Chronic	Randomized, double-blind, placebo-controlled	Placebo: 3 patients with HFpEF (67% female) 63 ± 8 yrPotassium nitrate: 9 patients with HFpEF (67% female) 62 ± 5 yr	Oral (potassium nitrate, 6 mmol 2× per day for 1 wk + 6 mmol 3× per day for 1 wk)	Supine maximal cycling exercise	↔V̇o2peak, ↑WRmax, ↑tlim, ↔BP
Zamani et al. (31)	Acute	Randomized, double-blind, placebo-controlled, crossover	17 patients of HFpEF (12% female) 65 ± 9 yr	Oral (beetroot juice, 12.9 mmol nitrate, single dose)	Supine maximal cycling exercise	↑V̇o2peak, ↑WRmax, ↑tlim, ↔BP
Heart failure with reduced ejection fraction (HFrEF)
Kerley et al. (47)	Acute	Randomized, double-blind, placebo-controlled, crossover	10 patients with HFrEF (64% male), 56 ± 11 yr	Oral (beetroot juice 12.9 mmol nitrate, single dose)	Incremental shuttle walk test	↑distance walked, ↔BP
Coggan et al. (48)‡	Acute	Randomized, double-blind, placebo-controlled, crossover	9 patients with HFrEF (56% male) 57 ± 10 yr	Oral (beetroot juice, 11.2 mmol nitrate, single dose)	6-min walk test and single leg isokinetic dynamometry	↔distance walked,↑maximal knee extensor power & velocity, ↔BP
Hirai et al. (49)	Chronic	Randomized, double-blind, placebo-controlled, crossover	10 patients with HFrEF (100% male) 63 ± 5 yr	Oral (beetroot juice, 12.9 mmol nitrate per day for 9 days)	Upright low- and high-intensity cycling exercise	↔V̇o2peak, ↔tlim, ↔BP
Coggan et al. (50)	Acute	Randomized, double-blind, placebo-controlled, crossover	8 patients with HFrEF (75% male) 52 ± 5 yr	Oral (beetroot juice, 11.2 mmol nitrate, single dose)	Semirecumbent submaximal and maximal cycling exercise	↑V̇o2peak, ↑tlim, ↔BP
Woessner et al. (51)	Chronic	Randomized, double-blind, placebo-controlled, crossover	16 patients with HFrEF (93.75% male) 63 ± 4 yr	Oral (beetroot juice, 16 mmol nitrate per day for 5 days)	Treadmill maximal exercise	↔V̇o2peak, ↔tlim, ↔BP

BP, blood pressure (i.e., mean arterial pressure and/or systolic blood pressure); tlim, time to exhaustion; V̇o₂peak, peak oxygen uptake; WR_max, maximal power output.

^*Protocol includes combined exercise training and nitrate intervention.

^†Protocol utilized nebulized nitrite intervention.

^‡Protocol includes resistance strength outcomes.

Two acute, single-dose studies have reported contrasting effects of beetroot juice supplementation on exercise tolerance in HFpEF. The higher nitrate dose (12.9 mmol nitrate) used by Zamani et al. (31) resulted in a modest increase in V̇o_2peak (i.e., ∼1 mL/kg/min) and exercise tolerance during supine cycling exercise partly due to reductions in systemic vascular resistance. Conversely, the lower dose (6.1 mmol nitrate) used by Eggebeen et al. (43) had no significant effects on V̇o_2peak or exercise tolerance during upright submaximal cycling. Apart from differences in experimental protocols (i.e., exercise mode), these findings suggest that relatively high inorganic nitrate doses may improve clinical status and exercise tolerance in patients with HFpEF.

Chronic inorganic nitrate and nitrite supplementation studies in patients with HFpEF have also produced mixed results. Short-term (1–2 wk) oral administration of beetroot juice (43) or KNO₃ (46) elevated exercise tolerance with no changes in V̇o_2peak. Of note, lower hemoglobin concentration with serial blood draws likely constrained improvements in V̇o_2peak in the latter study by Zamani et al. (46). Conversely, a long-term (4-wk) study using beetroot juice supplementation combined with exercise training (44) and another using inhaled nitrite (45) failed to improve V̇o_2peak or exercise tolerance. It is important to note, however, the following when interpreting these long-term interventional studies: 1) the relatively low nitrate dose supplementation in the aforementioned training study increased plasma nitrate but not nitrite concentration, and 2) plasma nitrite concentration was not measured following inhaled nitrite.

A recent meta-analysis evaluating the existing HFpEF studies (as listed in Table 1) suggested that inorganic nitrate and nitrite supplementation may not be associated with improvements in exercise performance (as evaluated by time to exhaustion and V̇O_2peak) in patients with HFpEF (56). However, as rightfully noted by the authors, interpretation of those results must be made with caution, given the small number of studies and sample sizes, differences in experimental protocols, and other potential confounding factors (e.g., clinical heterogeneity, polypharmacy, potential variability in oral bacteria, and unknown optimal supplementation doses). Compounding these technical challenges is the highly heterogeneous nature of the disease encompassing hypertension, diabetes/metabolic syndrome, obesity, and/or chronic kidney disease, among others, all under the same HFpEF diagnosis (28).

Despite the inconsistent effects on exercise tolerance described above, inorganic nitrate and nitrite have the potential to improve central hemodynamics in patients with HFpEF. Invasive cardiac catheterization studies by Borlaug et al. (53, 54) revealed that infused and inhaled inorganic nitrite lowered pulmonary capillary wedge pressure, mean pulmonary artery pressure, and right atrial pressures at rest and during low-intensity cycling exercise. It is interesting that dietary nitrate supplementation (with beetroot juice) seems to lower resting systolic blood pressure in patients with HFpEF but not in patients with HFrEF (Fig. 2), suggesting greater systemic activation of the nitrate-nitrite-NO pathway in HFpEF and/or fundamental differences in response to vasodilatory therapy between HF phenotypes (58).

Figure 2.

Effect of dietary inorganic nitrate supplementation (with beetroot juice) on mean resting systolic blood pressure from HFpEF and HFrEF studies published previously. See Table 1 and text for additional information. The following HFpEF studies were not included: Zamani et al. (31), no available resting systolic blood pressure data; Shaltout et al. (44), no within-subject data comparison (i.e., control vs. inorganic nitrate) preexercise training; and Borlaug et al. (45), protocol used inhaled (nebulized) nitrite. The following HFrEF study was not included: Coggan et al. (50), no available resting systolic blood pressure data. Standard deviation/error bars omitted for clarity. *P < 0.05 vs. control.

In summary, it is clear that additional studies with larger cohorts are critical to further understand the physiological responses to and potential clinical value of these interventions in patients with HFpEF. Encouraging results in some studies provide a compelling rationale to continue pursuing inorganic nitrate and nitrite clinical trials in HFpEF (see Fig. 1 for a summary of previous data). The nitrate-nitrite-NO pathway may constitute an important target to enhance exercise tolerance and, consequently, clinical outcomes in patients with HFpEF.

INORGANIC NITRATE SUPPLEMENTATION IN HFREF

Despite evidence of reduced NO bioavailability in individuals with HFrEF, inorganic nitrate supplementation studies have failed to show consistent benefits on cardiac hemodynamics and skeletal muscle vascular, metabolic, or contractile function in patients with HFrEF (Fig. 1) (31, 47, 49–51). As illustrated in Fig. 2, dietary nitrate supplementation with beetroot juice may lower resting systolic blood pressure in patients with HFpEF, but a similar, robust effect appears to be absent in patients with HFrEF. The limited body of research to date has provided mixed results on the effects of nitrate supplementation on exercise tolerance in patients with HFrEF (Table 1).

Dietary nitrate supplementation via beetroot juice in the studies by Hirai et al. (49) and Woessner et al. (51) did not improve central or peripheral vascular function, skeletal muscle microvascular oxygenation, mitochondrial respiration, or exercise tolerance (as evaluated by time to exhaustion and V̇o_2peak during treadmill and upright cycling protocols) in patients with HFrEF. In contrast, two other studies found modest improvements in aerobic exercise performance following dietary nitrate supplementation in patients with HFrEF. Coggan et al. (50) reported improved time to exhaustion and V̇o_2peak during semirecumbent cycling exercise, and Kerley et al. (47) showed improved walking performance to exhaustion as assessed via an incremental shuttle walk test. It is interesting that, in addition to methodological differences among studies (e.g., upright vs. semirecumbent cycling and related effects of gravity on hemodynamics), the heterogeneity of patients under the umbrella of HFrEF may also play a significant role in these mixed outcomes. In the two studies that did not show an improvement in aerobic performance with dietary nitrate (49, 51), most of the patients had ischemic cardiomyopathy. Conversely, the two studies reporting beneficial effects of dietary nitrate only included patients with nonischemic cardiomyopathy (47, 50).

A recent pilot study (59) found that chronic (∼13 days) nitrate supplementation may enhance cardiac output (assessed noninvasively via Doppler ultrasound) during submaximal exercise on a recumbent cycle ergometer in patients with HFrEF. However, that nitrate supplementation with beetroot juice (9 days) did not improve cardiac output (evaluated noninvasively via impedance cardiography) during upright cycling exercise in patients with HFrEF (49) further highlights the impact of exercise mode on these cardiovascular outcomes.

To our knowledge, only a single study by Coggan et al. (48) has reported that nitrate supplementation confers muscle strength benefits (i.e., increased peak torque and power during knee extension) in patients with HFrEF. Although these improvements in muscle contractile function were not translated into greater aerobic exercise performance (as assessed via the 6-min walk test), it is noteworthy that nitrate supplementation may retain important clinical implications for HFrEF. Diminished muscle power with HFrEF can result in functional limitations highly predictive of disability and dependence (i.e., reduced ability to perform activities of daily living) and mortality in this patient population (60, 61). Future studies are thus needed to determine whether nitrate-induced increases in muscle power translate into improved habitual physical activity, quality of life, and/or survival in HFrEF.

Based on the evidence summarized above and similar to the HFpEF condition, factors that likely influence the clinical effectiveness of nitrate supplementation in HFrEF include disease etiology and severity, associated comorbidities, prescribed medications, and nitrate/nitrite supplementation regimens. It is also feasible that unique aspects of HFrEF pathophysiology could explain, at least in part, the inconsistent effects of inorganic nitrate supplementation on exercise tolerance. Examination of plasma nitrite concentrations following oral inorganic nitrate consumption suggests a potential disruption within the nitrate-nitrite reduction pathway compared with HFpEF populations and healthy controls (51). Such derangements could drive the apparently distinct effects of dietary inorganic nitrate on resting systolic blood pressure in HFrEF versus HFpEF (Fig. 2). Moreover, central dysfunction in HFrEF could abrogate potential improvements in peripheral (skeletal muscle) Q̇o₂/V̇o₂ matching with inorganic nitrate supplementation. Additional work in these areas may provide important clues for therapeutic exploitation in the future. It is also worth noting that sex differences in cardiovascular (dys)regulation may contribute to the disparity in findings between HFrEF and HFpEF, as most investigations in HFrEF included predominantly male participants (with the opposite being true for HFpEF studies; vide Table 1). The studies investigating nitrate supplementation in HFrEF are in their infancy, with relatively small sample sizes and short dosing periods. The initial findings suggest that nitrate supplementation in the HFrEF population may not consistently improve exercise tolerance when used as an acute intervention (i.e., a single dose or up to 9 days). Future studies are needed to 1) uncover the mechanistic limitations of this intervention to maximize potential beneficial outcomes and 2) explore the combined effect of inorganic nitrate with exercise training to unveil potential additive or synergistic effects.

KEY CONSIDERATIONS AND FUTURE DIRECTIONS

Although a host of early investigations in healthy humans and animals suggested that a nitrate-based therapy may benefit patients with HF, the successful translation of these findings into the clinical population has been challenging. The specific reasons for these mixed findings are currently unclear but likely include differences in study methods and HF pathophysiology (i.e., HFpEF vs. HFrEF), as mentioned earlier. What is clear is that the underpinnings of nitrate therapy in HF remain to be fully elucidated and that the confounding effects of differing HF severity, pharmaceutical treatment strategies, and other clinical/environmental factors likely preclude consistent improvements in exercise tolerance following nitrate supplementation. Development of direct in vivo NO evaluation methods could provide valuable insights particularly in the context of elevated oxidative stress as seen in HF (57, 62). The central role of symbiotic bacteria in the bioactivation of nitrate must also not be overlooked, as alterations in bacterial loads and/or species could modulate physiological effects and, therefore, exercise tolerance (42). The acute blood pressure-lowering effects of orally ingested nitrite have been shown to be dependent on the acidic gastric environment in healthy individuals (63). Moreover, it is intriguing that infused nitrite has failed to reduce resting blood pressure in both healthy and HFpEF populations despite reaching similar plasma levels as when administered orally (53, 63). The variety of HF animal models and differences in rodent versus human physiology (e.g., higher distribution of type II muscles in rodents) further challenges our ability to directly translate inorganic nitrate and nitrite interventions into the clinical HF population.

Some of the critical questions that remain to be answered include 1) whether HF alters the oral microbiome and/or gastrointestinal environment, thereby reducing the performance-enhancing properties of nitrate treatment, and, if so, 2) whether the buccal flora and/or gastric milieu of HF patients can be modulated to use the nitrate-nitrite-NO pathway better and improve exercise tolerance. Considering the fiber-type specific effects of nitrate supplementation (35), investigations into fiber-type characteristics of patients with HF included in clinical trials may shed light on potential mechanisms and therapeutic applications of inorganic nitrate in HFpEF and HFrEF.

GRANTS

This work was supported in part by a PRF Summer Faculty Grant from Purdue University (to D.M.H.) and pilot funding (to S.K.F) as part of the IDeA Networks of Biomedical Research Excellence (INBRE), award P20GM103466.

DISCLOSURES

E.W. is a co-applicant on patents related to the therapeutic use of inorganic nitrate and nitrite.

AUTHOR CONTRIBUTIONS

S.K.F., M.N.W., M.J.H., M.D.B., M.C., E.W., J.D.A., and D.M.H. prepared figures; S.K.F., M.N.W., M.J.H., M.D.B., M.C., E.W., J.D.A., and D.M.H. drafted manuscript; S.K.F., M.N.W., M.J.H., M.D.B., M.C., E.W., J.D.A., and D.M.H. edited and revised manuscript; S.K.F., M.N.W., M.J.H., M.D.B., M.C., E.W., J.D.A., and D.M.H. approved final version of manuscript.