Dietary nitrate increases VO₂peak and performance but does not alter ventilation or efficiency in patients with heart failure with reduced ejection fraction

Abstract

Background

Patients with heart failure with reduced ejection fraction (HFrEF) exhibit lower efficiency, dyspnea, and diminished peak O₂ uptake (VO₂peak) during exercise. Dietary nitrate (NO₃^-), a source of nitric oxide (NO), has improved these measures in some studies of other populations. We determined the effects of acute NO₃^- ingestion on exercise responses in eight patients with HFrEF using a randomized, double-blind, placebo-controlled, crossover design.

Methods and Results

Plasma NO₃^-, nitrite (NO₂^-), and breath NO were measured at multiple time points and respiratory gas exchange was determined during exercise after ingestion of beetroot juice containing or devoid of 11.2 mmol of NO₃^-. NO₃^- intake increased (P<0.05-0.001) plasma NO₃^- and NO₂^- and breath NO by 1469±245, 105±34, and 60±18%, respectively. Efficiency and ventilation during exercise were unchanged. However, NO₃^- ingestion increased (P<0.05) VO₂peak by 8±2%, i.e., from 21.4±2.1 to 23.0±2.3 mL.min^-1.kg^-1. Time to fatigue improved (P<0.05) by 7±3 %, i.e., from 582±84 to 612±81 s.

Conclusions

Acute dietary NO₃^- intake increases VO₂peak and performance in patients with HFrEF. These data, in conjunction with our recent data demonstrating that dietary NO₃^- also improves muscle contractile function, suggest that dietary NO₃^- supplementation may be a valuable means of enhancing exercise capacity in this population.

Introduction

Tens of millions of men and women around the world suffer from heart failure (HF), a disabling and often deadly affliction (2). In approximately half of all such individuals, the ejection fraction (EF) of the heart is reduced (2). However, regardless of the precise nature or etiology of the disease, i.e., HF with reduced EF (HFrEF) or HF with preserved EF (HFpEF), patients with HF exhibit dyspnea and diminished peak oxygen (O₂) uptake (VO₂peak) during exercise (18,19). Along with declines in maximal muscle speed and power (12), these abnormalities in aerobic exercise responses play a major role in the disability, loss of independence, and reduced quality of life that accompany HF. Perhaps more importantly, elevations in ventilatory demand and decreases in VO₂peak (and in skeletal muscle contractile function (22)) are highly predictive of mortality in patients with HF (3,13,26,40).

One factor contributing to the exercise intolerance of HF – especially HFrEF - may be a reduction in nitric oxide (NO) signaling. Along with its well-recognized role as a vasodilator, NO modulates numerous other physiological functions relevant to exercise performance, e.g., muscle contractility (10,11,12,41). There is considerable evidence, however, that NO bioavailability is diminished in HFrEF, as a result of both reduced NO production via the NO synthase (NOS) pathway (45) and more rapid destruction of NO due to increased oxidative stress (33). For example, endothelial dysfunction is common in HFrEF (48), indicative of blunted NO activity. Breath NO levels, a biomarker of whole-body NO production, are also lower in patients with HFrEF (1,8,9), as are the circulating concentrations of nitrite (NO₂^-) (31,46), the immediate degradation product of NO. The conversion of ¹⁵N-labeled arginine to ¹⁵N-labeled nitrate (NO₃^-) has also been shown to be diminished in patients with HFrEF (24), demonstrating directly that NOS-mediated NO production is impaired. Finally, increasing NO bioavaiability via L-arginine supplementation has been shown to improve 6 min walk distance in patients with HFrEF (39). Collectively, these data suggest that reduced NO signaling in HFrEF may contribute to the altered exercise responses described above.

Although most of the NO in the body is produced via the NOS pathway, it is now recognized that production of NO from dietary NO₃^- is an important source as well (28). In this enterosalivary pathway, ingested NO₃^- is reduced to NO₂^- with aid of the mouth microbiota and then to NO in the tissues via a number of endogenous catalysts (43). This last step is enhanced at low pO₂ and low pH, conditions that regularly exist in exercising muscle. A number of studies have therefore examined the effects of dietary NO₃^- supplementation, often in the form of beetroot juice (BRJ), on physiological responses and performance during exercise (23). Many, but not all, of these studies have reported that NO₃^- ingestion can enhance efficiency, reduce ventilatory demands, and/or increase performance during exercise in at least some populations, including patients with HFpEF (15,50,51). On the other hand, results of previous studies of patients with HFrEF (20,25) have been equivocal.

The purpose of the present proof-of-concept study was to test the hypothesis that acute dietary NO₃^- intake would reduce ventilatory demands, increase VO₂peak, and improve exercise performance in patients with HFrEF. We chose to study patients with HFrEF instead of HFpEF because evidence of reduced NO bioavailability is strongest in this population (vida supra). We focused specifically on ventilatory responses and VO₂peak because of their importance as determinants of exercise capacity and predictors of survival in patients with HFrEF (3,13,26,40). Furthermore, since improvements in economy or efficiency are believed to be an important mechanism by which dietary NO₃^- enhances performance in other subject groups (5,36), we designed our study to carefully quantify not only gross but also delta efficiency during exercise, as the latter is a more direct indicator of muscle contractile efficiency (41).

Materials and Methods

Subjects

The subjects in this study were patients ≥18 y of age with HFrEF (i.e., EF <45%) who were on stable medical therapy (i.e., no addition, removal, or change in medication dose of >100% in the last 3 mo). Each underwent a physical exam, medical history, and blood tests for fasting chemistries. In addition, to document the presence of HFrEF a resting echocardiogram was obtained from those who had not undergone cardiac imaging for clinical purposes in the last 12 mo. Subjects were excluded if they had major organ system disease or dysfunction other than HF, were pregnant, smoked, or had significant orthopedic limitations or other contraindications to exercise. In addition, subjects using antacids or proton pump, xanthine oxidase, or phosphodiesterase inhibitors (e.g., sildenafil) were excluded, as these can affect reduction of NO₃^- and NO₂^- to NO (30). Finally, subjects treated with organic nitrates (e.g., trinitroglycerin) were also excluded. After screening of 33 subjects, 10 subjects were enrolled in the study, with eight completing the entire protocol as planned (Fig. 1). One subject was unable to achieve a steady-state at even 20 W, such that their gross and delta efficiency and VT could not be determined. Data from another subject were excluded when subsequent analysis of their plasma samples demonstrated that they inadvertently received NO₃^- during both trials. Approval for the study was obtained from the Human Subjects Office at Indiana University and the Human Research Protection Office at Washington University School of Medicine, and all subjects provided written, informed consent.

Figure 1.

CONSORT diagram illustrating flow of subjects through the study.

Experimental design and protocol

Upon enrollment, each patient was studied using a randomized, double-blind, placebo-controlled, crossover design (Fig. 2, top panel). During one trial, they were tested 2 h after ingesting 140 mL of a concentrated BRJ supplement (Beet It Sport^®, James White Drinks, Ipswich, UK) containing 11.2 mmol of NO₃^-. During another trial, they were tested after ingesting the same volume of NO₃^--depleted BRJ. This placebo is prepared by the manufacturer by extracting NO₃^- from BRJ using an ion exchange resin and is indistinguishable from the standard product in packaging, color, texture, taste, and smell, and does not alter plasma NO₃^- or NO₂^- concentrations or breath NO levels. There was a minimum 1 wk washout period between trials. To limit variation in baseline NO₃^-, NO₂^-, and NO levels, subjects were instructed to avoid high NO₃^- foods for 10 d prior to intervention and throughout the study. Subjects were also instructed to avoid food, caffeine, alcohol, and exercise for 12 h prior to each trial, and to not chew gum or use mouthwash on study days, as these products can block conversion of NO₃^- to NO₂^- and hence to NO via the enterosalivary pathway (17).

Figure 2.

Experimental design (top panel) and protocol (bottom panel).

Subjects arrived at the Clinical Research Unit of Washington University School of Medicine in the morning after fasting overnight. Baseline heart rate and blood pressure were first measured, after which an antecubital venous catheter was inserted and a blood sample was obtained. Plasma was rapidly separated via centrifugation and frozen at -80° C for subsequent determination of NO₃^- and NO₂^- concentrations using a dedicated HPLC system (ENO-30, Eicom USA, San Diego, CA). Briefly, plasma was thawed on ice, mixed 1:1 with methanol, and centrifuged at 4° C for 10 min at 10,000 g. A 10 μL aliquot of the protein-poor supernatant was then injected into the HPLC, wherein NO₃^- and NO₂^- were isolated via a separation column, NO₃^- reduced to NO₂^- on a cadmium column, and both reacted with Griess reagent then detected spectrophotometrically at 540 nm. Plasma NO₃^- and NO₂^- concentrations were calculated based on integrated peak areas compared to those of authentic standards. This method was highly reproducible, with test-retest correlation coefficients of 0.99 and 0.98 for NO₃^- and NO₂^-, respectively. To further reduce variability, all samples from a single subject were analyzed together. Breath NO level, a biomarker of whole-body NO production (1,8,9,35), was also measured once at this time using a portable electrochemical analyzer (NIOX VERO, Circassia Pharmaceuticals Inc., Chicago, IL) following American Thoracic Society guidelines. These measurements were repeated 1 and 2 h after the subject had ingested the BRJ, and also 10 min after completion of all exercise testing (i.e., at ∼3 h). The latter consisted of submaximal steady-state and maximal incremental exercise on a semi-recumbent cycle ergometer (Lode, Gronigen, The Netherlands) (Fig. 2, bottom panel). Semi-recumbent cycle ergometry was chosen to minimize use of upper body musculature, thus aiding interpretation of any observed changes in exercise efficiency. After adjustment of the seat position, subjects first pedaled the ergometer at 60 rpm for 6 min each at 20, 40, and 60 W while respiratory gas exchange was measured continuously using a ParvoMedics 2900 metabolic cart (ParvoMedics, Sandy, UT). Heart rate, blood pressure, and perceived exertion (7) were determined during the last 30 s of each stage. Following 10 min of rest, subjects resumed pedaling at 60 W for 1 min, after which the power output was incremented by 10 W/min (47) until volitional fatigue. Respiratory gas exchange and heart rate were monitored continuously and blood pressure was measured periodically throughout the test and also immediately following cessation of exercise.

Data analyses

Respiratory gas exchange data collected during the final 2 min of each stage of the submaximal exercise test were averaged and used in all subsequent analyses. Gross efficiency was calculated as the ratio of external power to metabolic power (37), multiplied by 100%. Delta efficiency, i.e., the slope of the relationship between external and metabolic power, and the metabolic cost of unloaded cycling, i.e., the y intercept of this relationship, were determined by regression analysis (40). Similarly, during the maximal exercise test the oxygen uptake efficiency slope (OUES; Ref. 4) was calculated by regressing VO₂ (in L/min) on the log of ventilation (Ve; also in L/min), both being measured at 15 s intervals. The Ve/VCO₂ slope (3) was calculated in a similar fashion. Ventilatory threshold (VT) was determined using the V-slope method (6). Peak power was defined as the average power during the last 1 min of exercise. VO₂peak was defined as the highest VO₂ measured over any 1 min period.

Statistical analyses were performed using GraphPad Prism version 7.02 (GraphPad Software, La Jolla, CA). Normality of data distribution was first tested using the D'Agostino-Pearson omnibus test. Data were subsequently analyzed using two-way (treatment × order) ANOVA, with subject as a repeated measures factor within treatment. A P value of <0.05 was considered significant. Primary outcome variables were changes in ventilatory responses and VO₂peak in response to dietary NO₃^-. Secondary outcome variables were changes in exercise performance and efficiency; all other variables measured were considered tertiary.

Results

Patient characteristics

Characteristics of the patients are shown in Table 1. All had mild-to-moderate nonischemic HFrEF (based on NYHA class, MLWHFQ score, and EF). All were under stable, standard-of-care therapy, including use a β-blocker and, in six out of eight, treatment with an angiotensin converting enzyme inhibitor (ACEi) or an angiotensin receptor blocker (ARB).

Table 1.

Patient characteristics.

N (M/F)	8 (6/2)
Age (y)	52 ± 5
Height (m)	1.79 ± 0.03
Body mass (kg)	107.6 ± 14.1
BMI (m/kg2)	33.1 ± 3.5
Duration of HF (y)	6 ± 3
NYHA class (I/II/III/IV)	3/2/3/0
MLWHFQ (score)	35 ± 8
Ejection fraction (%)	34 ± 2
B-blocker	8/8
ACEi/ARB	6/8
Spironolactone	6/8
Statin	2/8

Values are mean±S.E. for n=8. NYHA, New York Heart Association. MLWHFQ, Minnesota Living with Heart Failure Questionaire. ACEi, angiotensin converting enzyme inhibitor. AR, angiotensin receptor blocker.

Plasma NO₃^- and NO₂^- and breath NO

No changes in plasma NO₃^- or NO₂^- concentration (Fig. 3, top and middle panels) or in breath NO levels (Fig. 3, bottom panel) occurred during the placebo trial. In contrast, ingestion of NO₃^--containing BRJ elevated (P < 0.01) plasma NO₃^- concentrations approximately 10-fold after 1 h, with this increase being maintained for the remainder of the experiment (Fig. 3, top panel). Concentrations of the downstream metabolites of NO₃^-, i.e., plasma NO₂^- and breath NO, were also significantly elevated by NO₃^- intake, albeit to a much lesser degree (Fig. 3, middle and bottom panels). The increase in plasma NO₂^- also seemed to lag behind that of NO₃^-, achieving statistical significance only after 2 h and peaking at 10 min post-exercise. These findings are consistent with the important rate-limiting role played by oral bacteria in the enterosalivary pathway of NO production (28).

Figure 3.

Effect of acute ingestion of beetroot juice either devoid of (Placebo; open bars) or containing (Nitrate; solid bars) 11.2 mmol of NO₃^- on plasma NO₃^- (top panel) and NO₂^- (middle panel) concentrations and breath NO levels (bottom panel) in patients with heart failure with reduced ejection fraction. Values are mean ± SE for n=8. 10′ Post = 10 min post-exercise. Nitrate significantly higher than than Placebo at same time point: *P<0.05, †P<0.01, ‡P<0.001.

Responses to submaximal exercise

Despite the increase in NO bioavailability resulting from NO₃^- ingestion, no differences were observed in VO₂, ventilation, ventilatory equivalents (i.e., Ve/VO₂ and Ve/VCO₂), respiratory exchange ratio, or gross efficiency during submaximal steady-state exercise (Table 2). Delta efficiency was also unaffected by dietary NO₃^- intake, averaging 26.2 ± 2.5 and 24.9 ± 1.8% in the placebo and nitrate trials, respectively (P = NS). The metabolic cost of unloaded cycling was also unchanged, averaging 200 ± 27 W, or 1.87 ± 0.07 W/kg, in the placebo trial and 215 ± 27 W, or 2.06 ± 0.14 W/kg, in the nitrate trial (P = NS). Finally, no significant differences were observed in heart rate, systolic or diastolic blood pressures, or in perceived exertion (Table 2).

Table 2.

Cardiorespiratory and perceptual responses to steady-state exercise.

		Power output (W)
	Trial	20	40	60
VO2 (L/min)	Placebo	0.81 ± 0.09	1.00 ± 0.09	1.26 ± 0.12
	Nitrate	0.87 ± 0.10	1.08 ± 0.11	1.34 ± 0.13
VO2 (mL.min-1.kg-1)	Placebo	7.8 ± 0.4	9.8 ± 0.7	12.3 ± 1.0
	Nitrate	8.4 ± 0.5	10.5 ± 0.7	13.1 ± 1.0
% of VO2peak	Placebo	38.5 ± 3.9	48.1 ± 5.0	60.6 ± 6.6
	Nitrate	38.0 ± 3.2	47.5 ± 4.0	59.1 ± 4.9
Ve (L/min)	Placebo	22.5 ± 2.7	26.8 ± 2.9	33.9 ± 4.1
	Nitrate	24.1 ± 3.6	29.5 ± 4.6	36.1 ± 5.3
Ve/VO2 (L/L)	Placebo	27.5 ± 1.0	26.5 ± 1.0	26.6 ± 1.3
	Nitrate	27.2 ± 1.5	26.6 ± 1.7	26.4 ± 1.7
Ve/VCO2 (L/L)	Placebo	33.9 ± 1.2	31.8 ± 1.3	30.6 ± 1.2
	Nitrate	33.5 ± 1.7	32.2 ± 1.6	30.9 ± 1.6
Respiratory exchange ratio	Placebo	0.81 ± 0.01	0.83 ± 0.01	0.87 ± 0.02
	Nitrate	0.81 ± 0.02	0.82 ± 0.02	0.85 ± 0.02
Gross efficiency (%)	Placebo	7.6 ± 0.8	12.1 ± 1.2	14.4 ± 1.4
	Nitrate	7.2 ± 0.6	11.3 ± 0.9	13.6 ± 1.1
Heart rate (beats/min)	Placebo	85 ± 3	98 ± 7	103 ± 5
	Nitrate	96 ± 5	101 ± 4	107 ± 9
Systolic blood pressure (mmHg)	Placebo	136 ± 7	141 ± 7	143 ± 8
	Nitrate	132 ± 9	137 ± 9	136 ± 8
Diastolic blood pressure (mmHg)	Placebo	76 ± 5	75 ± 5	76 ± 5
	Nitrate	84 ± 5	81 ± 4	78 ± 5
Perceived exertion (units)	Placebo	8 ± 1	9 ± 1	10 ± 1
	Nitrate	8 ± 1	10 ± 1	12 ± 1

Values are mean±S.E. for n=8. VO₂, oxygen uptake. Ve, ventilation. VCO₂, carbon dioxide production.

Responses to maximal exercise

Ingestion of NO₃^- did not alter ventilatory responses during the incremental exercise test, regardless of whether the data were analyzed to determine the OUES, Ve/VCO₂ slope, or VT (Table 3). Respiratory exchange ratio, heart rate, and systolic and diastolic blood pressures at peak exercise were also unchanged (Table 3). The patients were, however, able to achieve a higher (P<0.05) peak power (Table 3) and exercise longer (P<0.05; Fig. 4, top panel) following acute dietary NO₃^- intake. This improvement in exercise performance was accompanied by a moderate, but potentially clinically-significant (see Discussion), increase in VO₂peak, expressed in either L/min (P<0.05; Table 3) or in mL.min^-1.kg^-1 (P < 0.05; Fig. 4, bottom panel). Notably, NO₃^- ingestion increased VO₂peak in seven out of the eight patients, with individual increases ranging from 0.8 to 3.9 mL.min^-1.kg^-1, or 5 to 19%. VO₂peak in the remaining patient, who weighed the most and hence received the smallest dose of NO₃^- per kilogram of body mass, was essentially unchanged. For the group as a whole, however, no statistically significant correlations were observed between the magnitude of the increase in VO₂peak and the dose of NO₃^- provided or the increase in plasma NO₃^-/plasma NO₂^-/breath NO. The highest correlation was between the relative increase in plasma NO₂^- and the relative increase in VO₂peak (r = 0.64; P = 0.09).

Table 3.

Responses to incremental exercise.

	Placebo	Nitrate
OUES (L/log L)	2.73 ± 0.39	2.77 ± 0.39
Ve/VCO2 slope (L/L)	25.6 ± 1.9	24.6 ± 2.5
VT (L/min)	1.42 ± 0.15	1.49 ± 0.19
VT (mL.min-1.kg-1)	14.4 ± 2.1	14.9 ± 1.8
VT (% of VO2peak)	66.1 ± 3.7	64.1 ± 2.5
Peak respiratory exchange ratio	1.05 ± 0.03	1.05 ± 0.02
Peak heart rate (bts/min)	134 ± 6	139 ± 7
Peak systolic blood pressure (mmHg)	158 ± 8	155 ± 10
Peak diastolic blood pressure (mmHg)	90 ± 11	82 ± 6
Peak power (W)	154 ± 14	160 ± 14*
Peak power (Wkg)	1.53 ± 0.24	1.57 ± 0.23*
VO2peak (L/min)	2.42 ± 0.34	2.60 ± 0.35*

Values are mean±S.E. for n=8. OUES, oxygen uptake efficiency slope (Ref. 4). Ve, ventilation. VCO₂, carbon dioxide production. VT, ventilatory threshold (Ref. 6). VO₂peak, peak oxygen uptake.

^*P<0.05 vs. Placebo.

Figure 4.

Effect of acute ingestion of beetroot juice either devoid of (Placebo; open bar or symbols) or containing (Nitrate; solid bar or symbols) 11.2 mmol of NO₃^- on time to fatigue (top panel) and peak O₂ consumption (VO₂peak; bottom panel) during an incremental exercise test in patients with heart failure with reduced ejection fraction. Values are mean ± SE for n=8; individual results are also shown (circles, men; squares, women). *Nitrate significantly higher than Placebo: P<0.05.

Discussion

The purpose of the present study was to determine the effects of dietary NO₃^- supplementation on the responses to aerobic exercise in patients with HFrEF. Using a double-blind, placebo-controlled, crossover design, we found that acute ingestion of 11.2 mmol of NO₃^- resulted in significant increases in exercise duration, peak power, and VO₂peak during an incremental cycle ergometer exercise test. Contrary to our initial hypothesis, however, this was not accompanied by any changes in the ventilatory response (i.e., ventilatory equivalents, OUES, Ve/VCO₂ slope, or VT) during submaximal or maximal exercise. There were also no changes in either gross or delta efficiency during steady-state exercise.

As stated above, we found that acute ingestion of NO₃^- enabled patients with non-ischemic HFrEF to exercise longer and to achieve a higher peak power output during incremental exercise. This improvement in performance was accompanied by an increase in VO₂peak. The former is in keeping with the results of Kerley et al. (25), who reported that acute NO₃^- intake enhanced performance during an incremental shuttle walk test in patients with non-ischemic cardiomyopathy. In contrast, Hirai et al. (20) found that repeated ingestion of NO₃^- did not improve performance or VO₂peak in patients with HFrEF primarily of ischemic origin. The reason for this discrepancy is not clear, but it may be due to this difference in disease etiology. On the other hand, it appears unrelated to disease severity, as even the three paients we studied with baseline VO₂peak values of ∼15 mL.min^-1.kg^-1, i.e., comparable to those studied by Hirai et al. (20), demonstrated increases in VO₂peak and in performance following NO₃^-ingestion.

Regardless of the above, an increase in VO₂peak of the magnitude that we observed, i.e., +1.6 ± 0.5 mL.min^-1.kg^-1, or +8 ± 2%, may prove to be clinically significant. In particular, in a previous cross-sectional study of patients with HFrEF one of us (LRP) found that for every 1 mL.min^-1.kg^-1 increase in VO₂peak there was a 5% decrease in the annual risk of death or transplantation (38). A quantitatively-similar relationship was observed between changes in VO₂peak and disease outcome in the longitudinal HF-ACTION trial (44). At least theoretically, then, the acute dietary NO₃^--induced increase in VO₂peak observed in the present study would translate into almost a 10% reduction in annual risk. Additional research will be needed to test this hypothesis, especially in those at greatest risk (such as the three patients mentioned above).

It is also worth noting that the magnitude of the improvement in VO₂peak that we observed is comparable to that typically resulting from standard-of-care drug therapies or from endurance exercise training in patients with HF, both of which provide salutatory effects. Specifically, a number of previous studies have demonstrated that chronic treatment of HFrEF patients with a beta blocker or ACEi/ARB increases VO₂peak by approximately 10% (e.g., 14,16,32). Improvements in VO₂peak with exercise training are also similar (22). Intriguingly, the 8% enhancement of VO₂peak that we found in response to acute dietary NO₃^- intake occurred in patients with HFrEF already on optimal medical therapy, including use of a beta blocker and, in most cases, an ACEi/ARB, indicative of an additive effect. Future studies will be required to determine whether the impact of dietary NO₃^- on VO₂peak is also additive (or perhaps even synergistic) to that of exercise training in patients with HF.

Although the present results indicate that acute dietary NO₃^- intake increases VO₂peak in patients with HFrEF, the specific mechanisms responsible for this beneficial response cannot be determined from the present data. From the perspective of the cardiovascular Fick equation, though, an increase in VO₂peak could only result from an increase in heart rate, stroke volume (SV), and/or arteriovenous O₂ difference (a-vO₂diff) at peak exercise. Indeed, at peak exercise heart rate tended to be higher and diastolic blood pressure tended to be lower, suggesting that the dietary NO₃^--induced increase in VO₂peak we observed may have been the result of a greater cardiac output in a setting of reduced total peripheral resistance. Given the direct effects of NO on arteriolar smooth muscle, the latter response might be expected. In addition, recent data indicate that dietary NO₃^- intake also enhances vasodilation in contracting muscle by reducing sympathetic nerve activity (36). Again, however, in the absence of direct measurements the mechanism(s) responsible for the increase in VO₂peak observed in the present study remain unknown.

Although acute dietary NO₃^- intake resulted in a significant increase in performance and VO₂peak, there were no changes in the ventilatory response to exercise, quantified as either Ve/VO₂ or Ve/VCO₂ during steady-state exercise or as OUES, Ve/VCO₂ slope, or VT during incremental exercise. The effects of dietary NO₃^- on these parameters in patients with HFrEF have not been previously reported. The present results, however, are generally comparable to previous similar studies of patients with HFpEF (15,51), although Zamani et al. (50) found that dietary NO₃^- supplementation resulted in a significant increase in VT. It should be noted, however, that the increase in VT in their study was only 0.5 ± 0.2 mL.min^-1.kg^-1, which is nearly identical to the 0.4 ± 0.5 mL.min^-1.kg^-1 difference (P=NS) that we observed. Thus, the effects of dietary NO₃^- on ventilatory responses in patients with HFrEF or HFpEF would at best seem equivocal.

As indicated previously, studies of dietary NO₃^- supplementation in healthy individuals have often, although not always, reported improvements in exercise economy or efficiency (36). The mechanism responsible for this O₂-sparing effect is not clear, however, with some data suggesting that it results from direct inhibition of mitochondrial respiration (29) and other data implicating a decrease in ATP utilization by contracting muscle (5). In any case, given the compromised circulatory function of HF patients, any reduction in the demand for delivery of O₂-carrying blood during exercise would seem beneficial. Hirai et al. (20), however, did not observe any dietary NO₃^--induced changes in submaximal VO₂ during exercise. Despite using a protocol carefully designed to account for the slower VO₂ kinetics found in HF, minimize involvement of non-active tissues, and allow assessment of not only gross but also delta efficiency, we also found acute dietary NO₃^- intake did not alter the energy requirements of submaximal exercise. As suggested by Zamani et al. (50), this may reflect differences between patients with HF and young, healthy control subjects in age or in the factors controlling mitochondrial respiration during exercise. Regardless, the present data demonstrate that, at least in patients with HFrEF, acute dietary NO₃^- intake can increase performance and VO₂peak even in the absence of any changes in energy demand at a given power output.

There are a number of limitations to the present study. First, we studied a relatively small number of individuals, and therefore may have failed to detect some true effects of NO₃^-supplementation, e.g., a decrease in blood pressure. However, our sample size was comparable to those of similar previous studies of dietary NO₃^- intake on exercise responses in patients with HFrEF (20,25), and was adequate to detect changes in one of our primary outcomes, i.e., VO₂peak. Second, as previously discussed we did not directly measure central or peripheral determinants of VO₂peak, and therefore cannot determine the mechanisms responsible for the improvement that was observed. This does not, however, negate our primary finding that dietary NO₃^- supplementation increases exercise capacity and VO₂peak in patients with HFrEF. Finally, we studied only the effects of acute ingestion of NO₃^- at a single, fixed dose, and therefore cannot draw any conclusions on the effects of longer-term treatment and/or other doses. Answering such questions will therefore require additional research.

To summarize, the results of this proof-of-concept study demonstrate that acute ingestion of 11.2 mmol of NO₃^- (in the form of a concentrated BRJ supplement) increases aerobic exercise performance and VO₂peak, but does not alter ventilatory responses or gross or delta efficiency during exercise, in patients with mild-to-moderate HFrEF. Along with our previous data demonstrating that acute dietary NO₃^- intake results in comparable improvements in muscle contractile function in this population (10), these suggest that dietary NO₃^- supplementation may be a valuable adjunctive treatment for exercise intolerance in this population. Larger, i.e., multi-center trials are needed to confirm the present findings and to determine whether longer-term dietary NO₃^- treatment improves physical activity levels, quality of life, and perhaps even survival in patients with HFrEF.

Highlights.

• Acute dietary NO₃^- intake increased VO₂peak in patients with HF by 8±2% (P<0.05).

• Time to fatigue during exercise improved by 7±3 % (P<0.05).

• Dietary NO₃^- may be a means of enhancing exercise capacity in patients with HF.