BEET-HF: The Effects of Dietary Inorganic Nitrate Supplementation on Aerobic Exercise Performance, Vascular Function, Cardiac Performance and Mitochondrial Respiration in Patients with Heart Failure with Reduced Ejection Fraction

Chronic heart failure (CHF) is characterised by an inability of the heart to pump enough blood to meet the body’s metabolic needs, resulting in exercise intolerance. A reduction in nitric oxide (NO) bioavailability has been implicated as an initiator and/or contributor to many of the peripheral skeletal tissue dysfunctions that contribute to the exercise intolerance in patients with CHF. Inorganic nitrate supplementation has been identified as an important mediator of exercise tolerance via increasing NO bioavailability, but the potential efficacy of this on patients with heart failure with reduced ejection fraction (HFrEF) as well as the effect on vascular function is not well understood and was the focus of Study 1. Additionally, to our knowledge, no previous study has examined the potential impact of nitrate supplementation on cardiac performance during submaximal exercise and mitochondrial respiration in individuals with HFrEF. These were the foci of Studies 2 and 3 respectively. Study 1: The effect of dietary inorganic nitrate supplementation on exercise tolerance and vascular function in patients with HFrEF. The primary aim of this study was to determine the effect of chronic inorganic nitrate supplementation on exercise tolerance, as measured by peak aerobic capacity (VO2peak) and time to exhaustion (TTE), during treadmill exercise in patients with HFrEF. A secondary aim was to determine the effect of chronic supplementation on vascular function (endothelial function) in these patients. Methods: Sixteen patients with HFrEF (15 men and 1 woman, 63 ± 4 y, BMI: 31.8 ±2.1 kg∙m-2) completed the primary outcome of this study (exercise tolerance), and 12 completed the vascular function component. Participants were randomly allocated, in a double-blind, crossover design, to consume either a nitrate rich beetroot juice (16mmol nitrate/day), or a nitrate-depleted placebo for five days prior to the first testing visit. Participants then continued daily dosing until they completed a cardiopulmonary exercise test (CPX) and a battery of vascular function assessments (peripheral and central blood pressure (BP) as well as aortic stiffness and brachial artery flow mediated dilation (BAFMD)). Results: There were significant increases in both plasma nitrate (p 0.05) changes in peripheral tissue oxygenation during exercise, as measured non-invasively with near-infrared spectroscopy (NIRS). There were no differences in the brachial blood pressure measurements including systolic blood pressure (SBP) (nitrate: 130 ± 4 mmHg, placebo: 132 ± 5 mmHg, p=0.58), diastolic blood pressure (DBP) (nitrate: 80 ± 3 mmHg, placebo: 81 ± 3 mmHg, p= .74) and mean arterial pressure (MAP) (nitrate: 96 ± 3 mmHg, placebo: 98 ± 4 mmHg, p=0.67). There were also no significant differences in aortic pressure or stiffness. BAFMD reactive hyperaemic percent change tended to improve (nitrate: 5.7% ± 1.1, placebo: 4.1% ± 0.7, (p=0.06), and this change had a moderate effect size (ES) (Cohen’s d 0.607). Conclusions: Results from this study indicate the nitrate appears ineffective at improving exercise tolerance and vascular function in HFrEF. Future studies should explore alternative interventions to improve peripheral muscle tissue function in HFrEF. Study 2: The effect of dietary nitrate supplementation on cardiac output and stroke volume during submaximal exercise in men with HFrEF: a pilot study. The primary aim of this exploratory study was to determine the effect of chronic inorganic nitrate supplementation on cardiac performance during three submaximal exercise bouts. Methods: Five male patients with HFrEF (61 ± 3y) completed this pilot study. Participants consumed either the nitrate-rich beetroot juice (16 mmol nitrate) or the placebo an average of 13 ± 2 days prior to the testing visit. They completed a three-stage (15-25 watts, 25-40 watts and 35-60 watts) discontinuous exercise protocol on an echo-compatible recumbent cycle ergometer with simultaneous Doppler echocardiography. Cardiac output (Q) and stroke volume (SV) were derived using the Doppler velocity time integral via the Huntsman method. Results: There were significant increases in both plasma nitrate (p=0.004, ES=3.54) and nitrite (p=0.01, ES=0.82) following nitrate supplementation. Although not statistically significant (all p>0.27), the differences in Qduring stage two and stage three had medium to large ES (stage two: nitrate: 6.4 ± .4 L∙min-1, placebo: 5.3 ±. 2 L∙min-1, ES=1.51; stage three: nitrate: 7.5 ± 0.6 L∙min-1, placebo: 6.4 ± 0.7 L∙min-1, ES=0.50) exercise. Changes in Q were accompanied by medium to large ES changes in SV (stage two: ES=0.97 and stage three: ES=0.57) and medium to large increases in heart rate (HR) at rest and all exercise stages. These differences were likely mediated by a reduction in total peripheral resistance (TPR) at stage two (ES=-1.62) and stage three (ES=-0.81). Conclusions: We report potentially clinically important improvements in measures of cardiac performance during submaximal exercise following nitrate supplementation in patients with HFrEF. The initial findings from this pilot study warrant further investigation in larger and more diverse samples in order to determine the efficacy of this intervention. Study 3: The effect of dietary nitrate supplementation on mitochondrial respiration in men with HFrEF. The primary aim of this exploratory study was to determine the effect of chronic inorganic nitrate supplementation on parameters of mitochondrial respiration in patients with HFrEF. Methods: Seven male participants (62 ± 2y) completed this invasive study. Participants consumed the nitrate rich beetroot juice (16mmol nitrate/day) or a placebo for an average of 15 ± 2 days prior to their muscle biopsy. Muscle samples were taken from the vastus lateralis. Mitochondrial respiration was assessed using high resolution respirometry. Western blot analysis was used to assess the protein content of mechanistic target of rapamycin complex 1 (mTORC1), p38 mitogen activated protein kinase (p38MAPK), protein kinase B (Akt), and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). Results: Plasma nitrate increased (831%, p 0.05). Similarly, there were no differences in mitochondrial content, as assessed by citrate synthase activity (p=0.73) and no differences were noted in total and phosphorylated forms of mTORC1, p38MAPK, Akt, or PGC-1α (all p>0.10). Conclusions: Short-term nitrate supplementation, as a standalone treatment, may not be an effective way to improve mitochondrial function in patients with HFrEF and, as such, it may be clinically important to combine nitrate supplementation with other interventions known to affect mitochondrial function, such as exercise training. General Conclusions. Short-term inorganic nitrate supplementation had no effect on exercise tolerance (Study 1-Chapter 4), peripheral tissue oxygenation (Study 1- Chapter 4), or mitochondrial respiration (Study 3- Chapter 6) in patients with HFrEF. However, it may have a meaningful clinical effect on Qand SV during submaximal exercise (Study 2- Chapter 5). It may also improve vascular function (Chapter 4), reduce TPR (Chapter 5) and reduce DBP and MAP during submaximal exercise (Chapter 5) in these patients. Overall the data suggest that nitrate supplementation may be used in conjunction with other pharmacological and non-pharmacological (exercise training) interventions to improve clinical outcomes in this population. This hypothesis should be explored in the future by conducting a large-scale clinical trial.