Effect of inorganic nitrate on exercise capacity, mitochondria respiration and vascular 1 function in heart failure reduced ejection fraction 2

Background: Chronic under perfusion of the skeletal muscle tissues is a contributor to a 29 decrease in exercise capacity in patients with heart failure reduced ejection fraction (HFrEF). 30 This under perfusion is due, at least in part, to impaired nitric oxide (NO) bioavailability. 31 Oral inorganic nitrate supplementation increases NO bioavailability and may be used to 32 improve exercise capacity, vascular function and mitochondrial respiration. 33 Methods : Sixteen patients with HFrEF (15 men, 63 ± 4 y, BMI: 31.8 ± 2.1 kg∙m -2 ) 34 participated in a randomised, double-blind, crossover design study. Following consumption 35 of either nitrate rich beetroot juice (16 mmol nitrate/day), or a nitrate-depleted placebo for 36 five days participants completed separate visits for assessment of exercise capacity, 37 endothelial function and muscle mitochondrial respiration. Participants then had a two week 38 washout prior to completion of the same protocol with the other intervention. Statistical 39 significance was set a priori at p<0.05 and between treatment differences were analysed via 40 paired- t-test analysis. 41 Results : both 42 and respiratory Conclusions Inorganic nitrate supplementation did not improve exercise capacity and skeletal muscle mitochondrial respiratory function explore alternative interventions improve


Recruitment and eligibility
Participants were identified through medical chart reviews and interested individuals were 147 provided a detailed description of the nature of the study and, if interested, were invited to 148 sign an informed consent and complete a screening cardiopulmonary exercise test that also 149 served as a familiarisation visit. Participants were screened either over the phone or in person 150 to ensure they met all inclusion criteria. The key criteria were for participants to have an EF 151 <40%, be on stable medications (for 3 months), and to have no existing injuries. While 152 individuals with comorbidities were invited to participate, CHF had to be considered their 153 primary condition (see Figure 2). In total, 882 medical charts were reviewed, nineteen 154 participants were recruited and sixteen individuals (62.6 ± 3.6 years) with diagnosed HFrEF 155 (EF 30.4 ± 1.8 %) completed the study. Participants consumed a total of 210 ml (16 mmol nitrate) per day. They were asked to 159 consume one 70 ml bottle with each meal. However, on testing days they were requested to 160 consume the morning dose exactly 2.5 hours prior to the appointment time (15,32). 161 Compliance to supplementation and conversion of nitrate to nitrite was confirmed by a blood 162 draw on each of the two interventional CPX testing visits. For the duration of the trial, all 163 participants were asked to refrain from the use of any type of mouthwash due to 164 demonstrated reductions in conversion of nitrate to nitrite via oral bacteria (33). They were 165 also asked to maintain their normal dietary and exercise patterns for the duration of the study. 166 While diet was not specifically monitored throughout the study, participants were given 167 instructions on certain high nitrate food items to avoid. 168 Aerobic capacity assessment 170 The CPX tests utilised a two-step treadmill protocol whereby all participants first completed 171 six minutes of low-intensity walking at 1.4 km/hour at a 4% grade. The protocol then 172 increased in speed or incline (in an individualised manner, with intensities replicated at 173 subsequent visits) every two minutes. All tests were continued until the participant reached 174 volitional exhaustion. The total time to exhaustion was recorded as the total exercise 175 duration. Expired respiratory gases were collected breath-by-breath via a facemask attached 176 to a gas analyser (Medgraphics,177 142090-001, Revia, Minnesota, USA) and heart rate (HR) was monitored continuously via a 178 12-lead ECG (Mortara, X-Scribe II, Milwaukee, WI, USA). The gas exchange threshold was 179 calculated via the V-slope method (2) Participants were asked to hold all morning medications until vascular post-testing.

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Following 10 minutes of supine rest, endothelial function was assessed via brachial artery 201 flow mediated dilation (FMD) using a high-resolution ultrasound (Terason,LifeHealthcare,202 New South Wales, Australia) with R wave trigger (35). Ten-second video clips were captured 203 in duplicate at baseline and during forearm occlusion and a continuous two-minute video was 204 captured after the occlusion cuff release (reactive hyperaemia). Peak change following 205 reactive hyperaemia was calculated as the percentage change in brachial artery diameter from 206 baseline to immediately following peak hyperaemia.

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For all BP measurements, the non-invasive SphygomoCor® (AtCor Medical, Sydney, NSW, 209 Australia) diagnostic system was utilised (12). A SphygomoCor® brachial blood pressure 210 (BP) cuff was fitted on the upper arm. The system recorded pulsations at the brachial artery 211 and produced (via a generalised transfer function) aortic pressure waveforms and predicted 212 central systolic BP, diastolic BP, mean arterial pressure, pulse pressure, augmentation index 213 and aortic pressure. Two measurements were captured, with the lower of the two readings 214 recorded. If the two blood pressure readings were >6 mmHg apart, a third measure was 215 recorded to ensure a true resting value and the average of the two lowest BP were recorded.  were separated with forceps and immediately placed in ice-cold preserving solution BioPS.

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The plasma membrane was permeabilised by agitation for 30 min at 4°C in BioPS containing 232 50 μg/ml saponin and subsequently washed in the respiration medium MIR05. Mitochondrial 233 respiration was measured in duplicate (from 2-4 mg wet weight of muscle fibres) in MiR05 234 at 37°C, using a high resolution respirometer (Oxygraph-2k, Oroboros, Innsbruck, Austria).

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A substrate-uncoupler-inhibitor titration (SUIT) protocol was utilised (27). The SUIT 236 sequence was as follows: malate (2 mM) and pyruvate (5 mM) in the absence of adenylates 237 were added for measurement of leak respiration (CI) L. ADP (5 mM) was added for 238 measurement of oxidative phosphorylation capacity(CI) P . Succinate (10 mM) was added for 239 the measurement of p through complex 1 and 2 combined (CI+II) P . Cytochrome c (10 mM) 240 was then added to test for outer mitochondrial membrane integrity (an oxygen flux increase 241 of <15% from (CI+II)p was considered acceptable). This was followed by a series of  (#5586). One antibody from Calbiochem for PGC-1α (#st1202) was also utilized. Following 267 TBST washes, samples were incubated at room temperature with the appropriate host 268 species-specific secondary antibody for 60 min, before being exposed to a chemiluminescence solution. Images were taken with a ChemiDoc Imaging System fitted 270 (Bio-Rad). Densitometry was performed with Image Lab 5.0 software (Bio-Rad).

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Nineteen patients commenced the trial, however, three dropped out prior to completion of 291 both rounds of testing due to reasons unrelated to the study. Anthropometric and clinical 292 characteristics of the 16 who completed the study are described in Table 1 p<0.001 and 94%, p< 0.05) respectively, Figure 3A-D. One participant's plasma nitrite data 302 was excluded from the analysis due to a concentration 4 standard deviations above the mean.   Twelve participants completed the vascular testing (four could not be analysed due to 315 insufficient image quality). There were no significant differences between interventions in 316 the resting brachial BPs (SBP, DBP and MAP) between the placebo and nitrate 317 interventions (∆= -2, -1, -2 mmHg, all p>0.30). There were also no significant differences in 318 the measures of aortic pressure or stiffness (Table 2).
Finally, there were no differences in resting brachial artery diameters (nitrate 3.92 ± 0.16 mm 321 and placebo 4.0 ± 0.13 mm, p=0.44) or peak reactive hyperaemic response (nitrate 5.7 ± 1.1 322 % and placebo 4.1 ± 0.68 %, p=0.06) between interventions. Previous studies in both healthy and clinical cohorts have indicated significant increases in 343 plasma nitrate and nitrite following supplementation (1, 7, 10, 21). In the present study, there 344 was a significant increase in plasma nitrate and nitrite following supplementation. However reported levels in HFpEF (795nM) and healthy (580nM) subjects. This is despite the present 347 study utilising a higher dose than the majority of previous clinical trials, (1,5,7,10,17,18,348 30). This suggests a potential poor conversion of nitrate to nitrite in HFrEF. The oral 349 microbiome has been shown to play a crucial role in the conversion of plasma nitrate to For the main outcomes of the study, there were no differences between peak or submaximal 358 aerobic capacities between treatments. These findings are in agreement with a previous study 359 in HFrEF which reported no improvement in exercise capacity following a smaller (12.9 360 mmol) chronic dose of inorganic nitrate (10). The present study also showed no differences in 361 gas exchange threshold or VO 2 during recovery. There have been two previous positive 362 findings for aerobic capacity in the HFrEF patient cohort, however, they employed varying 363 cutoffs for EF% (including patients with EF >40% in their samples) and one utilized a 364 recumbent cycle modality which may have increased venous return to the right atrium and 365 influenced central hemodynamics (4, 17). When these factors are controlled for, it appears 366 supplementation has no effect on aerobic exercise capacity in HFrEF. interventions. Our results corroborate and expand on the findings of previous smaller trials in 376 HFrEF showing no effect on BP.

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No previous studies which have utilised nitrate supplementation with patients with CHF have 379 examined the effects FMD (7,11,39,40). In the present study, the peak percent change in 380 brachial diameter from baseline following nitrate supplementation was 5.7% compared to 381 4.2% following placebo. This response is similar to another nitrate supplementation study in   HFrEF (10, 34, 40). In the current study, we report no effect of supplementation on 395 tissue oxygenation as measured by NIRS. We also report, for the first time in HFrEF, that mitochondrial respiration and mitochondrial-related protein expression following 397 supplementation did not change. At the onset of this clinical trial, a previous study in humans 398 had demonstrated that nitrate supplementation could improve mitochondrial efficiency via 399 increasing the capacity for ATP synthesis (19). However, to date these results have yet to be 400 replicated with nitrate or nitrite supplementation in mice nor human models (22,26). Herein  The current study has several potential limitations. While the study is the largest to date in 411 this population, it was still a relatively small sample size. The patient population was also 412 primarily male (n=15). This was not intentional as recruitment was open to both men and 413 women, but the lack of women participants does limit the applicability of the findings. In line 414 with some of previous studies assessing the effects of nitrate supplementation in cohorts of 415 patients with CHF, recruitment in the present study was inclusive of those individuals with 416 diagnosed chronic comorbidities (hypertension, diabetes and COPD). Participants with any 417 comorbidity that was either uncontrolled or that was identified as a primary contributor to 418 reduced exercise capacity or symptomology, however, were excluded. Additionally, dietary 419 logs were not a component of this trial. While participants were asked to maintain their 420 normal dietary habits and were given a list of high nitrate food items to avoid, the diet was not specifically controlled for beyond these measures. Another limitation of the study is that      Abbreviations: Akt, protein kinase, MAPK, mitogen-activated protein kinase, mTORC1, 640 mechanistic target of rapamycin complex 1, p, phosphorylated.