The Influence of Active Muscle Mass and Nitrate Supplementation on Fatigability in Individuals With Heart Failure
Phase: NA
Conditions: Heart Failure With Reduced Ejection Fraction
Sponsor: Newcastle University

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Short Title: Neuromuscular fatigability in individuals with heart failure 
 
Full Title: The influence of active muscle mass and nitrate supplementation on 
fatigability in individuals with heart failure 
 
Research Protocol 
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NCT Number:  
 
Research Team: 
Dr Callum Brownstein (Lecturer, Newcastle University) 
Dr Sarah Charman (Research Associate, Newcastle University) 
Miss Emily Dodd (PhD Researcher, Newcastle University) 
Prof Guy MacGowan (Consultant Cardiologist, Freeman Hospital) 
Prof Djordje Jakovljevic (Senior Lecturer, Newcastle University) 
Dr Kristian Bailey (Consultant Cardiologist, RVI) 
Dr Matthew Williams (Consultant Cardiologist, RVI) 
Dr Oscar Fernandez (Consultant Cardiologist, Freeman Hospital) 
Dr Katherine Nelson (Consultant Cardiologist, RVI) 
Dr Alisdair Blain (Biostatistician, Newcastle University) 
 
 
 
 
 
 
 
 

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Background 
Chronic heart failure (CHF) is a complex clinical syndrome associated with structural 
and functional abnormalities, which lead to reduced cardiac output at rest and/or in 
response to stress [1]. Exercise intolerance is a hallmark symptom of chronic heart 
failure (CHF) and is associated with reduced quality of life as well as being a strong 
prognostic indicator [2, 3]. Research in recent years has attempted to better 
understand the aetiology of exercise intolerance to provide therapeutic targets to 
improve physical capacity and quality of life, with studies primarily focusing on maximal 
oxygen uptake, metabolic thresholds and oxygen uptake kinetics [4-7]. Another 
important determinant of exercise intolerance is neuromuscular fatigability, defined as 
the reduction in neuromuscular function measured after exercise of a discrete time 
period [8, 9]. At present, the few studies that have assessed neuromuscular fatigability 
in individuals with CHF have utilised exercise involving a small muscle mass, such as 
isometric knee extension. However, one limitation of this approach is that it does not 
reflect the types of activity performed on a daily basis, and thereby lacks ecological 
validity. Specifically, activities of daily living (e.g. walking, gardening, housework or 
climbing stairs) involve dynamic, large muscle mass exercise and in turn a 
substantially higher cardiorespiratory demand relative to isometric tasks. At present, 
there is limited research assessing neuromuscular fatigability in individuals with CHF 
during large muscle mass exercise. Research characterising fatigability and 
determining its underlying mechanisms can help better understand the aetiology of 
exercise intolerance, and in turn provide therapeutic targets aimed at improving 
physical capacity and quality of life in individuals with CHF. 
One potential strategy to attenuate fatigability is through nitrate supplementation (i.e. 
beetroot juice). Specifically, the consumption of nitrate-rich beetroot juice promotes 
increased nitric oxide bioavailability, which in turn can enhance local perfusion and 
oxygenation, skeletal muscle contractility, and muscle efficiency [10]. Given that 
individuals with CHF have impaired nitric oxide bioavailability and reduced local 
perfusion and oxygenation [6], which likely contribute to impaired fatigability, nitric 
oxide represents an attractive intervention to mitigate fatigability and improve exercise 
tolerance. To date, no study has assessed the effect of nitrate supplementation on 
neuromuscular fatigability in individuals with CHF.  
 

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Aim and Objectives: 
The aims of this project are to, 1) characterise neuromuscular fatigability in individuals 
with CHF during exercise involving a small and large active muscle mass (Part I), 2) 
determine the effect of nitrate supplementation on neuromuscular fatigability during 
large muscle mass exercise in individuals with CHF (Part II), and 3) understand the 
impact of exercise intolerance on quality of life in individuals with CHF (Part III). 
 
Methods/Design: 
Study Design 
Part I is a cross-sectional, observational study, which will characterise neuromuscular 
fatigability in individuals with CHF. Part II is a randomised crossover interventional 
study, which will assess neuromuscular fatigability following nitrate supplementation 
and a sham-control in individuals with CHF. For part II, participants will consume 
nitrate-rich beetroot juice in the lead up to one visit, and beetroot juice with nitrate 
extracted as a sham-control in the lead up to another visit, with the order of the 
conditions randomised.  Part III is a qualitative study, which will evaluate the impact of 
exercise intolerance on quality of life in individuals with CHF.  
 
Sample size 
A total of 20 patients will be recruited into the study, with the same 20 patients 
participating in Parts I-III.  
 
Eligibility criteria 
Inclusion criteria: 
• Patients with a left ventricular ejection fraction < 40%; 
• Diagnosed for at least 3 months; 
• Classified according to New York Heart Association (NYHA) class II-III; 
• Clinically stable and receiving an optimal medical treatment; 
• Aged ≥ 45 years old; 

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• Willingness to participate in a semi-structured interview (this is optional and the 
participant will be able to participate in the study if they choose not to take part 
in the interview); 
• Ability to read, write and converse in English without the support of an 
interpreter; 
• Willingness to undertake physical activity; 
• No contraindications to physical activity and capable of performing activities of 
daily living independently, without the use of a walking aid; 
• Able to provide written informed consent. 
 
Exclusion criteria: 
• Electrically implanted device (e.g. pacemaker, left ventricular assist device); 
• Uncontrolled cardiac arrhythmias, myocardial infarction, percutaneous 
coronary intervention and/or bypass graft surgery up to 3 months previously; 
• Receiving antacids or proton pump, xanthine oxidase, or phosphodiesterase 
inhibitors which affect the reduction of nitrate to nitrite and nitrite to nitric oxide; 
• Treated with organic nitrates (e.g. trinitroglycerin) 
• Major multi-morbidity or other alternative diagnoses of no obvious acute and 
self-limiting cause (e.g. patients with a terminal diagnosis of cancer, patients in 
receipt of oxygen therapy or oxygen saturation at rest <92%); 
• Obesity (body mass index > 30 kg/m2); 
• Current smoker; 
• Presented with severe symptoms requiring urgent assessment and stabilisation 
(e.g. breathless at rest, hypotension, confusion); 
• Severe physical disability preventing them from functioning independently; 
• Unable to provide informed consent; 
• Currently taking part in any other study; 
• Unable to communicate in English. 
 

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Recruitment procedures 
Patients will be identified from the Heart Failure Diagnostic Clinic run at the Royal 
Victoria Infirmary and Freeman Hospital, Newcastle upon Tyne, by Consultant 
Cardiologists, Dr MacGowan, Dr Bailey, Dr Williams, Dr Fernandez and Dr Nelson, all 
of whom are part of the research team and play a vital role in identifying patients who 
meet the inclusion criteria. The consultant cardiologist will then invite the patient to talk 
to a member of the research team (Dr Callum Brownstein, Dr Sarah Charman, Miss 
Emily Dodd) who all have honorary contracts with the Newcastle upon Tyne Hospitals 
NHS Trust and sufficient level of competence and permission to assess patients’ 
medical records using computerised systems in place. This will take place at the end 
of the patient’s consultation, after which a member of the research team will meet the 
patient to explain the study. If the patient is interested, they will be provided with an 
information sheet. To avoid coercion, the patient will not be asked to make a decision 
in the presence of a member of the research team, and instead will be afforded 48 
hours to read the information sheet and decide whether they would like to participate. 
The research team will request to call the patient after this 48 hour period to discuss 
the study and answer any questions. If the patient is happy to proceed when they will 
be invited to the Clinical Research Facility for Visit 1, where they will provide informed 
consent if they are willing to become a participant in the study.  
 
Research Visits 
Parts I and II: Quantitative work package 
Individuals with CHF (n = 20) will visit the NIHR Clinical Research Facility on 4 
occasions across Parts I and II of the study. Visit 1 will involve screening and consent, 
completion of questionnaires, familiarisation with study procedures, and performing a 
non-invasive measurement of mitochondrial function using near-infrared spectroscopy 
(NIRS). Visits 2-4 will be randomised in order, and will include, 1) incremental single-
leg cycling to exhaustion with intermittent assessments of neuromuscular function, 2) 
incremental double-leg cycling to exhaustion with intermittent assessments of 
neuromuscular function, following a period of supplementation with nitrate-rich 
beetroot juice and, 3) incremental double-leg cycling to exhaustion with intermittent 

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assessments of neuromuscular function, following a period of supplementation with a 
nitrate-extracted beetroot juice placebo. For Visits 2-4, during which exhaustive 
exercise will take place, a consultant cardiologist will be present. The cardiologist will 
monitor ECG at rest prior to exercise and throughout the exercise protocol. Exercise 
will be discontinued if any abnormalities in the ECG trace are observed, or if the 
participant begins to feel unwell. Furthermore, the cardiologists involved in the study 
are trained in emergency care, and will be able to provide immediate assistance if the 
participant required medical care. Further details on these visits are provided below.  
 
Visit 1 – Screening, consent, questionnaires, familiarisation, and NIRS-derived 
measure of mitochondrial function (1 hour 30 min) 
The following procedures will be performed during Visit 1: 
1) An investigator will discuss aspects of the study with the participant, including 
the measurements involved and the supplementation process, and the 
participant will be provided with the opportunity to ask further questions and if 
they are happy to participate then they will be requested to provide written 
informed consent. A Medical History Questionnaire will be completed after 
written informed consent has been received (20 min). 
2) Physical examination performed, including body weight, height, and blood 
pressure (10 min). 
3) Participants will complete four validated questionnaires. These will include the 
Minnesota Living with Heart Failure (MLHF) Questionnaire [11], Short-Form 
Health Survey (SF-36) [12], Pittsburgh Fatigability Sale [13], Godin Leisure-
Time Physical Activity questionnaire [14] and food frequency questionnaire (20 
min).  
4) Participants will be familiarised with performing assessments of neuromuscular 
function on a customised semi-recumbent cycle ergometer, consisting of 
isometric maximal voluntary contractions (MVCs) of the knee extensors and 
supramaximal single (1 ms) electrical stimuli to the femoral nerve during and 
following MVCs. They will subsequently be familiarised with performing single-
leg cycling at low-intensity, followed by immediate re-assessments of 
neuromuscular function (20 min). 

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5) A measurement of mitochondrial function will be taken using NIRS. This will 
involve participants performing 2 × 10 s contractions of the knee extensor at 
40% MVCs separated by 10 s rest. Immediately following the second 
contraction, repeated, transient (5-20 s) occlusions of arterial circulation to the 
knee extensors will be performed to over a 3 min period using a rapid occlusion 
system (VenaPulse, VP-25, ACI Medical, USA) inflated to > 240 mmHg. This 
permits the measurement of muscle oxygen uptake recovery following muscle 
contractions by observing the slope of deoxygenation using NIRS, with the 
speed of oxygen uptake recovery well associated with gold-standard 
measurements of mitochondrial function [15] (20 min).  
Depending on the order of randomisation, participants will be provided with their 
supplement for the subsequent visit, and instructed on supplementation.  
 
Visits 2-4 – Neuromuscular fatigability during double-leg cycling following nitrate-
extracted beetroot juice placebo, double-leg cycling following nitrate-rich beetroot juice 
supplementation, and single-leg cycling (2 hours) 
Supplementation will only take place prior to the visits involving double-leg cycling (i.e. 
not prior to single-leg cycling). The supplementation will consist of 7 consecutive days 
of consuming either 2 × 70 ml of nitrate-rich beetroot juice (Beet It Sport, James White 
Drinks, UK) per day in the morning and evening, or 2 × 70 ml of nitrate-extracted 
beetroot juice as a placebo control (Beet It Sport, James White Drinks, UK). Testing 
will take place on the 7th day of supplementation, with participants asked to consume 
2 bottles of the supplement 2 hours prior to testing taking place. Participants will be 
asked to avoid chewing gum or using mouthwash throughout the study period, as this 
can interfere with the conversion of nitrate to nitric oxide. Furthermore, in the 7 days 
during the supplementation periods, participants will be asked to complete a beetroot 
juice intake diary. A minimum washout period of 7 days will be given between periods 
of supplementation. Confirmation of supplementation will be performed by two means, 
1) measurement of salivary nitrate concentrations and 2) having participants return 
empty, consumed bottles to the laboratory.  
The following procedures will be performed during Visits 2-4: 

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1) Physical examination performed, including body weight, height and weight (10 
min) 
2) Salivary nitrate measurement via chewing on a cotton ball for 2 minutes, to 
subsequent be placed in an Eppendorf and frozen for later analysis (only during 
double-leg cycling visits) (2 min). 
3) Participant set-up (45 min), including: 
a. Set-up for fatigability measurements, including placement of peripheral 
nerve stimulation electrodes on the femoral nerve (cathode) and gluteal 
fold (anode), and electromyography electrodes placed on the vastus 
lateralis. Stimulation of the femoral nerve via the cathode and anode 
permit the measurement of contractile function through resting twitch 
responses to supramaximal stimuli (i.e. stimuli eliciting a maximal twitch 
response) and voluntary activation of the quadriceps through 
supramaximal stimuli delivered during an isometric maximal voluntary 
contraction of the knee-extensors. These methods have been used 
extensively to quantify the neuromuscular mechanisms of fatigability in 
a range of populations [16], including individuals with CHF [17];  
b. Placement of electrocardiography electrodes; 
c. Placement of NIRS on the vastus lateralis (PortaLite, Artinis, The 
Netherlands). NIRS provides a non-invasive means of measuring 
muscle oxygenation by shining near-infrared light which penetrates the 
tissue. The degree of absorption of near-infrared light depends on the 
degree of oxygenation of haemoglobin, with non-absorbed light reflected 
back towards a receiver optode on the NIRS device. Accordingly, NIRS 
is able to measure relative changes in oxygenated and deoxygenated 
haemoglobin through changes in the absorption of near-infrared light 
[18].  
d. Placement of a bioreactance Non-Invasive Cardiac Output Monitor 
(NICOM, Cheetah Medical, USA) which we have previously evaluated 
[19-21] The method uses four pairs of electrodes applied at the front side 
of the upper and lower thorax (similar to ECG). Bioreactance is a novel 
method for continuous non-invasive cardiac function monitoring and 
estimates cardiac output by analysing the frequency of relative phase 
shift of electronic current delivered across the thorax.  

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e. Placement of face mask for measurement of gas exchange (i.e. oxygen 
consumption, carbon dioxide production) and standard metabolic 
analyser (Cortex, Germany). 
4) Arterial occlusion of the knee extensors for 5 min, followed by 10 min passive 
recovery. This is performed in conjunction with NIRS to provide a physiological 
calibration of the minimum and maximum NIRS signal through the lowest 
muscle oxygenation (during occlusion) and highest muscle oxygenation 
(following the hyperaemic response after the release of occlusion), respectively 
(15 min).  
5) Figure 1 below displays a schematic of the exercise protocol. The exercise 
protocol will consist of incremental cycling during single- or double-leg cycling, 
depending on visit and randomisation. Double-leg cycling will consist of 3 min 
stages of incremental cycling beginning at 16 W, with a 16 W increment each 
stage up until stage 5 (80 W). If stage 5 is reached, the increment will increase 
to 26 W. During the single-leg cycling visit, the protocol will be the same apart 
from the power output of each stage being half that of double-leg cycling (i.e. 8 
W increment for stages 1-5, 13 W beyond stage 5). At baseline, between each 
stage, and at task-failure, an assessment of neuromuscular function will be 
performed, comprising isometric MVCs of the right knee extensor, and the 
assessment of % voluntary activation (VA) of the knee extensors using 
supramaximal femoral nerve stimulation during the MVCs, and contractile 
function through the assessment of the force evoked following supramaximal 
femoral nerve stimulation delivered at rest (potentiated twitch force, Qtw,pot). 
Exercise will continue until participants are unable to achieve the target power 
output, or voluntarily disengage from the task. Following exercise cessation, the 
same neuromuscular function assessment will be performed at 1, 3, 5, 10, and 
15 min post-exercise in order to determine the rate of recovery of 
neuromuscular function (30 min).  
 
 
 
 

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Part III: Qualitative Work Package 
While Parts I and II will further our understanding on the aetiology of exercise 
intolerance and the effect of nitrate supplementation in improving exercise tolerance 
in individuals with CHF, it is important to clarify the impact of exercise intolerance on 
the lives of these individuals. As such, the aim of Part III is to understand the symptoms 
associated with performing exercise and the impact of exercise intolerance on social, 
functional and emotional functioning on individuals with CHF.   
One optional semi-structured interviews will be conducted with the participants who 
provide consent to participate in this part of the study (n = 20 or until saturation in 
findings) once they have completed the Parts I and II. Participants may choose to opt 
out from this part of the study and this option will be included in the consent form. An 
interview specific topic guide has been developed on symptoms associated with 
performing physical activity and on the impact of exercise intolerance on quality of life. 
Data from the semi-structured interviews will be analysed thematically using an 
inductive approach [13]. All interviews will be transcribed verbatim. The interviews will 
be conducted remotely and participants will be given the option to use the zoom 
platform or receive a telephone call. If the participant becomes distressed or upset 
from completing the interview then we have requested they contact the research study 
Figure 1. Protocol schematic displaying incremental cycling to exhaustion with stages 
interspersed with neuromuscular assessments, and post-exercise recovery 
measurements. DL, double leg; SL, single leg; MVC, maximal voluntary contraction. 

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team who will initiate referral to our clinical psychology department at the Freeman 
Hospital or Royal Victoria Infirmary, Newcastle upon Tyne. 
 
End of the Study 
At the end of the study all participants will be provided with information about the 
study’s major findings. This will be communicated by letter mailed to the participants’ 
home address and will also include an invitation to attend a volunteer feedback 
evening at the Clinical Research Facility where the overall results of the study will be 
presented. The study will be considered as completed when participants have 
completed the proposed clinical procedures. 
 
Data storage 
All digital data will be securely stored on the University’s Filestore Service and will be 
pseudonymised with study identification numbers. The data will be accessible only to 
authorised project staff and backed up daily with a highly available offsite mirror. The 
audio recordings for the qualitative work package will be destroyed once they have 
been transcribed. 
 
Data archive and sharing 
Research data that supports publications and unpublished data of value at project end 
will be archived with supporting documentation in data.ncl (https://data.ncl.ac.uk/), 
Newcastle’s Research Data Repository. The datasets will be made public under a 
Creative Commons licence to ensure credit is given when the data is reused and 
access provided for at least ten years. Data deposited will also be assigned a 
persistent identifier (i.e. DOI) that can be included in project outputs, including 
publications, to detail how and where the data can be accessed. At this stage all study 
identification numbers will be removed and all data will be anonymised before being 
archived and shared through the repository. Where there is a risk to data being re-
identified the dataset will be archived to make the record findable but access will be 
controlled and dependent on the future use of the data in question.   

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Sample size and statistical analysis 
The target sample size is based on a power calculation using the  expected effect 
moderate effect size for a positive effect of nitrate supplementation on exercise 
tolerance derived from previous research [22]. For an a of 0.05 and a power of 0.80, 
a total sample size of 24 is required. To account for potential drop-outs, an additional 
4 participants will be recruited, based on drop-out rate of ~20% in studies assessing 
the effect of nitrate supplementation on exercise tolerance [23, 24]. For Part I, the 
analysis will include a two-way repeated measures analysis of variance (ANOVA) to 
determine the effect of the magnitude of active muscle mass (small vs. large) on 
neuromuscular fatigability and other physiological variables measured during cycling. 
For Part II, the analysis will include a two-way repeated measures ANOVA to 
determine the effect of nitrate supplementation vs. placebo on neuromuscular 
fatigability and other physiological variables measured during cycling. For Part II, an 
inductive approach to analysis will be made and data saturation of themes will be 
determined at the analysis stage [25]. Two independent reviewers in the research 
study team will code and extract segments of the data to identify key themes. Inclusion 
of supporting quotes from each of the themes will be included in the write up and 
publication. 
 
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Swank, A.M., et al., Modest increase in peak VO2 is related to better clinical outcomes 
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McDonagh, T.A., et al., 2021 ESC Guidelines for the diagnosis and treatment of acute 
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Poole, D.C., et al., Muscle oxygen transport and utilization in heart failure: 
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5. 
Poole, D.C., et al., Exercise limitations in heart failure with reduced and preserved 
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Hirai, D.M., T.I. Musch, and D.C. Poole, Exercise training in chronic heart failure: 
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Haykowsky, M.J., et al., Determinants of exercise intolerance in patients with heart 
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Brownstein, C.G., G.Y. Millet, and K. Thomas, Neuromuscular responses to fatiguing 
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Riegel, B., et al., The Minnesota Living With Heart Failure Questionnaire: sensitivity 
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Godin, G. and R.J. Shephard, A simple method to assess exercise behavior in the 
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Ryan, T.E., et al., Assessment of in vivo skeletal muscle mitochondrial respiratory 
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Millet, G.Y., et al., Electrical stimulation for testing neuromuscular function: from 
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Weavil, J.C., et al., Heart failure with preserved ejection fraction diminishes peripheral 
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Barstow, T.J., Understanding near infrared spectroscopy and its application to skeletal 
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Perkins, R.E., et al., Relationship between bioreactance and magnetic resonance 
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Jones, T.W., et al., Bioreactance is a reliable method for estimating cardiac output at 
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Jakovljevic, D.G., Trenell, M.I., MacGowan, G.A., Bioimpedance and bioreactance 
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25. 
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