Heart Failure in Patients With Diabetes: Cells, Crosstalk and Consequences
Conditions: Diabetes Mellitus Type 2, Heart Failure, HFrEF - Heart Failure With Reduced Ejection Fraction, HFpEF - Heart Failure With Preserved Ejection Fraction
Sponsor: University of Leeds

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
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Heart failure in Patients with Diabetes: cells, crosstalk and consequences 
 
 
 
 
 
PROTOCOL VERSION NUMBER AND DATE Version 1.0 April 20th 2024 
 
RESEARCH REFERENCE NUMBERS 
 
IRAS number: 343489 
Funder’s code: BHF RE_23_130040-3 
Sponsor’s code: 2024-NCT37 
CPMS ID: 62251 
ClinTrials ID: NCTXXXXXXXX 
Edge ID: XXXXXXX 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
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SIGNATURE PAGE 
The undersigned confirm that the following protocol has been agreed and accepted and that the Chief 
Investigator agrees to conduct the study in compliance with the approved protocol and will adhere to the 
principles outlined in the Declaration of Helsinki, the Sponsor’s SOPs, and other regulatory requirement. 
 
I agree to ensure that the confidential information contained in this document will not be used for any other 
purpose other than the evaluation or conduct of the investigation without the prior written consent of the 
Sponsor. 
 
I also confirm that I will make the findings of the study publicly available through publication or other 
dissemination tools without any unnecessary delay and that an honest accurate and transparent account of the 
study will be given; and that any discrepancies from the study as planned in this protocol will be explained. 
 
 
 
 
Chief Investigator: 
Signature: .............................................................................. 
 
Date: 17/04/2024 
 
Name: (please print): 
Klaus Witte............................................................................. 
 
 
 
 
 
 
 
 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
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LIST of CONTENTS 
 
Page No. 
SECTION A 
 
LIST OF CONTENTS 
 
iii 
LIST OF ABBREVIATIONS 
v 
KEY STUDY CONTACTS 
vi 
STUDY SUMMARY 
vii 
FUNDING 
 
viii 
ROLE OF SPONSOR AND FUNDER 
viii 
ROLES & RESPONSIBILITIES OF STUDY STEERING GROUPS AND INDIVIDUALS 
 
 
viii 
KEY WORDS, CONTRIBUTORS AND STUDY FLOW CHART  
ix 
SECTION B: STUFY PROTOCOL 
1.   TITLE 
1 
2. 
ABSTRACT 
1 
3. 
BACKGROUND  
1 
4. 
RATIONALE 
 
2 
5. 
RESEARCH QUESTION/AIM(S)  
15 
6. 
STUDY SETTING AND DESIGN 
16 
7. 
PARTICIPANT RECRUITMENT AND ELIGIBILITY CRITERIA 
16 
8. 
STUDY CLINIAL PROCEDURES 
17 
9. 
CLINICAL TESTING PROCEDURES 
20 
10. STUDY TISSUE ANALYSIS METHODS 
21 
11. STUDY ENDPOINTS 
22 
12. STATISTICAL POWER AND ANALYSIS 
23 
13. LONG TERM PATIENT ORIENTATED OUTCOMES 
24 
14. RISK ASSESSMENT 
24 
15. PARTICIPANT COMPLIANCE AND SAFETY 
26 
16. ROLES AND RESPONSIBILITIES 
27 
17. DATA PROTECTION AND PATIENT CONFIDENTIALITY 
27 
18. WITHDRAWAL CRITERIA 
28 
19. STUDY CONDUCT (including safeguarding implications, ethical approval, 
regulatory review, amendments, protocol compliance, patients and public 
involvement, peer review, dissemination, monitoring and end of study criteria). 
28 
20. INDEMNITY 
30 
21. ACCESS TO THE FINAL DATASET 
30 
22. DISSEMINATION AND AUTHORSHIP 
30 
SECTION C 
 
APPENDICES  
31 
REFERENCES 
19 
 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
AR 
Adverse Reaction 
CI 
Chief Investigator 
CRF 
Case Report Form 
NHS R&D 
National Health Service Research & Development   
PI 
Principal Investigator 
PIS 
Participant Information Sheet  
REC 
Research Ethics Committee 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SSG 
Study Steering Group 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
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KEY STUDY CONTACTS 
Chief Investigator 
Dr Klaus Witte 
LIGHT building 
University of Leeds 
Woodhouse Lane 
Leeds 
LS2 9JT 
k.k.witte@leeds.ac.uk 
00447768254073 
00441133926642 
Study Co-ordinator 
As above 
Sponsor 
Head of Research Regulatory Compliance,  
The Secretariat,  
The University of Leeds,  
Woodhouse Lane,  
Leeds, LS2 9JT.  
Email: governance-ethics@leeds.ac.uk  
Telephone 0113 3437587 
Joint-sponsor(s)/co-sponsor(s)  
N/A 
Funder(s) 
British Heart Foundation 
Key Protocol Contributors 
Klaus Witte, John Gierula, Scott Bowen, Lee Roberts, 
Cedric Duval, Khalid Naseem, Mark Kearney. 
 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
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STUDY SUMMARY 
Study Title 
Heart failure in Patients with Diabetes: cells, crosstalk 
and consequences 
Internal ref. no. (or short title) 
IRAS 343489 
Study Design 
Observational cohort study 
Study Participants 
Six groups: people with and without heart failure with 
and without reduced ejection fraction, and with and 
without and type II diabetes mellitus. 
Planned Size of Sample (if applicable) 
600 
Follow up duration (if applicable) 
5 years of digital contact for vital outcomes 
Planned Study Period 
5 years of recruitment and 5 years for long term follow-
up 
Research Question/Aim(s) 
 
1. Conduct a systematic analysis of cardiac muscle, 
skeletal muscle, endothelial cells, and blood platelets 
from CHF patients to elucidate the alterations induced by 
T2DM; 
2. Investigate how T2DM influences intercellular 
communication to promote the progression of CHF; 
3. Establish correlations between abnormalities in these 
cells correspond to heart abnormalities, patient 
symptoms and clinical outcomes. 
 
 

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FUNDING AND SUPPORT IN KIND 
FUNDER(S) 
(Names and contact details of ALL organisations 
providing funding and/or support in kind for this study) 
FINANCIAL AND NON FINANCIALSUPPORT GIVEN 
British Heart Foundation  
£1,000,000 
 
ROLE OF STUDY SPONSOR AND FUNDER 
The University of Leeds will act as Sponsor for this study. As Sponsor, the University of Leeds assumes overall 
and final responsibility for the initiation and management of this study including study design, conduct, data 
analysis and interpretation, manuscript writing, and dissemination of results. 
 
The study will be funded through a grant from the British Heart Foundation (BHF). As funder the BHF will have 
no input on the conduct of the study, the analysis or write-up. 
 
ROLES AND RESPONSIBILITIES OF STUDY MANAGEMENT COMMITEES/GROUPS & INDIVIDUALS 
Study Steering Group 
The Study Steering Group will consist of the Clinical Co-PIs (Witte and Kearney), and the key collaborators 
(Professor Kalid Naseem, Dr Cedric Duval, Dr Scott Bowen and Professor Lee Roberts, and Dr John Gierula. 
The nurse assigned to this study (to be decided) will also form part of this group. The Study Steering Group will 
meet every three months and will coordinate recruitment and all study-related activities and will also be 
responsible for reviewing all adverse events and for declaring these within 24 hours to the Sponsor. 
 
Patient & Public Involvement Group 
A PPI Advisory Group consisting of two patients and one carer has been formed to aid study design, set-up, 
design and writing of the patient-facing documentation. In the preparatory phase, they have helped with patient-
facing documents and ethical approval application. During the recruitment phase, they will meet with the 
Steering Group every six months to review recruitment progress and participate in safety and adverse events 
monitoring. They will therefore be involved in the 6-monthly decision to continue the study or to cease 
recruitment.  

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PROTOCOL CONTRIBUTORS 
Dr Klaus Witte, Dr John Gierula, Dr Cedric Duval, Dr Scott Bowen, Prof. 
Lee Roberts, Prof M. Kearney, Prof. K. Naseem. 
 
KEY WORDS  
Chronic heart failure, type II diabetes mellitus, organ crosstalk 
 
STUDY FLOW CHART 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
STRATEGY OF THE OVERALL PROJECT 

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SECTION B: STUDY PROTOCOL 
TITLE: Heart Failure in Patients with Diabetes Mellitus: cells, crosstalk and consequences.  
 
ABSTRACT: Heart failure is a leading cause of mortality and morbidity worldwide1,2 and is characterized by 
symptoms such as shortness of breath, peripheral oedema and impaired exercise capacity due to structural or 
functional heart disease. The condition is associated with impaired longevity, high levels of morbidity (poor 
quality of life) and enormous costs for the healthcare system. In the western world, approximately 1-2% of the 
population suffer from heart failure, increasing to over 10% of people over the age of 70. The prognosis of 
patients with severe heart failure is similar to that of patients with cancer.  
 
Diabetes mellitus is reaching pandemic proportions across the globe. A condition previously characterised 
simply as a relative or absolute deficiency of insulin and diagnosed by elevated sugar levels in the bloodstream, 
it is now much better understood as a syndrome of impaired glucose tolerance accompanied by a range of 
metabolic abnormalities that can be observed in all tissues of the body. This metabolic remodelling is 
accompanied by higher risks of chronic disease particularly cardiovascular disease including atherosclerosis but 
also, even in the absence of coronary artery disease, a considerable increase in the risk of heart failure.  
 
These two morbid and life-limiting illnesses commonly occur together, each worsening the other in a synergistic 
relationship that leads to greater symptoms, resistance to therapies and shorter longevity.  
 
Further detail on how these diseases interact is urgently required. However, in addition to understanding the 
clinical impacts on patients, in order to focus existing therapies and develop new options, we need to understand 
how diabetes mellitus and heart failure interact at a metabolic level, which cellular-based pathways are affected 
and how these lead to abnormal cellular function, and thereby to tissue and organ dysfunction. We also need to 
understand how communication between tissues contributes to the overall pathophysiology.  
 
The present investigation will therefore be a single-centre non-randomised, observational study involving an 
unselected but highly phenotyped cohort of patients with and without heart failure and with and without type 2 
diabetes mellitus and controls without disease. Patients will be recruited from heart failure and diabetes clinics 
and controls will be identified from these patients’ carers and their relatives through direct contact and through 
advertisements in outpatient departments.  
 
Participants will undergo cardiac ultrasound, exercise testing, assessments of lung function, non-invasive 
haemodynamics, autonomic function, hand and leg muscle strength, and lung function assessment along with 
blood testing. At additional optional visits, unselected volunteers will be given the opportunity to participate in 
optional substudies where they will undergo steady state exercise with simultaneous heart ultrasound, 
assessments of autonomic function, longer term glucose monitoring, sampling of a small amount of fat and 
muscle, and MRI scanning of heart and thigh muscles. Participants’ direct involvement will end at that point 
although we will continue to monitor them using their digital records on an annual basis for up to 5 years to gain 
information on the prognostic value of the metabolic and haemodynamic testing. Tissue and blood samples will 
be coded and stored in a Human Tissue Authority-approved freezer until analysis following which they will be 
related to clinical variables with the aim of identifying mechanisms by which metabolic disease (in this case 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
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diabetes mellitus) influences the progression of heart failure with the long term objective of informing the 
development of more effective treatment strategies. 
 
The present investigation will allow us to advance our understanding of how metabolic disease and heart failure 
interact with the goal of developing targeted interventions that could open new treatment avenues.  
 

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BACKGROUND AND RATIONALE: Heart failure is a disease of heterogeneous aetiology, which is 
characterized by typical symptoms such as shortness of breath, peripheral oedema and inability to exercise due 
to structural or functional heart disease.3 Chronic heart failure (HF) results in high mortality and morbidity in 
patients as well as enormous costs for the healthcare system. Furthermore, the prognosis of patients with severe 
heart failure is similar to that of patients with cancer.4 In the Western world, around 1-2% of the population 
suffers from heart failure, while the figure rises to over 10% in people more than 70 years of age although recent 
figures from western Europe are projecting rates of more than 15%. 
 
Heart failure is classified into two main groups based upon the presence or absence of impaired left ventricular 
function. People with heart failure have reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF). 
Over the past decades, significant advances have been made in managing HFrEF through the development of 
evidence-based pharmacotherapies, implantable devices, and innovative care models. Until recently the medical 
management of HFpEF was limited to symptom control using diuretics and rate control of atrial fibrillation.  
 
Modern treatment of HF with reduced ejection fraction (HFrEF): Medical management of HFrEF has 
expanded to include four classes of agents proven to prolong life and reduce hospitalizations. Guidelines do not 
advocate any specific drug alone, but rather that optimal therapy for HFrEF should include all four medications.5 
When these agents are titrated to their optimal doses in a coordinated program, these agents together can 
achieve a considerable gain in life expectancy.6 However, the reality from observational studies is that whilst a 
dose response is apparent in patients with co-morbidities,7 the prognosis remains poorer for these patients, 
especially those with diabetes mellitus than those without co-morbidities in whom an almost normal life 
expectancy is possible.8 
 
Is the prognosis of heart failure really improving? The challenge facing people with heart failure and their 
families, carers and healthcare teams, is that although patient-orientated outcome 
rates in trials seem to be improving, this is not universally reflected in data from 
registries. Whilst recently published data from Denmark suggests that the 
mortality from HF is improving in younger patients, this was not seen in older 
people (figure 1).9 Recent data from the USA go further, suggesting that gains in 
prognosis made 20 years ago have reversed (figure 2). It is likely, that these data 
are partially due to the increasing proportion of those with HF who have HFpEF.10 
These people are generally older, have more co-morbdities, and have a limited 
response to standard medical therapies.   
 
The challenge of HF with preserved ejection fraction (HFpEF): HF has 
traditionally been viewed as a failure of LV systolic function, with reduced 
LV ejection fraction (EF) used to define systolic dysfunction, assess 
prognosis, and select patients for therapeutic interventions.11 During the 
last two decades a syndrome that includes symptoms of breathlessness 
and congestion but in the presence of ‘normal’ ejection fraction has been 
increasingly recognised and it is now well established that HF can occur in 
the presence of LVEF in the normal range. As discussed above HFpEF accounts for a substantial and 
Figure 1: The challenge facing heart failure patients, 
carers and healthcare teams. Reproduced from 9. 
Figure 2: The reversal of gains in prognosis in HF in the 
USA. Reproduced from 10. 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
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increasing proportion of clinical cases of HF.12,13 These individuals have similar symptoms and impairments in 
quality of life as those with heart failure with HFrEF.14  
 
Moreover, the clear understanding that we have gained about the pathophysiology of HFrEF has contributed to 
the development of therapies that extend longevity, and clinical trials of drugs targeting activation of the renin 
angiotensin aldosterone (RAAS) and sympathetic nervous system (SNS) have been shown to reduce the risk of 
death and hospitalisation due to progressive HF in patients with HFrEF. These agents have not however, shown 
such favourable results in patients with HFpEF.15 where (with the possible exception of sodium-glucose co-
transporter 2 inhibitors,16,17) the benefits of the agents are attenuated for those with higher LVEF, 18,19 and for the 
overall population with HFpEF no therapies have been shown to improve survival. 20,21 This suggests that 
mechanisms leading to the development and progression in patients with HFpEF may be different from patients 
with HFrEF. Fundamentally, perhaps HFpEF is either a condition involving different pathophysiological drivers, 
or our diagnostic criteria are incomplete and not sufficiently specific.  
 
The impact of metabolic disease: The development and persistence of both phenotypes of heart failure is 
increasingly appreciated to be contributed to by abnormal metabolism, not only of the cardiomyocytes but also of 
multiple tissues throughout the body where the heart failure syndrome and metabolic disease independently lead 
to a variety of structural and functional changes in muscle, fat, and vascular tissue by way of a series of as yet 
imprecisely identified cross-talk mechanisms. This might explain the adverse relationship between type II 
diabetes mellitus (T2DM) and heart failure, where in addition to being contributory to the development and 
persistence of HF,22 such that 25-40% if people with HF also have T2DM, the presence of T2DM as a co-
morbidity worsens considerably the prognosis of HF.23,24 
 
Relevance of diabetes mellitus: The pathophysiological causes of how T2DM contributes to the development 
and poorer prognosis of heart failure are not yet sufficiently understood. However, it is well known that metabolic 
abnormalities have functional and structural effects on the heart and peripheral muscles,25 which appear to be at 
least partially reversible.26 
 
The problem of persistent symptoms in HF: The significant advances in the treatment of heart failure in 
recent years have focused primarily on prognosis and long-term survival. The approval of new treatments is 
driven almost exclusively by the impact on long-term survival. Symptom-oriented approaches, on the other hand, 
have received much less attention over the last 30 years, despite the fact that most patients remain symptomatic 
despite optimal therapy, and that this persistence of symptoms drives the impaired quality of life. In a cohort of 
461 patients seen in a heart failure clinic, after one year of optimized treatment, we found that only a minority 
had improved symptoms.27 The majority remained symptomatic with ongoing daily limitations, and this was 
particularly the case with people with diabetes mellitus as a co-morbidity.  
 
Skeletal muscles in HF: Exercise intolerance is the major symptom in HFrEF, but only part of this can be 
explained by cardiac (central) dysfunction. 28  Peripheral skeletal muscle pathology is considered a key 
therapeutic target in HFrEF, as it directly exacerbates symptoms and independently predicts survival.29 Muscle 
pathology in HFrEF is characterised by fiber atrophy and weakness alongside early fatigue, consequent to 
impaired energy metabolism, and abnormal fiber type shifts (Type I to II).28 Underpinning this muscle pathology 

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are a variety of mechanisms including elevated protein degradation (e.g. via MuRF1), pro-inflammatory 
cytokines (e.g. via IL6, TNFα), reactive oxygen species, and mitochondrial dysfunction. Treatments that slow, or 
even reverse, the progression of skeletal muscle pathology could offer an opportunity to improve clinical 
outcomes in HFrEF. However, to date, there remains no established pharmacological treatment for skeletal 
muscle pathology in HFrEF. Many of the adverse changes in skeletal muscles seen in HFrEF are also present in 
HFpEF,30 and include abnormal skeletal muscle blood flow, not expected in HFpEF,31 suggesting that the 
pathways by which these changes are induced, based upon data from HFrEF models are more complex than 
originally assumed.32 
 
The lungs and diaphragm in HF: It is unsurprising that the function of lungs and diaphragm are related to 
symptoms in people with HF even in the absence of chronic airways disease. We have previously described that 
both expiratory function and inspiratory function are abnormal in people with HFrEF33 and also that the airways 
are abnormal even during non-forced assessment with impulse osscillometry.34,35 HFpEF is associated with both 
skeletal muscle and diaphragm weakness 36  in the presence of catabolism (breakdown of structural and 
contractile proteins).37 The origins of this catabolism are unclear but could stem from skeletal muscle-cardiac 
and diaphragmatic cross-talk.38  
 
Skeletal muscle-cardiac communication in HF: The heart has the highest energy consumption in the body, 
which ensures continuous contraction. The energy supply required for this is covered by different substrates 
such as fatty acids, glucose (sugar), lactate (lactic acid), ketone bodies and amino acids. When healthy, the 
heart is nourished primarily by fatty acids which are used by the mitochondria (the powerhouse of the cell) to 
generate energy in the form of ATP). In heart failure, cardiac metabolism is perturbed and associated with 
impaired mitochondrial function. This is accompanied by increased glucose utilization, which supplies 
mitochondrial-independent energy (ATP) via glycolysis. Furthermore, it is already known that brown and white 
adipose tissue and muscle,39 regulate systemic metabolism through a metabolite-inter-organ signalling axis that 
implies cross-talk between organs, so abnormal skeletal muscle or lipid metabolism could influence cardiac 
metabolism both in health and disease.40,41 It is quite possible, based on our previous data, that these changes 
could have a negative impact on cardiac metabolism. 
 
Where we are: Our group are global leaders in the field of tissue pathology in heart failure and diabetes mellitus 
as evidenced by our publication record.41,42,43,44,28,45,46,47,48,49 These data have thrown light on the problem relating 
skeletal muscle, fat, the endothelium, the diaphragm and cardiac dysfunction in HFrEF. The explosion of HFpEF 
and the development of metabolic therapies such as the sodium-glucose co-transporter 2 inhibitors have led to 
an even greater need to understand the disease processes and how they affect cellular energetics and cross-
talk. Our previous work has been undertaken exclusively in patients undergoing pacemaker implantation and this 
limitation has been a consistent feature in the above publications. Amongst other goals, this project will provide 
validation and stepwise progress in a wide range of people with and without heart failure and importantly, will 
also provide unique data in a group of people without disease. 
 
RESEARCH QUESTION 
How does the combination of HF and DM lead to the adverse prognosis, poorer response to therapies 
and worse symptoms? 

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The present project aims to define the relationship between HF and DM in order to provide an answer to the 
above question and also begin the process of developing interventions that could help improve the treatment of 
these most vulnerable subgroups.  
 
There are therefore several overarching primary objectives:  
1. Conduct a systematic analysis of the function of skeletal muscle, endothelial cells, fat cells and blood 
platelets from HF patients to elucidate the alterations induced by T2DM. 
2. Investigate how T2DM influences intercellular communication between organs (including platelets and 
coagulation factors) to promote the progression of the syndrome of HF. 
3. Establish correlations between abnormalities in these cells and organ-level cardiac, diaphragm, renal, and 
skeletal muscle function, patient symptoms and clinical outcomes. 
 
Secondary objectives include: 
4. What metabolic pathways are abnormal in people with heart failure, diabetes mellitus and both? 
5. Can we identify using metabolomics, a substrate or compound that underlies the metabolic abnormalities?  
6. Does organ 'cross-talk' have a negative influence on the functions of skeletal muscle, fat and cardiac function? 
7. Is peripheral autonomic activation at higher heart rates reflected in higher activation of cardiac sympathetic 
tone in HFrEF? 
8. Do the laboratory data describing muscle and fat metabolomics relate to clinical variables, response to 
therapy and prognosis? 
9. Can we confirm the skeletal muscle findings from pectoralis biopsies in people having pacemakers in groups 
with and without heart failure not having pacemakers? 
 
STUDY DESIGN AND SETTING 
This will be a single-centre UK-based, unblinded, observational, mechanistic study carried out in 600 people with 
and without the two phenotypes of heart failure, with and without diabetes mellitus and without either.   
 
PARTICIPANT RECRUITMENT AND ELIGIBILITY CRITERIA 
Identification, approach and consent: Participants will be identified from the clinical service at Leeds Teaching 
Hospitals NHS Trust.  
Group A - heart failure: Patients will be approached at specific points during their pathway of care at time 
points designed to make participation as easy as possible. Those newly referred but with a previous diagnosis of 
HF will be approached by the clinical team during the triage process. They will receive a letter and information 
offering them direct entry to a research-orientated, one-stop clinic which will be followed-up by the clinical team 
with a telephone call to answer questions and confirm their interest. Only when they have agreed to have their 
information passed to the research team, will their details be forwarded in order to make the appointment for the 
baseline visit. The conduction of baseline tests during the routine clinical visit can avoid a second visit.  
Patients with signs and symptoms and a raised NT-pro-BNP but with a possible diagnosis of heart failure will be 
approached in the NHS clinic by the clinical team, once the diagnosis has been confirmed. They will be offered 
an information sheet, the opportunity to ask further questions and, if during a telephone call, they are interested 
a further appointment at the research clinic. 

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Patients attending a HF clinic with an existing diagnosis will also be approached at the time of their routine visit, 
offered an information sheet and the opportunity to ask questions. If they are interested, an appointment for the 
baseline research visit will be made. 
The source of Group A patients will be recorded to allow us to differentiate those with de novo HF from those 
with a previous diagnosis. This will also be facilitated by the documentation of the date of diagnosis. 
 
Group B - diabetes mellitus: Patients attending the diabetes service at Leeds Teaching Hospitals NHS Trust 
will be approached by the clinical team, offered an information sheet and given a week to digest this. Their 
details will be passed to the research team who will contact them after one week to confirm interest and organise 
the baseline visit to the research unit where written consent will be taken.  
 
Participation requirements for groups A and B will include the diagnoses (either HF or DM) AND fulfilling 
the inclusion and exclusion criteria below: 
 
Group C – control subjects: These will be sourced in two ways. The clinical teams will approach carers and 
relatives of people with heart failure and diabetes attending the clinics. We will also prepare posters to be 
displayed in outpatient departments making visitors aware of the study, offering the opportunity for self-referral. 
These posters will describe the study, the tests that would be done, and telephone numbers for direct contact. 
There will also be a QR code that will link to an online Form hosted by the University of Leeds to make contact 
easier. Potential participants will only be required to enter their names and telephone numbers to allow the 
research team to make contact. Upon self-referral, we will contact them, arrange to post out the information 
sheets, and offer the opportunity to ask questions. We will either make the appointment immediately or offer 
them another call at one week to answer further questions. 
 
Participation in Group C will require the subject to be older than 50 years, have neither HF nor DM, nor 
any long term chronically disabling condition as determined by the investigators (for example chronic 
airways disease or arthritis limiting day-to-day activity) AND fulfil the inclusion and exclusion criteria as 
follows:  
Inclusion criteria:  
Exclusion criteria: 
•  Age >18 years 
• Ability to provide written informed consent  
• Persons who are legally competent and mentally able to 
follow the instructions of the study staff  
• Anemia Hb <8 mg/dl  
• Patients with acute infectious diseases (e.g. pneumonia)  
• Patients with heart failure due to sepsis  
• People with acute myocardial ischemia, which is manifested, for example, 
by angina pectoris or ECG changes under stress  
• Patients with acute liver or kidney failure or severe COPD (FEV1<1.0)  
• Pregnant and breastfeeding women  
• People who are institutionalized on official or court orders  
• People who are dependent or employed by the sponsor or investigator  
• Taking study medication (of an investigational drug) 30 days before the 
start of the study  
 
Participants will not be paid for participation, although reasonable travel costs will be refunded according to 
guidelines laid out by INVOLVE for those attending for research purposes only. 
 
STUDY CLINICAL PROCEDURES:  

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Baseline visit:  
At the baseline visit the following tests will be done in the order in which they are listed here: 
1) Demographic variables including address postcode, medical history, co-morbidities, aetiology of heart failure if 
relevant, and year of diagnosis of HF (and/or DM), current medication, activity assessment, date of diagnosis of 
HF and HFA-PEFF50 score will be recorded:  
2) Two quality of life questionnaires (the Kansas 
City Cardiomyopathy Questionnaire and EQ5D-
5L) and one cognitive function assessment score.  
3) Frailty assessment: Frailty is an important risk 
factor for adverse outcomes in heart failure.[51] 
One component contributing to frailty is muscle 
strength. Hence, we will include an assessment of 
frailty known as the clinical frailty scale (CFS). 
This is not a questionnaire and requires no input from the patients. We will also however, include a questionnaire 
specific for sarcopenia to improve the identification muscle loss,52 and, using the baseline variables of body 
height, weight and serum albumin (from the blood tests) we will document the geriatric nutritional index tool 
which is related to prognosis in HF (GNRI = (14.89 × serum albumin [g/dL]) + (41.7 × weight [kg] / ideal body 
weight [kg]).53,54 We will also ask the patient to answer 5 questions that form the sarcopenia assessment (SARC-
F).55 
4) Blood tests for kidney function, glucose levels and HbA1c, platelet and clotting function and metabolic 
assessment. We will collect 50mls of venous blood through a single blood draw during the visit which will be 
divided into tubes for clinical and research purposes. The 40mls required for research purposes will be aliquoted 
and coded for participant anonymisation and transferred to the University of Leeds for analysis of platelet 
function, metabolomics and proteomics as described below. The blood sample will be collected through a small 
cannula through which, after blood sampling, a small wire will be passed through the cannula up the vein in 
order to collect some of the cells lining the inner surface of the vein.[56][57] This is painless and takes less than 
one minute. If the participant is to undergo an exercise test (see below), the cannula will be left in to allow us to 
take a small (2.5ml) blood sample at the immediate cessation of exercise.  
5) Handgrip strength: Lower handgrip strength is a marker of adverse outcome in HF,[58] and we 
have previously demonstrated a relationship to exercise capacity. As a non-invasive marker of 
skeletal muscle function we will ask each patient to perform maximal handgrip strength assessment 
using a standard handgrip dynamometer (Jamar). Maximal strength will be recorded as the best of 
three attempts on the non-dominant arm. Endurance will be assessed by the duration of the ability to 
hold a minimum of 80% power (best of three attempts). 
6) Bioimpedance to assess lean mass: Higher lean muscle mass and higher fat mass are associated with better 
outcomes in people with heart failure.[59] Body composition assessment may help define 
and clarify the obesity paradox in heart failure where, despite a higher rate of heart failure 
in obese people, worse rates of diabetes (an adverse factor in CHF) and lesser exercise 
tolerance, obesity is associated with better outcomes in cohort studies of people with 
prevalent heart failure. We will use a portable bioelectrical impedance device 
(ImpediMedSFB7) to assess the proportion of muscle and fat. 
Figure 3: The Heart Failure Association preserved ejection fraction score. 
Figure 4: The 
handgrip 
dynamometer 
Figure 5: Bioimpedance to 
assess body composition. 

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17 
7) Lung and diaphragmatic function assessment. All participants will undergo formal spirometry to measure 
inspiratory and expiratory capacity. We will also use impulse osscillometry to measure non-
forced airways resistance.34,35 
8) Echocardiography: This has been described previously and is routinely in use in our 
research projects.Error! Bookmark not defined.,Error! Bookmark not defined.,60 All 
participants will undergo complete echocardiography with two-dimensional grayscale and 
tissue Doppler images recorded in two- and four-chamber. The images are stored in the 
“Echopac” digital imaging system and analyzed offline (GE, Milwaukee, WI, USA). This analysis includes a 
calculation of LV end-diastolic and end-systolic volumes using the biplane disk method (modified Simpson 
method). The frame at the R wave is taken as end diastole and the frame with the smallest LV volume is taken 
as end systole. The LV end-systolic volume (ESV) index (ESVi) is calculated as ESV/body surface area (BSA) in 
each phase. Each measurement is averaged from three beats. BSA is calculated using the Mosteller equation. 
Our data to date have confirmed good reproducibility. This 
method 
of 
LV 
volume 
calculation is not only widely used and accepted,61,62 but 
the key variable is the 
ESV, which has a particularly high reproducibility.Error! 
Bookmark not defined.  
9) Non-invasive cardiac output measurement: This will be 
performed 
using 
the 
Finapres Nova hemodynamic monitor (Finapress Medical Systems, Enschede, Netherlands) during 
the pacemaker programming protocol described above. The device has been validated using 
invasive methods of hemodynamic assessment. It continuously measures stroke volume, cardiac output and 
peripheral resistance using finger plethysmography.  
10) An exercise ECG with metabolic gas analysis (cardiopulmonary exercise test). This is a common functional 
test established in the clinic to assess a patient's performance. The patient will walk continuously on a treadmill 
until exhaustion. The aim is to evaluate the patient's maximum performance. As part of the examination, blood 
pressure, pulse and oxygen saturation will be measured at three minute intervals. A stress ECG is usually 
carried out to assess prognosis, confirm diagnosis and measure response to therapy. It is a routine test in 
people with heart disease. Immediately upon ceasing exercise, a small blood sample (2.5ml) will be taken from 
the cannula used for the initial blood test. 
 
Clinical care: All data will be entered into a bespoke eForm on the Leeds Teaching Hospitals NHS trust 
electronic patient record system (PPM+) and will be available for all clinicians with access to view. Any abnormal 
findings will be discussed with the participant and passed to the participant’s general practitioner or a secondary 
care appointment will be made with the relevant team. This eForm will allow the input of a unique number that 
will link to the tissue and blood samples and allow the results of these to be linked with the clinical data. 
 
Substudies: 
We will offer participation in six optional substudies and one for patients with a pacemaker). We recognise that 
not all participants will wish to be involved with these but they will be included in the information sheet and 
consent forms as optional. 
10) Optional substudy 1 - thigh and pectoralis muscle biopsy: We have previously undertaken biopsy samples 
from many hundreds of patients (without and without heart failure) without complication. These have all been 
collected in people having a pacemaker. Doing this during a pacemaker procedure makes the sampling entirely 
Figure 7: The monitoring system 
(Finapress) measures cardiac 
output and peripheral resistance 
using finger cuffs. 
Figure 6: Impulse 
osccillomtery to assess 
airways resistance. 

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18 
pain free and easy to undertake. However, our hypotheses now need confirming in a true control group and also 
in a wider range of people with the two phenotypes of HF including those that do not need a pacemaker. 
We aim to sample tissue from pectoralis muscle and quadriceps muscle in around 25 people in each of the six 
groups. Previous studies that have assessed skeletal muscle function in HF patients have used muscle biopsies 
from the vastus lateralis muscle of the thigh. This particular muscle is more susceptible to the effects of training 
and detraining. The pectoralis area provides easy access to fat and, as a muscle not involved in locomotion, the 
pectoralis muscle gives a better estimation of muscle changes in people irrespective of their lifestyle (sedentary 
or not). We would like to be able to account for this in our laboratory-based assessments of muscle function.  
Pectoralis fat sampling: Under local anaesthetic and using aseptic technique we will make a 5mm incision under 
the collar bone on the non-dominant side. This reveals the subcutaneous adipose tissue beneath which the 
pectoralis muscle is easily visible. Three small samples of fat (<3mm in diameter) and three small samples of 
muscle (<3mm diameter) will be taken and immediately stored as outlined below 
in liquid nitrogen or other medium depending upon the testing to be done.47 The 
incision will be closed with skin adhesive and a dressing applied if required. 
Thigh muscle biopsy: The point of incision will be determined by asking the patient to tense their leg so that the 
correct muscle can be identified. This point will then be marked on them with a 
marker pen. In each case, the biopsy will be taken from the middle third of the 
outer part of the thigh. The area surrounding the incision point will be shaved 
with a disposable razor (if necessary) and covered with iodine. Lidocaine will be 
injected to anaesthetise the area where the biopsy is to be taken. We have 
previously used a Bergstrom needle to take samples. Since our early work, 
where we used the Bergstrom needle, a new technique (microbiopsy) which is 
more acceptable to patients has been proved to achieve adequate samples 
allowing metabolic activity to be assessed.63 This is done with a 16-guage needle (16-gauge Magnum® core 
disposable biopsy needles (1.65-mm diameter, Magnum® Needle, MN1610; Bard, Covington, GA, USA) and 
does not require an incision (Figure 8). The clinical tolerance in a study of people with COPD was excellent with 
pain scores of 0-1 and we expect the pain scores to be similar in people with (and without) heart failure. The 
total weight of 1–3 microbiopsy samples in these studies ranged from 11–20 mg. These tissue samples will be 
treated exactly the same as outlined above and below for the pectoralis major biopsy. Following the procedure, 
the biopsy wound will be dressed in several layers, including adhesive skin closure tape, a soft adhesive sterile 
dressing, a waterproof vapour permeable dressing and a pressure dressing comprising gauze pads and an 
elastic bandage.  
The absolute contra-indications to these biopsy procedures are: 
1) 
Participants with a known bleeding disorder,  
2) 
Participants still taking formal anticoagulation (warfarin or NOACS),  
The relative contra-indications to this procedure are: 
1) 
Participants who have demonstrated a tendency to keloid (hypertrophic) scarring,  
2) 
Participants on medications that affect the immune system (e.g. steroids),  
3) 
Participants on aspirin-like drugs (which are known to prolong bleeding),  
4) 
Post-biopsy management: Patients will receive advice on how to look after the dressings in the days 
following the procedure.  
Figure 8. The Magnum microbiopsy needle. 

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19 
11) Optional substudy 2 - Submaximal exercise on a stress echocardiography ergometer: 
We will invite participants for a second visit to allow us to assess how their heart adapts to 
exercise. Participants will be asked to exercise on a recumbent bicycle at a steady 
workload of 40-50% of that achieved during the baseline peak exercise test. During this 
time, we will perform an echocardiogram and the Finapress cardiac output assessment to 
collect information on contractility (aortic velocity time integral for cardiac output), diastolic 
function (mitral filling), LV volumes and peripheral vascular resistance64 at rest and moderate submaximal 
exercise. This could be particularly important for people with HFpEF who we propose might fall into two groups 
with and without impaired contractility on exercise.  
12) Optional substudy 3 - Sympathetic nervous system activity assessed by microneurography and heart rate 
variability. The autonomic nervous system is responsible for controlling heart rate, blood pressure and digestion 
and can be divided into two systems, the sympathetic (‘fight or flight’) system and the parasympathetic (‘rest and 
digest’) system. With advancing age and heart problems especially heart failure, the balance between the 
sympathetic and parasympathetic branches of the autonomic nervous system alters with increased sympathetic 
and decreased parasympathetic activity. This change is detrimental for heart function and contributes to 
decreased quality of life, breathlessness during exercise65 and shortened life.  
The sympathetic nervous system plays a large role in maintenance and progression of heart failure. Treatments 
that improve heart failure status whilst also lowering sympathetic activation are often associated with improved 
outcomes, but lowering sympathetic activity also improves heart failure status and outcomes – rather like a 
chicken and egg scenario that leads to a vicious cycle of deterioration. We want to explore whether heart rate 
increases lead to increased sympathetic tone, and whether heart rates above the critical heart rate augment this. 
This would give some mechanistic information about the adverse effects of higher heart rates on outcomes.  
In order to assess the sympathetic nervous system activation we will invite a subgroup of participants to undergo 
microneurography for Motor Nerve Sympathetic Activity (MSNA) assessment. 
The Leeds Cardiovascular Clinical Research Facility and the PI independently have experience of this 
technique,65,66,67,68,69,70 but briefly, microneurography uses small electrodes with a tip diameter of 1-3µm (less 
than the diameter of a hair), meaning that there is no need for local anaesthetic. The electrode is inserted into a 
nerve just under the skin of the leg. As the electrode is inserted, participants may report a brief sensation of ‘pins 
and needles’ or itching along the course of the nerve when the electrode is inserted, but pain is rare. Fewer than 
10% of subjects who underwent microneurography experienced symptoms such as muscle weakness or a 
sensation of numbness, but these subsided within hours and a maximum of two weeks.71  
To prevent infection, all electrodes used in microneurography experiments will be sterilised before insertion and 
used only once. If skin lesions or infections are present on the area to be recorded from, microneurography will 
not be performed. Recordings will be made for 15 minutes. We plan to invite 100 patients to undergo 
microneurography at baseline (25 from each group) aiming for 20 complete datasets from 
each group. 
These participants will also be invited to carry a heart rhythm monitor for 24 hours to 
document the natural rise and fall of their heart rate (heart rate variability) over 24 hours. This is a routine test in 
cardiac patients.  
13) Optional substudy 4 – Force Frequency Relationship assessment (for participants with pacemakers) force 
frequency relationship (FFR) 
Figure 9. The recumbent 
echocardiography bench to 
perform exercise 
echocardiography. 

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The echocardiographic approach will be the same as for the baseline resting echocardiography as outlined 
above. In those people with pacemakers we will undertake echocardiography at three or four pacemaker-
induced heart rates in order to subsequently calculate the FFR. This study requires no patient effort. 
PACING PROTOCOL: Echocardiographic images are acquired at rest with intrinsic atrial rhythm or a baseline rate 
of 40 beats per minute, after which atrial pacing is initiated in DDD mode (or VVI in patients with atrial fibrillation 
(AF)) for CRT patients. For subjects without CRT, we will program AAI mode (or DDD with long AV delays to 
avoid RV pacing). We do not exclude patients with single-chamber VVI pacemakers to avoid the deleterious 
effects of RV pacing. Basic heart rate programming will be the next highest multiple of ten over the intrinsic heart 
rate. After four minutes, another series of images is recorded and then the stimulation frequency is increased at 
stepwise intervals of 15 bts/min, with images recorded every four minutes. This is repeated until the maximum 
predicted heart rate set by Åstrand (200 years old) is reached or the patient develops discomfort (angina or 
palpitations). This point is considered peak and stimulation is reset to basal settings. For safety reasons, the 
subjects are asked to stay in the clinic for another 30 minutes. 
BLOOD PRESSURE MEASUREMENT: Calculating the end-systolic pressure-volume ratio requires measuring the LV 
pressure at end-systole . Systolic blood pressure (SBP) is used as a surrogate. Blood pressure is measured 
according to Riva Rocci.72 
FORCE-FREQUENCY CALCULATION: Dividing systolic pressure by LVESVi (SP/LVESVi) yields a surrogate of 
contractility73,74,75,76,77 which has been validated against invasive methods.73,77 We will plot heart rate versus 
contractility for each patient and at those with a parabolic curve define the 'critical HR' as the HR where the 
relationship between SBP/LVESVi reaches its maximum value. For those where multiple increases in HR induce 
a decrease in SBP/LVESVi, we will define the resting or baseline HR as “critical HR”.78 
14) Optional study 5: Cardiac Magnetic Resonance for cardiac contractility and ischaemia assessment 
An MRI scan is standard of care for heart failure patients to identify the severity of the left ventricular dysfunction, 
the involvement of the right heart, the presence of myocardial scar and ischaemia. We have previously used it to 
describe contractility at higher heart rates.  
We will invite all participants in each of the 6 groups to have a cardiac MRI scan unless they have had one of 
sufficient quality in the preceding 6 months in which case we will ask them if we can use their MRI data for 
research purposes. 
An MRI scan does not involve ionising radiation. Exclusion criteria for a clinical MRI scan at Leeds Teaching 
Hospitals NHS Trust will be followed. If participants have a pacemaker, standard operating protocols will be 
followed according to the MRI unit at Leeds Teaching Hospitals NHS Trust to ensure that the device is MRI-
conditional and programmed accordingly.  
15) Optional study 6: Continuous ambulatory blood glucose measurement 
Continuous blood glucose monitoring (CGM) is increasingly routine in people with type 1 diabetes mellitus. As 
described above, it is our hypothesis that impaired glucose tolerance in people with heart failure is a driver of 
adverse outcomes. However, it is also possible that even in people with heart failure but without diabetes 
mellitus, impaired glucose tolerance and metabolic changes associated with this contribute to the progression of 
the condition. We therefore plan to add a period of two weeks of CGM to around 30 people in each of the six 
groups. The diabetes team in Leeds Teaching Hospitals Trust use the Libre Freestyle device routinely and we 
plan to use this device in this study. If participants are interested, this sensor can be applied at the baseline visit. 
The nurses in the diabetes centre and those in the CV-CRF will work together to provide the sensor and the 
limited education required. The data are stored in the senor up to every minute and can be downloaded to either 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
21 
a bespoke monitoring device or through a mobile phone application. No participant interaction is required. If the 
participant wishes not to connect their mobile phone to the sensor, they can simply send us the sensor and we 
can download the data. On the other hand, we can provide a reader that connects to the sensor or the 
participant can download the application. In either of these circumstances, we would provide the participant with 
an NHS email address to which they can connect their reader or their application so that we can safely view and 
download the information. After two weeks, we will telephone the participant and organise that the sensor and 
reader are posted to us (in the event of no planned further optional visits), or advise the participant that the 
sensor can be discarded (in the event that the participant has used the mobile telephone application and no 
further visits are planned). Should the participant have planned a further optional visit, we would organise al of 
this at that visit. Complications are rare and limited to cutaneous reactions.79,80 Since the device will only be used 
once for two weeks by each participant we do not expect to have any long term skin problems.  
 
Timing and combination of the substudies 
It is not expected that all participants would undertake all substudies although it is entirely possible to do these 
within four visits since some can be combined. The baseline visit is expected to last around 2 hours.  
Participants with a pacemaker could have the force frequency assessment at the baseline visit (substudy 4). 
Participants willing to have the continuous blood glucose assessment (optional substudy 6) could have this 
placed during the baseline visit. The biopsies are likely to require an additional visit of their own, but the steady-
state exercise test and the microneurography and Holter heart rhythm monitor (substudies 2 and 3) can all be 
accomplished during a single visit of 60 minutes. The MRI scan of the heart and thigh muscles (optional study 5) 
will require a further separate visit.   
 
STUDY TISSUE ANALYSIS METHODS 
Subcutaneous adipose tissue (SAT) analysis: SAT will be subdivided into 5 pieces, with one piece being 
placed into MACS tissue storage solution, one into 4% PFA (for histology), one into PBS (for studies of 
angiogenesis and vascular density), one to BIOPS for respiratory assessment, and a final piece flash frozen in 
liquid nitrogen and stored at -80ºC (for RNA, protein  and metabolite/lipid extraction and qPCR and 
transcriptomics, immunoblotting, metabolomics, lipidomics and proteomics).  
 
Skeletal muscle sampling: Muscle samples will be split into approximately four portions. One portion of muscle 
will be immediately frozen in liquid nitrogen and stored thereafter at -80ºC for further molecular analyses e.g. for 
assessing changes in protein content that regulate muscle growth and atrophy, muscle regeneration, and 
metabolite and lipid content whereas another portion will undergo microscopy imaging to assess muscle 
structure. A further portion of muscle will be put into a specialised buffer that will allow functional measures of 
mitochondrial respiration (for energetics) and contractility (forces) to be performed within fresh isolated fibres. A 
final portion of muscle will also be prepared in another buffer that will allow isolation of other cells such as 
satellite or endothelial cells, which will allow cell cross talk to be explored. 
 
Endothelial cells from the venous cannula: These will be separated into two batches, one of which will be 
immediately frozen for RNA analysis and the other placed in medium to allow culture.  
 

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22 
Blood sampling: The 50mls of blood will be processed, half will be spun to provide plasma and both whole 
blood and plasma will initially be stored at -80°C. To investigate cardiac and peripheral metabolism, 
“metabolomics/lipidomics” analyses will be carried out as a first step. The “metabolome” includes molecules up 
to 2kDa such as sugars, lipids, amino acids and their derivatives. For further “biomarker” identification, peptides 
or proteins will be analysed using non-targeted “peptidomics”/“proteomics” as well as specific ELISA analyses, 
and determination of circulating microRNAs, non-coding RNA sections, which play an important role play in gene 
regulation, as well as circulating immune cells using FACS analysis.  
One aliquot will be used to explore platelet and clotting function in the six participant groups.  
 
It is likely that of the samples taken, analysis will not use all of the tissue available. Any remaining tissue or blood 
(we estimate 5-10mls) will be stored appropriately at the University of Leeds in a HTA approved environment 
until the end of the study. If our work has provided new avenues for which we require the tissue to be stored for 
longer than the current funding (5 years), we will apply for an amendment to allow longer storage and additional 
testing. We have therefore asked participants to consent for 10 years of storage. 
 
STUDY ENDPOINTS:  
Study primary endpoint: Are there differences in skeletal muscle cellular metabolism assessed by mass 
spectrometry-based metabolomics and lipidomics between the two phenotypes of heart failure compared with 
controls and what influence does diabetes mellitus have? 
 
Secondary and exploratory objectives: 
1. 
How does T2DM influence intercellular communication to promote the progression of HF. 
2. 
Are there correlations between abnormalities in these cells and cardiac abnormalities, patient symptoms 
and clinical outcomes? 
3. 
What metabolic pathways are abnormal in people with heart failure, diabetes mellitus and both? 
4. 
Can we identify using metabolomics, a substrate or compound that underlies the metabolic abnormalities? 
5. 
Does organ 'cross-talk' have a negative influence on the functions of skeletal muscle, fat, platelets and 
clotting function, and cardiac function?  
6. 
Is autonomic activation reflected in metabolic abnormalities in HFrEF or HFpEF? 
7. 
Is lung function, especially that of the diaphragm and smooth muscle of the medium-sized bronchioles 
reflective of the metabolic abnormalities and how do these relate to the two major HF phenotypes? 
8. 
Do the laboratory data describing muscle and fat metabolomics relate to clinical variables and prognosis? 
9. 
Is cardiac contractility impairment as measured by cardiac magnetic resonance related to the tissue-based 
metabolic abnormalities and can we identify different subgroups of contractile function within the heart failure 
and diabetes groups.  
 
STATISTICAL ANALYSIS AND POWER CALCULATION:  
Participants will be divided into 6 groups. Patients with HF will be categorised using the European Society of 
Cardiology 2016 guidelines on the diagnosis of HFrEF or HFpEF.81 Participants with HFpEF will be classified 
according to the presence of primary or secondary HFpEF according to the Heart Failure Association 
classification.82 The documentation in participants with HFpEF will include whether the condition is secondary 
(restrictive cardiomyopathy, hypertrophic cardiomyopathy, constrictive pericarditis or valvular heart disease)  or 

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23 
primary with the presumed leading phenotype and contributors highlighted (by a tick box) according to the recent 
statement from the Heart Failure Association (age, sex, type 2 diabetes mellitus, obesity, sleep apnoea, arterial 
hypertension, arterial hypotension, pulmonary hypertension, chronic obstructive pulmonary disease, iron 
deficiency, coronary artery disease, atrial fibrillation, high heart rate, chronotropic incompetence, atrial functional 
mitral regurgitation, functional tricuspid regurgitation, cachexia and sarcopenia, very high ejection fraction 
(>65%/>70%), LVEF between 50% and 55%, HFpEF in patients with cancer). 
The presence of diabetes will be determined according to European guidelines as a documented history of one 
of a fasting glucose value of ≥126mg/dL (7.0mmol/L), a 2-hour plasma glucose after a 75g oral glucose 
tolerance test of ≥200mg/dL (11.1mmol/L), a HbA1c ≥ 6.5% (48mmol/L) or a random blood glucose level of 
≥200mg/dL (11.1mmol/L).  
Key primary analyses will be the comparison between the groups in terms of skeletal muscle cell metabolism. 
Laboratory data will first be analysed for its distribution to determine whether parametric or non-parametric 
testing should be done. Data with a wide distribution might also be log-transformed to facilitate analysis. The 
data will be mean centred and scaled (either auto-scaled or pareto scaled). We expect the metabolomic data to 
be normally distributed with similar means and medians. The data will then by analysed using multivariate 
techniques (principal component analysis, partial least squares – discriminant analysis) and where appropriate 
the robustness of the models will be validated (permutation testing) as we have performed previously and is best 
practice for metabolomic data analysis.83 
 
Power calculations for metabolomic analyses were made using pilot metabolomic analyses. These data were 
generated from plasma of age and sex-matched eligible patients with or without HFrEF undergoing routine 
pacemaker implantation (Leeds Teaching Hospitals NHS Trust). HFrEF patients had stable HFrEF >3 months 
with LVEF <50%. Controls had pacemaker-requiring bradycardia, without confounding conditions (e.g. CVD, 
diabetes, dyslipidaemia, exercise intolerance, CHF or LV dysfunction) (Ethical approval: Leeds West Research 
Ethics Committee 11/YH/0291; Leeds Teaching Hospitals R&D committee CD11/10015). From these data we 
used a key metabolite, β-aminoisobutyric acid (BAIBA) to calculate power. Plasma BAIBA concentration is lower 
in age, sex and BMI-matched CHF patients (Figure 10). Based on this data plasma BAIBA concentration in 
control patients is 1 +/- 0.5 (SD) (Figure 10). To detect a 0.59-fold change (Fig X) in BAIBA concentration with 
Beta=0.8 and alpha=0.05 requires n = 23 per group. We have based the power calculations for the muscle 
biopsy studies on this.  
Appropriate sample sizes for the histology were determined based on our pilot 
and/or published data. For human experiments, primary outcome measure will be 
a difference in muscle fibre size. To detect a change of 20% with 80% power at an 
alpha level of P<0.05, assuming a standard deviation of 28%, requires n=18 
patients. To account for a 10% potential attrition rate, a final sample size of a 
minimum of n=20 patients is required. 
 
LONG TERM PATIENT ORIENTATED OUTCOMES: 
Understanding the adverse profile of diabetes mellitus in HF could lead to optimizing treatments for patients with 
and without diabetes mellitus. This could include metabolism-based targeted therapy to achieve an improvement 
in metabolism and a personalisation of anti-platelet and anti-coagulation approaches for those with both 
*
*
Figure 10: LC-MS analysis of plasma from 
CHF and control patients. Plasma BAIBA 
concentration is lower in CHF (n = 10). *, p 
< 0.05 

Heart failure in Patients with Diabetes: cells, crosstalk and consequences: Witte et al. 343489 (20th April 2024: V1.0)  
24 
conditions. The present investigation will also allow us to expand our understanding of heart-muscle crosstalk 
with the aim of developing targeted interventions that could open up new avenues of treatment.  
 
RISK ASSESSMENT: 
For the patients with HF, this study is additional to standard medical care due to the tissue sampling, the 
autonomic assessments and the exercise echocardiogram. The other tests are commonly but less 
rigorously undertaken during the clinical assessment of heart failure. For the group with diabetes 
mellitus and no HF and the controls, the HF assessments throughout will be research. We will not use 
additional ionising radiation.  
 
Complications of the clinical procedures: The clinical tests (echocardiography, blood tests and exercise testing) 
are routine in clinical practice. Exercise testing can leave patients feel a little tired the following day. The 
questionnaires ask no intrusive questions. and are all validated and in routine use in clinical settings. Any 
questions that the participants feel uncomfortable with can be left blank.  
For the convenience of the participants, the blood tests would occur through a small cannula inserted into a vein. 
This allows sampling at baseline and a small additional sample after the end of the exercise test. Prior to 
removing the cannula, a small guide wire can be passed up the cannula around 1cm into the vein. When this is 
withdrawn it takes some endothelial cells from the inner surface of the vein. This is painless but allows us to 
explore the function of these cells that form the 'teflon' lining of the veins which is often dysfunctional in people 
with diabetes and heart failure. This is a routine assessment in our research into the vascular changes in 
diabetes mellitus. The passing of this wire is painless and is not associated with any additional risk either of 
bruising or infection. 
 
Risks of the MSNA testing: 
This has been done for more than 20 years at Leeds Teaching Hospitals NHS Trust with no long term 
complications. Some participants can feel a little tingling over the lateral aspect of their calf in the first hours after 
the test, and very rarely this can last two weeks (but has never lasted longer).  
 
Risks of the pectoralis and thigh muscle biopsy: While this procedure is generally not harmful, some patients 
may experience some discomfort during the following aspects of the procedure: 
• 
During the injection of local anaesthetic; 
• 
During the extraction of the muscle tissue with the needle, some patients may experience a pushing and 
pulling sensation within the muscle although this is not painful.  
• 
Very occasionally if the needle catches some of the fascia covering the muscle a sharp pain may be 
experienced; this is very much like the discomfort of a “dead leg” felt when struck on the leg at sport. It 
eases after a short period of time and does not interfere with subsequent exercise. 
Following the biopsy, patients will not generally experience much discomfort as the area around the biopsy site 
will have been numbed by the local anaesthetic. Once the local anaesthetic wears off, they may find that the 
muscle is sore or bruised. After about four hours the muscle can feel rather stiff. However, this will only persist 
for about 24 hours and then ease off. We would advise against engaging in any contact sports, swimming, or 
explosive/high-intensity exercises for at least four to five days after the procedure.  
The microbiopsy procedure is very low risk with side effects identified as: 

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25 
• 
Bleeding from the skin wound. 
• 
Bruising (which can take several days to appear). 
• 
Soreness lasting several days. 
Previous research groups using the Bergstrom needle (which we do not plan to use) have reported some of the 
following which I have listed here simply to point out that firstly the microbiopsy technique is far safer than the 
technique that we used previously (albeit we had no complications in 25 patients) and also that it is conceivable 
although unlikely that these events could occur: 
• 
Bleeding from the skin wound. 
• 
Bruising (which can take several days to appear). 
• 
Soreness lasting several days. 
• 
Bleeding within the muscle—can present either as swelling within the muscle or extensive bruising.  
• 
Altered sensation or numbness of an area of skin adjacent to the biopsy scar.  This is caused by injury to 
a small sensory nerve branch and after a few months awareness of this disappears. 
• 
Denervation of a part of the muscle distal to the biopsy.  This is caused by damage to a motor nerve 
branch and will result in atrophy of the affected segment of muscle.  This will not be initially obvious but 
will only become apparent after several months.  Although this may be cosmetically visible this 
complication has never been demonstrated as having any effect on muscle function or performance. 
• 
Superficial wound infection—this is extremely rare and is unlikely to require any treatment. The following 
are signs/symptoms of infection: 
o 
Malaise - ususual feelings of triedness/lethagry  
o 
Fever - feeling atypically warm and/or cold 
o 
Swelling/hardness of the infected area 
o 
Pain, tenderness and redness around the infected area 
o 
Drainage/pus eminating from the infected site 
• 
Deep wound infection—there are no reported cases of this complication with needle muscle biopsies but 
it can occur in open muscle biopsy in immuno-compromised patients. 
These complications have not been reported with the microbiopsy technique, but we will monitor these 
very closely and provide the ethics committee with an annual update of complications.  
 
Risks of the CMR scan 
Participants can feel claustrophobic in which case the scan can be stopped. The stress part involves the use of 
an agent to make the heart go faster which can cause the sensation of palpitations and flushing. The contrast 
used to show blood supply to the heart muscle is routinely used clinically, is not iodine based and rarely causes 
a contrast reaction. The MRI unit has standard operating protocol for this situation.  
 
Risks of the long term glucose monitoring sensor 
These are very uncommon and usually involve a local skin reaction. This resolves within a few days. The sensor 
(patch) rarely falls off and participants will be taught what to do if this is the case: simply to post it as arranged or 
bring it to the research unit at their next visit.  
 
Abnormal results: 

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It is possible that in control participants, or participants without diabetes (or participants without known heart 
failure) we will find abnormal results. These will be dealt with along usual clinical pathways of care either through 
their primary care physician or directly to the secondary care service.  
 
PARTICIPANT COMPLIANCE AND SAFETY:  
Definitions 
Term 
Definition 
Adverse Event (AE) 
Any untoward medical occurrence in a participant involved in this research 
with a clinically relevant event not necessarily caused by or related to the 
research. 
Serious Adverse Event 
(SAE) 
A serious adverse event is any untoward medical occurrence that: 
• results in death 
• is life-threatening 
• requires inpatient hospitalisation or prolongation of existing hospitalisation 
• results in persistent or significant disability/incapacity 
• consists of a congenital anomaly or birth defect 
Other ‘important medical events’ may also be considered serious if they 
jeopardise the participant or require an intervention to prevent one of the 
above consequences. 
NOTE: The term "life-threatening" in the definition of "serious" refers to an 
event in which the participant was at risk of death at the time of the event; it 
does not refer to an event which hypothetically might have caused death if it 
were more severe. 
Related and Unexpected 
Serious Adverse Event 
(RUSAE) 
An SAE occurring to a research participant which, in the opinion of the 
Principle Investigator was: - ‘Related’ that is, it resulted from the 
administration of any of the research procedures, and - ‘Unexpected’ that is, 
the type of event is not listed in the protocol as an expected occurrence. 
 
All SAEs and RUSAEs will be reported to the Sponsor within one working day of the study team becoming aware of 
them using the governance-ethics@leeds.ac.uk email. All RUSAEs will also be reported by the CI to the REC within 
15 working days. 
 
ROLES AND RESPONSIBILITIES: 
Chief Investigator (CI) / delegate or independent clinical reviewer: 
1. Clinical oversight of the safety of patients participating in the study, including an ongoing review 
of the risk / benefit. 
2. Using medical judgement in assigning the SAEs seriousness, causality and whether the event 
was anticipated (in line with the Reference Safety Information) where it has not been possible to 
obtain local medical assessment. 
3. Using medical judgement in assigning whether and event/reaction was anticipated. 
4. Review of specific SAEs and RUSAEs in accordance with the trial risk assessment and protocol. 
Sponsor: 
1. Central data collection and verification of AEs, SAEs, RUSAEs according to the trial protocol onto 
a database.  
Study Steering Group (SSG):  

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In accordance with the Trial Terms of Reference for the SSG, reviewing recruitment and safety data every 
three months. 
 
Reporting death 
For most studies, we would report all deaths, including deaths deemed unrelated to the study, if they occur 
earlier than expected to the sponsor. However, given the population involved, including the fact that one of our 
aims is to assess the prognostic value of the tests being undertaken, it is likely that over 5 years, around 30-40% 
of patients (and around 10% of controls) will die. Furthermore, the observational nature of the study and the very 
benign nature of the testing being undertaken, we propose to report only deaths deemed by the SSG to be 
due to the study. 
 
Pregnancy 
This will lead to immediate withdrawal from the study. 
 
DATA MANAGEMENT AND PATIENT CONFIDENTIALITY: 
All investigators and study site staff will comply with the requirements of the Data Protection Act 2018 with 
regards to the collection, storage, processing and disclosure of personal information and will uphold the Act’s 
core principles.  
 
Personal information will be collected through direct patient contact during the study visits and through the 
electronic health records systems of the participating NHS organisation.  
Data will be maintained on NHS computers and will only leave this secure site when collated into a single file 
anonymised (deidentified). This can be done automatically using the Leeds Teaching Hospitals PPM+ system. 
  
Prior to a paper copy of the consent form being given to the participant, it will be scanned and stored in the 
electronic CRF and the patients electronic health record.  
 
Only the CI, members of the Steering Group, the Sponsor and direct study team members will have access to 
the password protected folder in the NHS system containing the identifiable dataset. 
 
A unique identifier added at the time of the blood tests and tissue sampling into the PPM+ electronic patient 
record system will allow the samples to be pseudonomyised prior to transfer to the University of Leeds in order 
to maintain the connection between clinical information and the blood/tissue analysis.  
 
The Leeds Teaching Hospitals PPM+ system will hold all the records of all patients and participants. Since all 
data will be collected as part of that system, the clinical testing results will be stored according to NHS criteria. 
Any electronic dataset produced by collation of these data (for example from the excel product of the eForm will 
be stored in its usable form for 5 years after the end of the study procedures such that it is possible that some 
participants’ data will be available in that form for up to 10 years. 
 
The data from the continuous blood glucose monitoring system and the heart rhythm monitors in the research 
participants (including the controls) will be handled in the same way that the clinical data are currently handled. 

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They can be downloaded into the patient management system of Leeds Teaching Hospitals NHS Trust and will 
be available to clinicians and researchers.   
 
Dr Witte (the CI) will be the data custodian. 
 
Archiving will be carried out along SOP for the Clinical Research Facility at Leeds General Infirmary and data will 
be stored for 5 years following the end of the follow-up phase. 
 
WITHDRAWAL CRITERIA: 
Participants will be free to withdraw from the study at any time. Anonymised baseline samples and data collected 
will be kept and analysed as planned. Withdrawal from the study will be communicated to the participant’s usual 
care provider. The reasons for withdrawal will be recorded on the eCRF. We have accounted for a 15% drop out 
rate in the sample size. Patients withdrawing from the study will be followed through the Leeds electronic patient 
record system as though they had completed the study for up to 5 years after the date of their withdrawal. 
We do not expect the investigation to lead to a deterioration of the clinical condition. Hence we do not plan any 
early stopping criteria based upon clinical testing or risks. However, should recruitment prove challenging, each 
year we will review whether the study will reach its goals. It will be the responsibility of the SSG to decide to 
continue recruitment or to change the protocol to improve same. 
 
STUDY CONDUCT:  
Study conduct throughout will be according to the principals of the Declaration of Helsinki. All patients will 
provide written formal consent prior to enrolment. The study is registered on ClinTrials.gov (NCTXXXXXXX). 
 
Safeguarding implications: The usual processes for which the investigators and the clinical teams receive 
annual training will be activated should information with safeguarding implications come to light.  
 
Research Ethics Committee (REC) and other Regulatory review & reports: The study protocol and all patient 
facing material have been submitted for HRA approval. The study will only begin at each site when HRA approval 
and local R&I approval have been secured. All correspondence with the REC will be retained in the Study Master 
File.  
 
For NHS REC reviewed research: Substantial amendments that require review by NHS REC and HRA 
approval will not be implemented until those reviews and approval are in place and other mechanisms are in 
place to implement them at sites. All correspondence with the REC will be retained. The CI will notify the REC of 
the end of the study. If the study is terminated prematurely, the CI will notify the REC, including the reasons for 
the premature termination. Within one year after the end of the study, the Chief Investigator will submit a final 
report with the results, including any publications/abstracts, to the REC.  
 
Regulatory Review & Compliance: Before any site can enrol patients into the study, the Chief 
Investigator/Principal Investigator or designee will ensure that appropriate approvals from participating 
organisations are in place.  
 

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For any amendment to the study, the Chief Investigator or designee, in agreement with the sponsor will submit 
information to the appropriate body in order for them to issue approval for the amendment. The Chief 
Investigator or designee will work with sites (R&D departments at NHS sites as well as the study delivery team) 
so they can put the necessary arrangements in place to implement the amendment to confirm their support for 
the study as amended. 
 
Amendments: All amendments will be submitted by the CI following Steering Group approval to the Sponsor for 
initial review. It is the sponsor’s responsibility to decide whether an amendment is substantial or non-substantial for 
the purposes of submission to the REC. all amendments both, substantial and non substantial will be submitted to 
the HRA using the HRA amendment tool following sponsor review. All amendments will also be notified to the local 
R&D office of participating organisations.  
 
Peer review: This study has undergone several stages of peer review. Initial scientific peer review was carried out 
by the local research team and then by the funder as part of the decision to support the project.  
 
Patient & Public Involvement: The study has been reviewed in a two-stage process by members of the public 
and patients. An initial proposal was presented to the Leeds PPI group and subsequently, the final protocol was 
reviewed and discussed by self-selected members of the PPI group that formed the PPI-advisory group for this 
study. Patients and carers were involved in shaping the design, methodology of the research. The PPI-AG will be 
involved in writing up the results for lay members and the dissemination of the findings.  
 
Dissemination: A dissemination plan will be developed in collaboration with the PPI advisors, sponsor and funder. It 
is likely that this will include formal scientific publication in peer-reviewed speciality medical and translational science 
journals to coincide with a press release. We will also provide an annual information sheet for participants and a final 
summary document at the end of the study.  
 
Protocol compliance: The Study Steering Group (SSG) will monitor compliance with the protocol and record all 
instances of protocol deviations, non-compliances, or breaches. 
A serious breach in this setting will be defined as a breach of the protocol or of the conditions or principles of Good 
Clinical Practice (or equivalent standards) which is likely to affect to a significant degree the safety or physical or 
mental integrity of the trial subjects, or the scientific value of the research.  
All deviations and serious breaches of the protocol will be documented in the patient records and in the electronic 
CRF and immediately reported to the Chief Investigator and Sponsor (governance-ethics@leeds.ac.uk) within one 
working day of the team becoming aware of them. 
Reports of serious breaches will give details of when the breach occurred, the location, who was involved, the 
outcome and any information given to participants. An explanation will be given, and the CI will work with the 
sponsor to prepare this report which will also include what further action the sponsor plans to take. 
Serious or recurrent deviations will lead to a ceasing of study recruitment until an investigation has been completed 
to determine the cause and measures are in place to avoid repetition.  
 

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End of the study: The study will end once 600 participants have been recruited and the last patient has completed 
5 years of follow-up. However, the steering group together with the patient advisory group can decide to terminate 
the study early in the event of safety issues.  
 
Continuation of the study: We believe, based upon preliminary data that 200 people in each group will be 
sufficient to deliver clinically and statistically significant outcomes. However, a comprehensive assessment of this 
nature including the metabolic and clotting assessments has never been undertaken previously. Should we require 
greater numbers to achieve more accurate information (or for example, we find that fewer people than expected 
agree to the tissue sampling) we will approach the ethics committee (with agreement of the Leeds Ethics and 
Governance team) to increase the sample size for all or specific groups as required.  
 
INDEMNITY: 
The University, when acting as Sponsor, has insurance cover in force, which meets claims against it and where 
those claims arise from the Universities own negligence in its role and activities relating to the study (and which 
is subject to the terms, conditions and exceptions of the relevant policy). Clinical negligence indemnification will 
rest with the participating NHS Trust under standard NHS arrangements. 
 
ACCESS TO THE FINAL DATASET: 
The final dataset will initially be available to members of the Steering Group and subsequently an anonymised 
version will be available to other researchers including outside of the University of Leeds in response to a 
reasonable request and confirmation of appropriate data protection policies and a mutual data sharing 
agreement.  
 
DISSEMINIATION POLICY AND AUTHORSHIP POLICY: 
We will disseminate the results through publications in peer-reviewed journals, at conferences and we will also 
work with the PPIE group to produce a document aimed at participants. Authorship eligibility will be decided at 
the end of the study, but it is expected that it will include the Steering Group Members and other team members 
who have contributed to the data collection, analysis and write up according to the international guidelines. 
 

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APPENDICIES: 
 
Appendix 1 – Schedule of Clinical Procedures 
 
Baseline 
Optional 
visit 
Optional 
visit 
Optional 
visit 
Informed consent 
X 
 
 
 
Demographics 
X 
 
 
 
Medical history 
X 
 
 
 
Quality of life questionnaire 
(KCCQ) 
X 
 
 
 
Quality of life questionnaire 
(EQ5D-5L) 
X 
 
 
 
Cognitive function assessment 
X 
 
 
 
Clinical Frailty Score 
X 
 
 
 
Sarcopenia (SARC-F) 
questionnaire 
X 
 
 
 
Blood tests 
X 
 
 
 
Exercise test 
X 
 
 
 
Echocardiogram 
X 
 
 
 
Pacemaker check (where 
relevant) 
X 
 
 
 
Handgrip strength 
X 
 
 
 
Bioimpedance assessment 
X 
 
 
 
Lung function, diaphragm and 
airways resistance assessment 
X 
 
 
 
Non-invasive cardiac output 
assessment (Finapress) 
X 
 
 
 
FFR assessment (optional & 
for people with pacemakers) 
X (optional) 
 
 
 
Ambulatory continuous blood 
glucose assessment (optional) 
X (optional) 
 
 
 
Pectoralis fat and muscle 
biopsy (optional) 
 
X 
 
 
Thigh muscle biopsy (optional) 
 
X 
 
 
MSNA and 24 hour heart 
rhythm assessment (optional) 
 
 
X 
 
Echocardiogram during 
submaximal exercise (optional) 
 
 
X 
 
Cardiac (and thigh) MRI scan 
(optional) 
 
 
 
X 
 

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Appendix 2 – Tissue and blood sample processing and disposition 
Thigh muscle sampling:  
1) Confirm identification 
2) Confirm consent and review planned procedure with participant 
3) Ensure appropriate tissue transportation available (for muscle tissue: Eppendorf tube with liquid 
nitrogen, BIOPS and OCT solutions) 
4) Check subject’s unique identifier for the samples is prepared and tubes are labelled (also with ‘T’, 
‘P’ or ‘F’) 
5) Subject is to be made comfortable on the bed. 
6) Left knee to be supported by a pillow with a pad below it.  
7) Items needed:  
Dressing pack 
Fenestrated drape 
Iodine or similar sterilisation liquid 
10 mls lignocaine 1% 
Blue needle x 1 and green needle x 1 
Magnum device 
Skin adhesive 
Dressing and bandage 
11 blade scalpel 
8) Identify lower lateral aspect of the quadriceps muscle and mark area to be biopsied with pressure 
9) Sterilise the area using swabs in dressing pack 
10) Cover with fenestrated disposable drape  
11) Inject 5mls lignocaine to the hub of the blue needle and exchange for the green needle injecting a 
further 2-5mls as necessary 
12) Make a small nick in the skin with the scalpel if necessary 
13) Pass the Magnum microbiopsy needle as directed in the instructions for this device x 3 passes 
checking that sufficient muscle has been sampled each time. 
14) Place each muscle and fat sample into a cryo, biops, or OCT tube and label with identification 
number from PPM+ eForm 
15) Close the puncture site with skin adhesive if required 
16) Dress appropriately and wrap with bandage. 
 
 

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Pectoralis muscle and fat sampling 
1) Confirm identification 
2) Confirm consent and review planned procedure with participant 
3) Ensure appropriate tissue transportation available (for muscle, Eppendorf tubes containing liquid 
nitrogen, BIOPS and OCT, for adipose tissue, Eppendorf tubes containing 4% PFA, BIOPS, liquid 
nitrogen, MACS and PBS) 
4) Check subject’s unique identifier for the samples is prepared and tubes are labelled (also with ‘T’, 
‘P’ or ‘F’) 
5) Subject is to be made comfortable on the bed. 
6) Items needed:  
Dressing pack 
Fenestrated drape 
Iodine or similar sterilisation liquid 
10 mls lignocaine 1% 
Blue needle x 1 and green needle x 1 
Skin adhesive 
Dressing and bandage 
11 blade scalpel 
Small self-retaining retractor 
Small needle holder 
Forceps  
Small artery clamp x1 
7) Identify location to be incised and mark area to be biopsied with pressure 
8) Sterilise the area using swabs in dressing pack 
9) Cover with fenestrated disposable drape  
10) Inject 10mls lignocaine to the area injecting a further 10mls as necessary 
11) Make a small incision (5-6mm) deep enough to allow access to muscle and dilate as standard to 
visualise pectoralis muscle 
12) Sample fat (x 3) and muscle (x3) 
13) Place each muscle and fat sample into a cryo, biops, or OCT tube and label with identification 
number from PPM+ eForm 
14) Close the incision site with skin adhesive 
15) Dress the wound appropriately. 
 
Following the biopsy procedure(s), subjects are to be asked to rest for a minimum of one hour prior to 
mobilising. Simple pain relief to be offered (paracetamol).  
 
 
 
 
 

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Sample disposition 
• 
Fat tissue (aiming for four equal pieces) to be labelled with unique identifier and ‘F’: 
MACS tissue storage solution x 1 
4% PFA x 1  
PBS x 1 
BIOPS x 1 
Liquid nitrogen x 1  
 
• 
Muscle tissue (three equal samples of quadriceps and three equal samples of pectoralis) to be 
labelled with unique identifier and ‘P’ or ‘T’: 
PBS x 1 
BIOPS x 1 
Liquid nitrogen x 1  
 
• 
Blood sampling 
50mls of venous blood will be drawn according to quality standards using a single needle stick 
following aseptic techniques. The blood will be aliquoted following usual procedures for the handling of 
blood samples as follows:  
 
One ‘gold’ (routine biochemistry – renal function, liver function, albumin, haematinics, NT-pro-BNP, 
thyroid function, troponin), one ‘grey’ (for glucose) and two ‘lavendar’ (HbA1c, blood count) tubes will 
be transferred to the pathology department using the subject’s NHS number as ID. 
  
 

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Appendix 3 - Amendment History 
Amendment 
No. 
Protocol 
version no. 
Date issued 
Author(s) of 
changes 
Details of changes made 
 
 
 
 
 
 

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