Manganese-enhanced Magnetic Resonance Imaging in Takotsubo Cardiomyopathy
Phase: NA
Conditions: Takotsubo Cardiomyopathy
Sponsor: University of Edinburgh

TAKO MEMRI 
04/12/2025 V7.0 
IRAS Project ID 348948 
 
 
CR007-T02 v3.0 
1 of 15 
 
 
 
 
Non-CTIMP Study Protocol 
TAKO MEMRI 
Manganese-enhanced Magnetic Resonance Imaging  
in Takotsubo Cardiomyopathy 
 
Co-sponsors 
The University of Edinburgh and Lothian Health Board 
ACCORD 
Usher Building  
The University of Edinburgh  
5-7 Little France Road  
Edinburgh BioQuarter- Gate 3 
Edinburgh EH16 4UX 
Protocol authors 
Dr Jennifer Ramsay 
Prof Dave Newby 
Prof Marc Dweck 
Funder 
British Heart Foundation 
Funding Reference Number 
PG/24/11930 
Chief Investigator 
Prof Dave Newby 
Sponsor number 
AC24155 
REC Number 
25/NS/0005 
Project registration 
Clinicaltrials.gov 
Version Number and Date 
Version 7.  4th December 2025.  
 
 
 
 
 
 
 
 
 

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04/12/2025 V7.0 
IRAS ID 348948 
 
 
 
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CONTENTS 
 
1 
INTRODUCTION ........................................................................................... 7 
1.1 
BACKGROUND .................................................................................... 7 
1.2 
RATIONALE FOR STUDY ................................................................. 10 
2 
STUDY OBJECTIVES ................................................................................ 10 
2.1 
OBJECTIVES ..................................................................................... 10 
2.1.1 Primary Objective ............................................................................ 10 
2.1.2 Secondary Objectives ...................................................................... 10 
2.2 
ENDPOINTS ...................................................................................... 11 
2.2.1 Primary Endpoint ............................................................................. 11 
2.2.2 Secondary Endpoints ...................................................................... 11 
3 
STUDY DESIGN ......................................................................................... 11 
4 
STUDY POPULATION ................................................................................ 14 
4.1 
NUMBER OF PARTICIPANTS ........................................................... 14 
4.2 
INCLUSION CRITERIA ...................................................................... 14 
4.3 
EXCLUSION CRITERIA ..................................................................... 15 
4.4 
CO-ENROLMENT .............................................................................. 15 
5 
PARTICIPANT SELECTION AND ENROLMENT ...................................... 16 
5.1 
IDENTIFYING PARTICIPANTS ......................................................... 16 
5.2 
CONSENTING PARTICIPANTS ........................................................ 16 
5.3 
WITHDRAWAL OF STUDY PARTICIPANTS .................................... 16 
6 
INTERVENTIONS ....................................................................................... 17 
6.1 
STUDY MEDICATION ........................................................................ 17 
6.2 
CONCOMITANT AND POST-STUDY TREATMENT(S) .................... 18 
6.3 
TREATMENT COMPLIANCE ............................................................. 18 
6.4 
DISCONTINUATION OF STUDY MEDICATION ............................... 18 
6.5.      IMAGING CONTRAST MEDIA………………………………………….19 
7 
STUDY ASSESSMENTS ............................................................................ 20 
7.1 
STUDY ASSESSMENTS ................................................................... 20 
7.2 
STUDY VISITS ................................................................................... 22 
7.3 
STUDY SCREENING ......................................................................... 25 
7.4 
DEMOGRAPHIC DATA ...................................................................... 25 
7.5 
CLINICAL ASSESSMENT .................................................................. 25 
7.6 
BLOOD SAMPLES ............................................................................. 25 
7.7 
URINE PREGNANCY TEST .............................................................. 25 
7.8       PATIENT QUESTIONNAIRES…………………………………………..26 
7.9 
12-LEAD ECG .................................................................................... 26 
7.10  6-MINUTE WALK TEST ..................................................................... 26 
      7.11    ECHOCARDIOGRAPHY………………………………………………….26 
      7.12    CORONARY ANGIOGRAM AND LEFT VENTRICULOGRAPHY……26 

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      7.13    GADOLINIUM-ENHANCED CARDIAC MAGNETIC RESONANCE 
IMAGING………………………………………………………………………………..26 
      7.14.   MANGANESE-ENHANCED CARDIAC MAGNETIC RESONANCE 
IMAGING………………………………………………………………………………..27 
      7.15 STORAGE AND ANALYSIS OF SAMPLES………………………………27 
8 
DATA COLLECTION .................................................................................. 27 
8.1 
SOURCE DATA DOCUMENTATION ................................................. 28 
9 
DATA MANAGEMENT ............................................................................... 28 
9.1 
DATA STORAGE ............................................................................... 28 
9.2 
PERSONAL DATA ............................................................................. 29 
9.3 
DATA INFORMATION FLOW ............................................................ 29 
9.4 
EXTERNAL TRANSFER OF DATA  .................................................. 29 
9.5 
DATA CONTROLLER ........................................................................ 29 
      9.6      DATA BREACHES………………………………………………………   30 
10 
STATISTICS AND DATA ANALYSIS ........................................................ 30 
10.1 
SAMPLE SIZE CALCULATION .......................................................... 30 
10.2 
PROPOSED ANALYSES ................................................................... 30 
11 
ADVERSE EVENTS ................................................................................... 31 
      11.1.   DEFINITIONS……………………………………………………………...31 
      11.2    IDENTIFYING AEs and ARs……………………………………………..32 
      11.3.   RECORDING AEs and ARs……………………………………………...32  
      11.4.   ASSESSMENT OF AEs, SAEs, ARs, SARs and SUSARs………….  32 
      11.5.   REPORTING OF SARs/SUSARs………………………………………..33 
      11.6.   REPORTING REQUIREMENTS…………………………………………34 
      11.7.   FOLLOW-UP PROCEDURES……………………………………………34 
12 
OVERSIGHT ARRANGEMENTS ............................................................... 34 
12.1 
INSPECTION OF RECORDS ............................................................ 34 
      12.2 STUDY MONITORING AND AUDIT……………………………………….34 
13 
GOOD CLINICAL PRACTICE .................................................................... 35 
13.1 
ETHICAL CONDUCT ......................................................................... 35 
      13.2    INVESTIGATOR RESONSIBILITIES……………………………………35 
14 
STUDY CONDUCT RESPONSIBILITIES ................................................... 37 
14.1 
PROTOCOL AMENDMENTS ............................................................. 37 
14.2 
MANAGEMENT OF PROTOCOL NON COMPLIANCE ..................... 38 
14.3 
SERIOUS BREACH REQUIREMENTS ............................................. 38 
14.4 
STUDY RECORD RETENTION ......................................................... 38 
14.5 
END OF STUDY ................................................................................. 39 
      14.6     INSURANCE AND INDEMNITY………………………………………...39 
15      AUTHORSHIP POLICY………………………………………………………..39 
16.     REFERENCES………………………………………………………………….40 

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LIST OF ABBREVIATIONS 
 
ACCORD 
Academic and Clinical Central Office for Research & 
Development - Joint office for University of Edinburgh and 
NHS Lothian 
AE 
Adverse Event 
AR 
Adverse Reaction  
CI 
Clinical Investigator 
ECG 
Electrocardiogram  
HASTE 
Half Fourier Acquisition Single Shot Turbo Spin Echo 
ISF 
Investigator Site File 
MEMRI 
Manganese Enhanced Magnetic Resonance Imaging  
ML 
Millilitres  
MRI 
Magnetic Resonance Imaging  
MG 
Milligrams  
NHS 
National Health Service  
PROBE 
Prospective Randomised Open-label Blinded Endpoint 
PI 
Principal Investigator  
SAR 
Serious Adverse Reaction  
SOP 
Standard Operating Procedure 
SUSAR 
Suspected Unexpected Serious Adverse Reaction  
 

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INTRODUCTION 
1.1 
BACKGROUND 
 
1.1.1 Takotsubo Cardiomyopathy  
Takotsubo cardiomyopathy is an acute potentially fatal cardiac emergency 
that presents with sudden severe left ventricular dysfunction. It predominantly 
affects middle aged (50-74 years) women (90%) [Pattisapu et al, 2021] and is 
triggered by emotional or physical stress. Its symptoms are indistinguishable 
from acute myocardial infarction and 1 in 10 will die in hospital. However, 
patients with takotsubo cardiomyopathy have unobstructed coronary arteries, 
transient severe acute left ventricular dysfunction with “ballooning” of the 
ventricular cavity and absence of any myocardial infarction or scarring. 
Greater diagnostic recognition has led to a 4-5-fold rising incidence of 
takotsubo cardiomyopathy in both the United Kingdom and North America. 
With an incidence of 5,000 cases/year in the United Kingdom, it now 
represents 5% of all admissions for presumed myocardial infarction. [Singh et 
al, 2022]  
  
Patients who recover from an acute takotsubo episode have substantially 
reduced long-term survival [Templin et al, 2015; Tornvall et al, 2016; Uribarri 
et al, 2019; Redfors et al, 2021]. Our own 5-year data of 620 patients in the 
Scottish Takotsubo Registry [Rudd et al, 2024] demonstrated a major excess 
of all-cause mortality compared with a 1:4 age and sex-matched general 
population (25% versus 15%; hazard ratio 1.78, p<0.0001). Importantly, this 
reduced survival was principally driven by cardiovascular mortality. In addition 
to mortality, there is also a substantial burden of morbidity with many patients 
experiencing hospitalisation for a recurrent takotsubo episode, stroke or 
myocardial infarction. In the international multicentre InterTak Registry, 
patients with takotsubo cardiomyopathy experienced a rate of major adverse 
cardiac and cerebrovascular events of 9.9% per patient-year, and a rate of 
death of 5.6% per patient-year [Templin et al, 2015]. This is consistent with 
our own data which showed that patients with takotsubo cardiomyopathy have 
5-fold higher rates of major adverse cardiovascular events than the general 
population: hospitalisation for heart failure (9.8%), myocardial infarction 
(7.1%), stroke (3.9%) and recurrent takotsubo cardiomyopathy (10.2%).   
  
Despite left ventricular ejection fraction recovering to normal during 
convalescence, persistent sub-clinical cardiac dysfunction has been 
consistently demonstrated [Scally et al, 2018]. Some patients continue to have 
debilitating symptoms like those with heart failure (fatigue, breathlessness, 
chest pain) which reduce their quality of life and cause at least one further 
hospital admission each year [NHS England]. Many have multiple recurrent 
episodes of takotsubo cardiomyopathy. A Swedish study reports an average 
cost of €10,360 to the health system in the first 6 months after a takotsubo 
diagnosis, with patients using repeated hospital, outpatient and primary care 

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resources [Wallstrom et al, 2019]. This would equate to over £50 million per 
annum across the United Kingdom, but the total burden to the NHS will be  
much higher considering the ongoing elevated risk of events beyond 6 
months.  
   
1.1.2 Detection of subclinical myocardial dysfunction   
The observation that left ventricular dysfunction and reduced left ventricular 
ejection fraction rapidly recovers after an acute takotsubo episode (a 
characteristic feature of this condition) is often falsely reassuring for both the 
patient and the clinician. It also means that detection of residual left ventricular 
dysfunction despite apparent normal standard clinical assessments of 
contractile function can be challenging. We have previously demonstrated 
impaired myocardial cardiac energetic status using magnetic resonance 
spectroscopy [Scally et al, 2018]. We then went on to explore whether this 
perturbation in energetics influenced myocardial contractile performance 
through alterations in myocardial calcium handling.     
  
1.1.3 Manganese-enhanced magnetic resonance imaging  
Manganese is a naturally occurring essential trace element, required by the 
human body as a co-factor and enzyme activator [Frayn, 2010; Santamaria, 
2008]. Its role has been demonstrated in the normal metabolic functioning of 
multiple organs in addition to the protection of mitochondrial membranes from 
oxidative stress via superoxide dismutase [Santamaria, 2008]. Manganese 
exhibits certain advantageous properties that make it a potentially powerful 
tool in magnetic resonance imaging. First, its paramagnetic properties reduce 
the T1 relaxation times of water, providing positive contrast in tissues where it 
accumulates, and excellent delineation of anatomical structures [Massaad & 
Pautler, 2010; Pan et al, 2010]. Second, it is a calcium analogue and is taken 
up by voltage-gated calcium channels and sodium-calcium exchangers on 
active viable myocytes [Du et al, 2001]. This is an active process which allows 
manganese to differentiate regions of altered myocyte function and, in 
combination with T1 mapping protocols, can be used to quantify myocardial 
calcium handling. Manganese therefore presents attractive opportunities both 
in terms of the practicalities of imaging and the tracking of active biological 
processes.  
  
As part of a previous British Heart Foundation-funded Clinical Research 
Training Fellowship (Dr Nicholas Spath, FS/17/19/32641), we have shown 
that manganese-enhanced magnetic resonance is a better measure of 
myocardial infarction size than late gadolinium enhancement [Spath et al, 
2018] and, using T1 mapping, we have described altered manganese uptake 
in patients with dilated cardiomyopathy [Spath et al, 2020] (Figure 1).   
  
1.1.4  Magnetic resonance imaging in takotsubo cardiomyopathy  
Using magnetic resonance imaging, we have previously reported the 
presence of severe global oedema of both the left and right ventricles 

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associated with profound cardiac energetic impairment in the acute stages of 
takotsubo cardiomyopathy [Dawson et al, 2015; Scally et al, 2016]. As part of 
a multicentre study, we have demonstrated persistent abnormalities of cardiac 
inflammation and energetics after an acute episode of takotsubo 
cardiomyopathy using magnetic resonance spectroscopy [Scally et al, 2018; 
Scally et al, 2019]. This occurred despite a normalisation of the resting left 
ventricular ejection fraction and was associated with some persistent 
symptoms and cardiac limitation during cardio-pulmonary exercise testing 
[Scally et al, 2018; Scally et al, 2019].   
  
In a previous Medical Research Council Clinical Research Training Fellowship 
(Dr Trisha Singh, MR/T029153/1), we have demonstrated that there was a 
profound reduction in myocardial manganese uptake in patients with 
takotsubo cardiomyopathy, and this was most marked immediately after the 
acute episode [Singh et al, 2022b]. Whilst there was some recovery in the 
longer term, there remained a marked residual reduction in myocardial 
manganese uptake (Figure 1). This suggests that perturbations in myocardial 
calcium handling may underlie the pathophysiology of takotsubo 
cardiomyopathy. Moreover, given the absence of a simple measure of 
ongoing left ventricular dysfunction in patients with takotsubo cardiomyopathy, 
manganese-enhanced magnetic resonance imaging represents an excellent 
potential quantitative surrogate biomarker to determine candidate pathways 
that could then be taken forward for further assessment.  
 
Figure 1 
Manganese-enhanced magnetic resonance imaging and T1 maps in healthy 
volunteers (A) and patients with takotsubo cardiomyopathy during the acute phase 
(B) and at 6-12 months after recovery (C). Compared to healthy volunteers, 
myocardial manganese uptake (Ki) is markedly inhibited in patients with takotsubo 
cardiomyopathy, being nearly halved in the acute phase and remaining substantially 
reduced in the longer term (D). 

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4.3.3 Manganese-enhanced magnetic resonance imaging and role of 
sodium-glucose co-transporter  
We recently assessed the effect of sodium-glucose co-transporter 2 inhibition 
on myocardial manganese uptake in patients with heart failure. In this 
preliminary pilot double-blind randomised study, dapagliflozin but not placebo 
appeared to improve myocardial manganese uptake in these patients. We are 
currently extending this work, but together with the time-course studies in 
patients with takotsubo cardiomyopathy, these promising early data suggest 
that the sodium-glucose co-transporter 2 may have a role in regulating 
myocardial calcium handling. We now need to confirm this in cohorts of 
patients with takotsubo cardiomyopathy.  
 
1.2 
RATIONALE FOR STUDY 
 
We have recently demonstrated the proof-of-concept that manganese uptake 
is markedly impaired in patients with takotsubo cardiomyopathy, particularly in 
the acute phase but also in the longer term. This detection of covert left 
ventricular dysfunction is important because it provides a common 
pathophysiological indicator of the underlying dysfunction as well as providing 
a quantitative measure of disease activity.  
 
We hypothesise that in patients with takotsubo cardiomyopathy:  
  
 
1. Acute renin-angiotensin system activation but not beta-adrenoreceptor 
stimulation, causes impaired myocardial manganese uptake.  
2. Chronic angiotensin receptor stimulation combined with vasoactive 
peptidases or sodium-glucose co-transporter 2 channels cause 
impaired myocardial manganese uptake.  
2 
STUDY OBJECTIVES 
2.1 
OBJECTIVES 
2.1.1 Primary Objective 
We propose to undertake experimental medicine studies in patients with acute 
or chronic takotsubo cardiomyopathy to explore whether neurohumoral 
activation or sodium-glucose transporters are implicated in the abnormal 
myocardial calcium handling of the left ventricle.   
2.1.2 Secondary Objectives 
As part of a planned subgroup analysis, we will undertake exploratory 
analyses to see if there are other factors that may determine myocardial 

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manganese uptake including cardiovascular risk factors. We will also explore 
the proportionate changes with acute versus chronic changes as well as 
compare the effectiveness of any reversal of altered myocardial calcium 
handling. We will also examine effects on cardiac and neurohumoral 
biomarkers to assess potential pathways of benefit. We will also explore 
correlations between myocardial manganese uptake and self-reported 
symptoms and objective assessments of exercise capacity.  
 
2.2 
ENDPOINTS 
2.2.1 Primary Endpoint 
Left ventricular myocardial manganese uptake.  
2.2.2 Secondary Endpoints 
1. Assessing effects of renin-angiotensin system inhibition and beta-
adrenoreceptor blockade on myocardial manganese uptake.  
2. Assessing effects of angiotensin receptor/neprilysin inhibition and 
sodium-glucose co-transporter 2 inhibition on myocardial manganese 
uptake.  
3. Assessing the effects of cardiovascular risk factors on myocardial 
manganese uptake.  
2.2.3 Exploratory Endpoints 
1. Correlation of neurohumoral markers and myocardial manganese 
uptake. 
2. Correlations between myocardial manganese uptake and self-reported 
symptoms and objective assessments of exercise capacity in 
Takotsubo Cardiomyopathy.   
 
3 
STUDY DESIGN 
This will be a prospective randomised open-label blinded endpoint (PROBE) 
study design with additional use of a cross-over design to improve statistical 
power. The following flow charts summarise what each study visit will involve 
for each different group of study participants.  
 
 
 
 
 
 
 

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Study 1: 
 
Visit 1 
Consent, clinical history and examination.  
Patient questionnaire, walking test and blood tests. 
Heart tests: ECG, heart ultrasound and MRI scan. 
Allocation to Bisoprolol, Valsartan or no medication. 
3 hours. 
 
 
 
 
Patients allocated to Bisoprolol and Valsartan will need to return for two 
additional visits 1 week and 2 weeks after starting medication. This is to check 
their heart rate, blood pressure and blood tests and to have their medication 
dose adjusted (10 minutes). Once on the highest dose, patients will not require 
to attend for these fortnightly visits. Patients randomised to no medication will 
not need to attend for these visits.  
 
 
 
Visit 2 (4 weeks later) 
Clinical history, examination, questionnaire and walking test.  
Heart tests: ECG, heart ultrasound and MRI scan. 
Adjustments to your medication and pill counting. 
3 hours 
 
 
 
Visit 3 (3 months later) 
Clinical history, examination, questionnaire, blood tests, walking test.  
Heart tests: ECG, heart ultrasound and MRI scan.  
Stopping your medication and pill counting. 
3 hours 
 
 
 
 
 

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Study 2: 
Visit 1. 3 hours.  
Consent, clinical history and examination.  
Patient questionnaire, walking test and blood tests.  
Heart tests: ECG, heart ultrasound and MRI scan.  
Allocation to Sacubitril/Valsartan or Dapaglifozin. 
 
 
 
 
 
Patients allocated to Sacubitril/Valsartan will be brought back for an additional 2 visits 
every 2 weeks to monitor their blood pressure and blood tests (10 minutes). The dose 
of Sacubitril/Valsartan may be increased depending on the results of these tests. Once 
on the highest dose, patients will not require to attend for these fortnightly visits. 
Patients allocated to Dapaglifozin do not need to attend for these visits and will remain 
on the same medication dose. 
 
 
 
Visit 2 (3 months later). 3 hours. 
Clinical history, examination, questionnaire and walking test 
Heart tests: ECG, heart ultrasound and MRI scan. 
Pill counting. Stop study medication for 1 month. 
 
 
 
Visit 3 (1 month later). 10 minutes. 
Clinical history and examination.  
Start alternative study medication. If previously on Sacubitril/Valsartan will start 
Dapaglifozin. If previously on Dapaglifozin will start Sacubitril/Valsartan. 
 
 
 
 
Patients allocated to Sacubitril/Valsartan will be brought back for additional visits 
every 2 weeks to monitor their blood pressure and blood tests (10 minutes). The dose 
of Sacubitril/Valsartan may be increased depending on the results of these tests.  
Patients allocated to Dapaglifozin do not need to attend for these visits and will remain 
on the same medication dose. 
 
 
 
 
Visit 4 (3 months later). 3 hours. 
Clinical history, examination,questionnaire,  blood tests and walking test. 
Heart tests: ECG, heart ultrasound and MRI scan.  
Pill counting. Stop study medication. 
 
 
 
 
 
 

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Healthy volunteers: 
 
Visit 1. 3 hours.  
Consent, clinical history and examination.  
Patient questionnaires, walking test and blood tests. 
Heart tests: ECG, heart ultrasound and MRI scan.  
3 hours. 
 
4 
STUDY POPULATION 
4.1 
NUMBER OF PARTICIPANTS 
We aim to recruit 60 participants with an acute diagnosis (within 3 months) of 
takotsubo cardiomyopathy and 40 participants with a prior diagnosis (>6 
months) of takotsubo cardiomyopathy as well as 20 age and sex-matched 
healthy volunteers. The study will end when the final participant attends for their 
final study visit. The expected length of recruitment period will be 24-30 months.  
 
4.1.1 Study 1: Patients with Acute Takotsubo Cardiomyopathy 
We will recruit 60 patients with acute takotsubo cardiomyopathy and 20 age 
and sex matched healthy volunteers. 
 
4.1.2 Study 2: Patients with Prior Takotsubo Cardiomyopathy 
We will recruit 40 patients with prior takotsubo cardiomyopathy (>6 months 
previously) for this second cohort of patients. We will recruit these patients 
contemporaneous with Study 1 so that we can use the same 20 age and sex 
matched healthy volunteers as a comparator. 
 
4.2 
INCLUSION CRITERIA 
 
Takotsubo cardiomyopathy patients: 
- 
Males and females >18 years of age 
- 
Clinical presentation of takotsubo cardiomyopathy  
- 
Have capacity to give formal consent  
 
Healthy volunteers: 
- 
Males and females >18 years of age 
- 
Normal LV function 
- 
No major co-morbidities such as ischaemic heart disease, heart failure, 
cancer or stroke  
- 
Have capacity to give formal consent 

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4.3 
EXCLUSION CRITERIA 
- 
Unable to tolerate or contraindication to magnetic resonance imaging  
- 
Renal failure (estimated glomerular filtration rate <30 mL/min/1.73 m2) 
– this would be assessed by performing a blood test  
- 
Prior history of cardiomyopathy 
- 
Current pregnancy  
- 
Weight greater than 250kg 
- 
Severe asthma for study 1 (given Bisoprolol is contraindicated in this 
condition)  
- 
Type 1 diabetes mellitus for study 2 (given Dapaglifozin is 
contraindicated in this population)  
- 
Significant hypotension (given the medications prescribed as part of the 
study can also cause hypotension as a side effect)  
- 
Significant bradycardia for study 1 (given Bisoprolol can exacerbate 
any pre-existing significant bradycardia)  
- 
Prescribed Digoxin (this medication interacts with the Manganese)  
- 
Unable to give formal consent  
 
4.4 
CO-ENROLMENT 
Co-enrolment with other studies may be considered in line with ACCORD co-
enrolment policy. Co-enrolment between studies will be agreed by the 
respective PIs prior to beginning of the study. Individual patients who are 
eligible for co-enrolment will be discussed between research fellows and PIs as 
necessary. This will ensure co-enrolment: 
1. Is appropriate (i.e. there are no contra-indications) 
2. Would not jeopardise the integrity of either study 
3. Would not compromise patient care and safety  
 
5 
PARTICIPANT SELECTION AND ENROLMENT 
 
5.1 
IDENTIFYING PARTICIPANTS 
For Study 1, potential participants will be identified by the co-investigator, who 
will be part of the patients’ cardiology clinical care team. This will happen either 
whilst the participant is still in hospital or shortly after discharge. The co-
investigator will provide an information sheet if the participant is interested in 
taking part in the study. 
 

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For Study 2, the co-investigator will identify participants via the Scottish 
National Takotsubo registry. PBPP and REC approvals are in place to obtain 
the contact details of patients identified as part of the Scottish Takotsubo 
Registry so that we can invite them to take part in research. Participants will 
also be identified by the patients’ usual clinical care team when they attend for 
cardiology clinic follow-up. If they meet inclusion criteria they will be invited to 
take part in the study and their information and contact details passed on to the 
co-investigator who will contact them to arrange an initial study visit.  
 
In addition to NHS Lothian, participants can be approached within the NHS 
Greater Glasgow and Clyde health board. Participants will be approached at 
the Golden Jubilee National Hospital which will act as a Participation 
Identification Centre (PIC). Participants will be approached in the usual way, by 
their usual clinical care team either whilst they are in hospital as an inpatient or 
at an outpatient clinic. All study activities including patient consent will still take 
place at the original research site at the University of Edinburgh. There is 
funding in place to ensure that patients travelling to Edinburgh from Glasgow 
are offered reimbursement for the cost of travel to the study site.  
 
Healthy volunteers will be recruited via an advertisement process.  
 
5.2 
CONSENTING PARTICIPANTS 
Participants will be enrolled following an opportunity to read the patient 
information sheet and ask questions. Informed consent can be taken by any of 
the study investigators and they will also decide if patients are eligible. Once 
written informed consent is obtained, screening for eligibility can begin. A 
screening log will be maintained which will contain anonymised data and will 
be kept in on Excel spreadsheet on a password-locked University of 
Edinburgh laptop. Consent will be confirmed at the beginning of any study 
activities. 
 
5.3 
WITHDRAWAL OF STUDY PARTICIPANTS  
Participants are free to withdraw from the study at any point or a participant 
can be withdrawn by the Investigator. If withdrawal occurs, the primary reason 
for withdrawal will be documented if possible. The participant will have the 
option of withdrawal from all aspects of the study but continued use of data 
collected up to that point. To safeguard rights, the minimum personally 
identifiable information possible will be collected. If participants withdraw from 
the study, they will not need to be replaced.  
 
 
 
 
 

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6 
INTERVENTIONS 
 
6.1 
STUDY MEDICATION 
6.1.1 Identity of study medication  
 
Study Medication 
Medication form, dose and 
usual indication 
Manufacturer 
Bisoprolol 
Oral tablet 
2.5 mg once daily 
Dose increased to 5mg 
after 1 week then increased 
to 10mg after another 1 
week 
Usual indication: Heart failure, 
hypertension, arrhythmias 
Alliance Healthcare 
Valsartan 
Oral tablet 
80 mg twice daily 
Dose increased to 160mg 
twice daily after 1 week 
Usual indication: Heart failure, 
hypertension 
Alliance Healthcare 
Sacubitril/Valsartan 
Oral tablet 
24/26 mg twice daily 
Dose increased to 49/51mg 
after 1 week then increased to 
97/103mg after 1 week 
Usual indication: Heart failure 
Novartis 
Dapaglifozin 
Oral tablet 
10 mg once daily 
Usual indication: Heart failure, 
type 2 diabetes mellitus  
AstraZenaca 
 
6.1.2 Doses and treatment regimens 
Patients in study 1 will be randomised 1:1:1 to no treatment, or open-label 
valsartan or bisoprolol.  
 
Patients in study 2 will receive 3 months treatment with combination sacubitril 
and valsartan and dapagliflozin 10 mg in random order. This will be separated 
by a one-month medication “wash out period.” 
 

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Medications will be prescribed on a research specific prescription form by a 
co-investigator in the Clinical Research Facility. The medication will then be 
dispensed “off the shelf” from the Royal Infirmary of Edinburgh pharmacy 
department.  
 
Randomisation will take place by block randomisation. An unbiased colleague 
will create the blocks for randomisation and the study investigators will be 
blinded to the blocks until the point of randomisation. 
 
6.1.3 Storage 
Study drugs will be stored in the pharmacy department, as per an agreement 
with the lead research pharmacist at the Royal Infirmary of Edinburgh. The 
pharmacy department will ensure that medications are stored as per 
manufacturer instructions.  
 
6.2 
CONCOMITANT AND POST-STUDY INTERVENTIONS 
Following completion of the study, the study medications will be discontinued. 
For bisoprolol, the dose will be slowly down titrated before complete 
discontinuation. We aim to reduce the Bisoprolol dose by 2.5mg each week. 
All other study medications will be safe to stop immediately.  
 
If participants are already prescribed medication which is the same, similar to 
or interacts with the study medication then one or more of their current 
medications may be changed. This would only be considered if it was safe to 
do so and after an informed discussion with the participant. In some cases, an 
alternative medication may be offered. At the end of the study, we will have a 
further discussion with participants regarding their medication. If we have 
discontinued a medication at the start of the study, this may be restarted 
again, but we will assess this on a case-by-case basis. We will inform the 
participants’ GPs which medication they should be prescribed at the end of 
the study.  
 
6.3 
COMPLIANCE WITH STUDY INTERVENTIONS 
The investigators will assess treatment compliance at attendance of each of 
the study visits by pill counting at each visit after randomisation.  
 
6.3.1 Accountability 
Compliance will be assessed by interview and pill counting. If the patient is 
suspected to have taken a medication overdose they will undergo immediate 
clinical assessment by one of the co-investigators. This will include checking 
their heart rate, blood pressure and performing a clinical examination. They 
may also require blood tests. If there is any concern, the co-investigator will 
arrange for the patient to be admitted to hospital. If an overdose occurs this 
will be recorded as a Significant Adverse Event.   
 

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6.4 
DISCONTINUATION OF STUDY MEDICATION 
The study medication will be discontinued under the following 
circumstances: 
 
1. 
At the request of the patient or if the patient withdraws from the 
study. 
2. 
By the investigator or the responsible clinician if this was felt to be in 
the best interests of the patient. 
3. 
On completion of the study. 
The patient will be asked to return any unused study medication to the 
pharmacy department at the Royal Infirmary of Edinburgh.  
 
6.4.1 Procedures for discontinuation of a subject from study 
medication 
Brief interruptions of therapy will be permitted (<14 days) where there is a 
clinical need. If medication needs to be stopped for longer than 14 days then 
the patient will be withdrawn from the study. This will be documented in patients’ 
medical records. Reasons for interruption or discontinuation of study 
medications include side effects of medications. The table below summarises 
some side effects associated with the study medication: 
 
Study Medication 
Potential side effects 
Bisoprolol 
Hypotension, bradycardia, erectile 
dysfunction, rash, cold peripheries  
Valsartan 
Hypotension, impaired renal function, 
nausea, diarrhoea, rash  
Sacubitril/Valsartan 
Hypotension, impaired renal function, 
nausea, diarrhoea, rash 
Dapaglifozin 
Diabetic 
ketoacidosis, 
genital 
infections (only applies to patients 
with type 2 diabetes mellitus)  
 
6.5 IMAGING CONTRAST MEDIA 
 
Mangafodipir  
Mangafodipir trisodium (manganese dipyridoxyl diphosphate, MnDPDP; IC 
Targets, Norway) 0.05 mmol/mL solution for intravenous infusion. Each mL 
contains 37.85 mg of anhydrous mangafodipir trisodium, 0.05 mmol (50 μmol), 
equivalent to 35.55 mg of mangafodipir. 10 mL contains 378.5 mg of anhydrous 
mangafodipir trisodium, 0.50 mmol (500 μmol), equivalent to 345.5 mg of 
mangafodipir. The standard dose of Mangafodipir will be 5ml although this will 

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be subject to the results of an ongoing comparison study being undertaken at 
the Edinburgh Royal Infirmary.  
 
The manganese will be administered by intravenous infusion over a period of 
approximately 10-15 minutes (0.4-1.2mL/min), during the cardiac MRI scan.  
 
7 
STUDY ASSESSMENTS 
7.1 
STUDY ASSESSMENTS 
All patient’s eligible for the study will have informed consent taken as well as a 
standardised clinical assessment. For patients in study 1 this will be carried 
out in the Clinical Research Facility or on the participants’ inpatient ward. For 
patients in study 2 this will be carried out in the Clinical Research Facility. 
Participants will be reimbursed for travel costs to the Clinical Research 
Facility, up to a value of £50 per participant.  
 
The different aspects of the study assessments are summarised in the table 
below:  
 
Assessment 
Description 
Time 
required 
Associated risks 
Consent 
The co-investigator will explain the 
study to potential participants with 
adequate opportunity for the 
participant to ask questions. If the 
participant agrees they will be asked 
to sign a consent form. 
10 minutes 
Nil significant 
Medical history 
and physical 
examination 
The co-investigator will check that 
patients don’t have any study 
eligibility contraindications. They will 
also check the patients’ past medical 
history, medications and allergies and 
will record their height, weight, heart 
rate and blood pressure and perform 
a physical cardiovascular 
examination. 
15-20 
minutes 
Nil significant 
Blood tests 
A needle will be inserted to take 
blood tests to ensure the patient is 
eligible to take part in the study as 
well as to assess their current clinical 
cardiac status and for storage of 
samples. 
10 minutes 
Pain from needle used 
for taking blood tests 

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Patient 
questionnaire 
Participants will be asked to complete 
a questionnaire about their symptoms 
and quality of life 
10 minutes 
Nil significant 
ECG 
We will perform an electrical tracing 
of the participants’ hearts. 
10 minutes Irritation from sticky pads 
used for ECG 
Cannula 
A small needle will be inserted into 
the participant’s arm, This will allow 
us to inject the manganese contrast. 
10 minutes 
Pain related to needle 
Urine 
pregnancy test 
This will be performed in female 
participants of child-bearing potential 
to ensure study eligibility. 
5 minutes 
Nil significant 
Echocardiogram We will perform an ultrasound scan of 
the participants’ hearts.  
20 minutes 
Nil significant 
6-minute walk 
test 
We will ask the participant to walk on 
the flat for 6 minutes whilst monitoring 
their oxygen saturations via pulse 
oximetry. 
6 minutes 
Nil significant 
MEMRI scan 
An MRI scan of the heart will be 
performed and the patient will be 
injected with manganese contrast. 
1 hour 
Small risk of allergic 
reaction to manganese 
Claustrophobia 
Randomisation 
to medication 
Participants will be randomised to 
medication 
10 minutes 
Nil significant 
Pill counting 
We will count the participants’ study 
medications. 
5 minutes 
Nil significant  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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7.2 
STUDY VISITS 
Study 1 participants will undergo 4 study visits if they are randomised to no 
medication and 6 study visits if they are randomised to medication. Study 2 
participants will undergo 7 study visits in total. Healthy volunteers will attend 
for 2 study visits. The below tables summarise the different assessments 
participants will undergo at each study visit: 
Study 1 
 
 
 
 
 
 
 
 
 
Assessment  
Screening and  
Randomisation 
visit 
1 week 
(Patients on 
Bisoprolol/ 
Valsartan) 
 
 
2 weeks 
(Patients on 
Bisoprolol/ 
Valsartan) 
 
 
 
4±1 weeks 
 
 
3±1 months 
Assessment of Eligibility 
Criteria  
  x 
   
 
 
 
Written informed consent  
  x 
   
 
 
 
Demographic data, contact 
details  
x  
  
 
 
 
Clinical assessment as per 
study protocol  
x 
x 
x 
x 
x 
Blood samples as per study 
protocol 
x 
 x  
x 
 
x 
Patient questionnaires as 
per study protocol 
x 
 
 
x 
x 
12-lead ECG 
 x  
 
 
x 
x 
Transthoracic 
Echocardiogram 
 x  
 
 
x 
x 
6-minute walk test 
x 
 
 
x 
x 
Urinary pregnancy test 
x 
 
 
x 
x 
Manganese-enhanced 
Cardiac Magnetic 
Resonance Imaging 
x 
 
 
x 
x 
Randomised allocation to 
Bisoprolol, Valsartan or no 
medication 
x 
 
 
 
 
Adjustment of medication 
dose 
 
x  
x 
x 
 
Stop medication 
 
 
 
 
x 
Pill counting 
 
 
 
x 
x 

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Study 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment  
Screening and  
Randomisation  
Visit 
1 week 
(Patients 
on 
sacubitril/ 
valsartan) 
 
 
2 weeks 
(Patients 
on 
sacubitril/ 
valsartan) 
 
 
3±1 
months 
 
4±1 
months 
 
1 week 
(Patients 
on 
sacubitril/ 
valsartan) 
 
 
2 weeks 
(Patients 
on 
sacubitril/ 
valsartan) 
 
 
7±1 months 
Assessment of 
Eligibility Criteria  
  x 
   
 
 
 
   
 
 
Written informed 
consent  
 x  
   
 
 
 
   
 
 
Demographic data, 
contact details  
x  
  
 
 
 
  
 
 
Clinical assessment as 
per study protocol  
x 
x 
x 
x 
x 
x 
x 
x 
Blood samples as per 
study protocol 
x 
 x  
 x  
x 
x 
 x  
 x  
x 
Patient questionnaires 
as per study protocol 
x 
 
 
x 
 
 
 
x 
12-lead ECG 
 x  
   
   
x 
 
   
   
x 
Transthoracic 
Echocardiogram 
 x  
  
  
x 
 
  
  
x 
6-minute walk test 
x 
 
 
x 
 
 
 
x 
Urinary pregnancy test 
x 
 
 
x 
 
 
 
x 
Manganese-enhanced 
Cardiac Magnetic 
Resonance Imaging 
x 
 
 
x 
 
 
 
x 
Randomised allocation 
to Sacubitril/Valsartan 
or Dapaglifozin 
x 
 
 
 
 
x 
 
 
 
Adjustment of 
medication dose 
 
x  
x  
 
 
x  
x  
 
Stop medication 
 
 
 
x 
 
 
 
x 
Pill counting 
 
 
 
x 
 
 
 
x 

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Healthy Volunteers 
 
Assessment 
Visit 2 (2 weeks) 
Assessment of Eligibilty Criteria 
x 
Written informed consent 
x 
Demographic data, contact details 
x 
Clinical assessment as per study protocol 
x  
Blood samples as per study protocol 
x  
Patient questionnaires as per study protocol 
x  
12-lead ECG 
x  
Transthoracic echocardiogram 
x  
6-minute walk test 
x  
Urine pregnancy test 
x  
Manganese-enhanced Cardiac Magnetic Resonance 
Imaging 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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7.3 
STUDY SCREENING 
For Study 1, potential participants will be identified by the co-investigator either 
whilst the participant is still in hospital or shortly after discharge. A member of 
the study team will provide an information sheet if the participant is interested 
in taking part in the study. 
 
For Study 2, the co-investigator will identify patients within the NHS Lothian 
Health Board via the Scottish National Takotsubo registry. Participants will 
also be identified within the NHS Greater Glasgow and Clyde Health Board by 
their usual clinical care team.  
 
Healthy volunteers will be recruited via an advertisement process.  
 
An anonymised screening log will be maintained by the co-investigator.  
 
7.4 
DEMOGRAPHIC DATA 
This will include name, initials, age, sex, height, weight, CHI number, telephone 
number, address, postcode and email address if available.  
 
7.5 
CLINICAL ASSESSMENT 
This will involve checking participants’ height, weight, heart rate and blood 
pressure. The co-investigator will perform a physical clinical examination of the 
participants’ cardiovascular system.  
 
7.6 
BLOOD SAMPLES 
This will include blood sampling for standard biochemical and haematological 
analyses (Urea and Electrolytes, Full Blood Count and Liver Function Tests) to 
check participants are eligible and safe to take part in the study. Further 
samples will be taken for U and Es for safety monitoring purposes for 
participants randomised to Valsartan and Sacubitril/Valsartan. An N-terminal 
pro-brain natriuretic peptide will be taken for research purposes.  
 
Furthermore, three blood samples will be taken from each participant and 
stored for potential future biomarkers.   
 
7.7 
URINE PREGNANCY TEST 
This will be performed in female participants of child bearing potential.  

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7.8 
PATIENT QUESTIONNAIRES 
Quality of life, symptoms, physical and social function will be recorded using 
the Kansas City Cardiomyopathy Questionnaire (KCCQ-12), Health Status 
(EQ-5D-5L) and the Patient-Reported Outcomes Measurement Information 
System short modules on Anxiety, Depression, Fatigue and Physical Function. 
 
7.9 
12-LEAD ECG 
An electrical recording of the participants’ hearts will be undertaken.  
 
7.10 6-MINUTE WALK TEST 
Participants will be asked to walk on the flat for 6 minutes whilst having their 
oxygen saturations monitored via a pulse oximeter.  
 
7.11 ECHOCARDIOGRAPHY 
All study participants including healthy volunteers will be studied with a 
standard 3 MHz transducer and 2- dimensional, M-mode, Doppler and colour 
Doppler measurements taken. Ejection fraction will be quantified using the 
modified Simpson’s technique and regional wall motion abnormalities 
assessed. If not performed as part of standard of care (whilst the participant is 
in hospital), we will organise a study specific transthoracic echocardiogram to 
be performed at visit 2 or as soon as possible as when is convenient for the 
participant. 
 
7.12 MANGANESE-ENHANCED CARDIAC MAGNETIC RESONANCE 
IMAGING 
Magnetic resonance imaging will be performed at the Edinburgh Imaging 
Facility in the Queen’s Medical Research Institute, University of Edinburgh.  
7.13 STORAGE AND ANALYSIS OF SAMPLES 
Blood samples for storage will be frozen (-70 degrees Celcius) and kept for a 
minimum of five years. They will be stored in a locked freezer in appropriately 
secured premises in the Laboratory at Edinburgh Royal Infirmary. The 
retained blood samples and participants data (including MRI images) will be 
used for secondary studies separate to this study as hypotheses arise. 
Anonymised blood samples may be sent to other academic institutions within 
the UK for analysis. Participants will be asked for their consent for storage of 
samples and use in other studies at the first study visit as part of the consent 
process.  

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8 
DATA COLLECTION 
 
Clinical Assessment 
Any changes in medication, any hospital admission or any clinical symptoms 
will be assessed at the study visit by the investigator. 
 
Blood Sampling 
Blood samples will be collected as per standard clinical care. The results will 
be uploaded onto the patients electronic NHS Lothian medical record. 
 
Electrocardiogram 
Electrocardiograms will be recorded as per standard clinical care and 
uploaded onto the patient’s electronic NHS Lothian medical record. 
 
Echocardiography 
Echocardiography will be performed according to standard clinical care. 
Echocardiograms will be uploaded to the EchoPAC data storage system as 
part of the patient’s electronic NHS Lothian medical record. 
 
Cardiac Magnetic Resonance Imaging with Gadolinium 
Cardiac magnetic resonance imaging will be performed according to standard 
clinical care and uploaded onto the National PACS system.  
 
Cardiac Magnetic Resonance Imaging with Manganese 
All images will be acquired with a 3T Siemens scanner. A 60-channel cardiac 
coil will be used to perform standard cardiac imaging sequences (breath-held, 
electrocardiogram-gated) for 2-chamber, 4-chamber, long axis and short axis 
views of the heart. This will provide standard assessment of cardiac volumes, 
mass, and function including left ventricular ejection fraction and regional wall 
motion abnormalities. Myocardial T1 will be measured serially for 40-60 min 
after intravenous infusion of mangafodipir (5 mmol/kg, 1 mL/min; IC Targets, 
Norway). T1 imaging will be performed with Modified Look-Locker Inversion 
recovery (Siemens Healthineers) in both short axis (base, mid-cavity) images  
of the heart (4-chamber and 2-chamber views to capture the apex). Scanner-
generated T1 maps will be analysed to quantify T1 within regions of interest.  
 
8.1 
 SOURCE DATA DOCUMENTATION 
The study database will be used to document all data collection which will 
include consent forms, patient questionnaires, case report forms and 
information about participant study visits. All patient-related data will be 
recorded in an pseudonymised way. Each patient will be unequivocally 
identified by a trial subject number, attributed at recruitment into the study. 
Key data from blood results, echocardiography and MRI reports will be 
documented in the study database. The original reports and images will be 
retained by the imaging core laboratory for future reference. 

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9 
DATA MANAGEMENT 
 
9.1 
DATA STORAGE  
 
Patient NHS electronic records will be accessed following formal and explicit 
patient consent. Pseudonymised data will be made available for other 
researchers to use where appropriate.  
 
All identifiable participant information (name, age, sex, address and postcode, 
CHI number, telephone number, email address, height, weight, clinical 
condition, date of study enrolment and dates of MRI scan) will be stored in a 
secure manner accessible only to members of the research team on an NHS 
Lothian device. CHI number is required to review participants relevant medical 
history, medications, medical imaging and standard of care intervention 
results. This will be stored in a password protected Excel file on an NHS 
Lothian device.   
 
All NHS Lothian computers used in the study will be encrypted, and password 
protected. Electronic data will be stored on (2-step) password-protected 
computers and only accessible to the study investigators. No personal 
identifiable information including CHI numbers will be accessed by members 
outside the research team or NHS Lothian. 
 
Only study subject number will be transferred to the password protected 
University laptop.  
 
The study will be guided by the principles of Good Clinical Practice and Data 
Protection Act in ensuring the confidentiality of personal data. Participant 
consent forms will contain personal data and a linked participant identification 
number. Hard copies of consent forms will be stored in a secure filing cabinet 
in a locked University Office (Room SU 305, Chancellor's Building). Study 
data will be kept on university password protected computers as linked 
anonymised data. This will enable the identification of each participant's data 
during the study, for any participant to withdraw and have personal data 
destroyed if they wish. All data will be anonymised after the study period. 
 
9.2 
PERSONAL DATA  
The following personal data will be collected as part of the research: 
- Name, age, sex, address and postcode, CHI number, height, weight, clinical 
condition, date of study enrolment and date of MRI scan  
- Personal data will be stored or accessed for a period of 6 to 12 months after 
the study has ended on a password protected Excel file to 

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allow participants to be identified so we can follow up on their management 
and clinical course. Thereafter, pseudonymised data may be stored for a 
period of up to five years.  
 
9.3 
DATA INFORMATION FLOW 
Patient’s initials, name, signature, age and date of birth will be on the consent 
form which will be kept in locked cupboards in the clinical research facility. 
This will be pseudonymised with a unique patient code, and this will be stored 
on a password-protected Excel document.  
 
9.4 
EXTERNAL TRANSFER OF DATA 
Data collected or generated by the study could potentially be transferred to 
any external individuals or organisations outside of the Sponsoring 
organisation(s) in a fully pseudonymised or linked pseudonymised format. 
With consent, anonymised data collected may be shared with other academic 
or commercial institutions.  
 
9.5 
DATA CONTROLLER 
A data controller is an organisation that determines the purposes for which, 
and the manner in which, any personal data are processed. 
The University of Edinburgh and NHS Lothian are joint data controllers along 
with any other entities involved in delivering the study that may be a data 
controller in accordance with applicable laws (e.g. the site). 
9.6 
DATA BREACHES  
Any data breaches will be reported to the University of Edinburgh 
(dpo@ed.ac.uk) and NHS Lothian (Lothian.DPO@nhslothian.scot.nhs.uk) 
Data Protection Officers who will onward report to the relevant authority 
according to the appropriate timelines if required. 
10 
STATISTICS AND DATA ANALYSIS 
10.1 SAMPLE SIZE CALCULATION 
 
In our prior study of manganese-enhanced magnetic resonance imaging in 
patients with takotsubo cardiomyopathy, we observed that manganese uptake 
was markedly suppressed during initial hospital admission (5.1±0.5 mL/100 g  
of tissue/min) and this partially recovered after 3 or more months (6.6±0.5 
mL/100 g of tissue/min) but remained below that observed in matched healthy  
volunteers (8.2±1.1 mL/100 g of tissue/min; P<0.001) [Singh et al, 2022]. For 
study 1, this gives a 100% power to detect a similar difference between healthy  

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volunteers and patients not receiving any treatment (n=10) at both baseline and 
3 months. In terms of treatment effects for studies 1 and 2, we anticipate an 
improvement of at least 0.6 mL/100 g of tissue/min which equates to just over 
a third of the difference between healthy volunteers and patients beyond 3 
months (8.2 versus 6.6 mL/100 g of tissue/min). At 90% power and 2-sided 
P<0.05 this requires a sample size of 18 per group. To account for some, drop 
out, we have increased the number of healthy volunteers recruited to a sample 
size of 20. For study 2, we are exploring novel therapies with uncertain impact, 
but the detectable effect size will be much smaller (approximately half) given 
the paired comparisons, cross-over methodology and effective group sizes of 
40.  
 
10.2 PROPOSED ANALYSES 
 
10.2.1 Imaging Analysis 
Pseudonymised participant MRI data will be sent electronically to the 
University of Edinburgh for analysis. The first MRI scan will serve as a 
baseline for comparison, and the pre-contrast imaging will serve as control for 
each scan individually. Images will be analysed using commercially available 
cardiac MRI software (Circle Cardiovascular Imaging software, cvi42®, Circle 
Cardiovascular Imaging Inc. Calgary, Alberta, Canada) through which 
myocardial contouring and T1 map creation and analysis is performed. 
 
Analysis will be performed in the Image Analysis Core of the Edinburgh 
Imaging facilities. A standardised approach to T1 image analysis will be 
employed for manganese uptake [Singh et al, 2022]. Briefly, the basal, mid-
cavity, and long axis T1 maps will be divided into segments according to the 
standard 17-segment model recommended by the American College of 
Cardiology/American Heart Association [Cerqueira et al, 2002]. After 
contouring epi- and endocardial borders and defining right ventricular insertion  
points, regions of interest will be derived for each of the 16 segments with a 
10-20% epi- and endocardial offset to reduce risk of blood pool contamination. 
Manganese uptake will be quantified using a two-compartment Patlak model 
as described previously [Singh et al, 2022b], and this has excellent 
repeatability and reproducibility [Singh et al, 2023]. All image analysis will be 
performed blinded to group, timing and treatment allocation. 
 
10.2.2 Statistical Analysis 
Summary data will be presented as number and percentage, mean ± standard 
deviation or median and interquartile range as appropriate. Based on our prior 
data, we anticipate that the data will be normally distributed. Within group and 
between group comparisons will be made with paired or unpaired Student’s t-
test or analysis of variance as appropriate. For study 2, we will explore 
whether there is any evidence of a time-order effect before proceeding to pool 
the data from all participants. This will greatly enhance our power to detect 

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meaningful differences. Repeated measures will be assessed by analysis of 
variance. Statistical significance will be taken as a two-sided P<0.05. 
11 
ADVERSE EVENTS 
 
The Investigator, or a delegated researcher, is responsible for the detection 
and documentation of adverse events that may be related to participating in 
the study and that meet the criteria and definitions detailed below.  
 
11.1 Definitions 
 
An adverse event (AE) is an untoward medical occurrence in a study 
participant.  
 
An adverse reaction (AR), in the context of this study, is any untoward and 
unintended response which is related to study medication or manganese 
contrast. 
 
A serious adverse reaction (SAR) is any AR that: 
• results in death of the clinical trial participant; is life threatening*; 
• requires in-patient hospitalisation or prolongation of existing hospitalisation; 
• results in persistent or significant disability or incapacity; 
• consists of a congenital anomaly or birth defect; 
• results in any other significant medical event not meeting the criteria above. 
 
A suspected unexpected serious adverse reaction (SUSAR) is any AR 
that is classified as serious and is suspected to be caused by the study 
medication or manganese contrast.  
 
*Life-threatening in the definition of an SAE or SAR refers to an event where 
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. 
 
11.2 Identifying AEs and ARs 
All AEs and ARs related to the imaging procedures will be identified from the 
time an MRI scan related procedure commences until 7 days after scan 
completion. All ARs to study medications will be identified from the time of 
medication commencement until 7 days after completion. 
 
11.3 Recording AEs and ARs 

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AEs will be recorded in the electronic health record. There is no requirement 
to complete an additional AE form. When an AE occurs, it is the Investigator 
will review all documentation (e.g. hospital notes, laboratory and diagnostic 
reports) related to the event. Any AEs that qualify as an adverse reaction (AR) 
to study procedures (imaging or medication) that meets seriousness criteria 
will be recorded and reported on an ACCORD SAE form, which will then be 
sent to the Sponsor via email (safety@accord.scot). 
 
Pre-existing medical conditions (i.e. existed prior to informed consent) should 
be recorded as medical history and only recorded as AEs/ARs if medically 
judged to have unexpectedly worsened during the study. Events that are 
consistent with the expected progression of underlying disease should not be 
recorded as adverse events. 
 
11.4 Assessment of AEs, SAEs, ARs, SARs and SUSARs 
Seriousness, causality, severity and expectedness will be assessed by the PI. 
The Investigator is responsible for assessing each adverse event. The CI may 
not downgrade an event that has been assessed by an Investigator as a SAR 
or SUSAR, but can upgrade an AR, SAR or SUSAR if appropriate. 
 
11.4.1 Assessment of causality 
The Investigator will make an assessment of whether an AE is likely to be 
related to the study procedures (imaging or medication) and therefore be 
considered an AR according to the definitions below. 
• Unrelated: Where an event is not considered to be related to the study 
procedures (imaging or medication)  
• Possibly Related: The nature of the event, the underlying medical condition, 
concomitant medication or temporal relationship make it possible that the AE 
has a causal relationship to the study procedures (imaging or medication). 
 
11.4.2 Assessment of seriousness 
Subsequent to the assessment causality, the Investigator will make an 
assessment of seriousness as defined in Section 10.1 
 
11.4.3 Assessment of severity 
The Investigator will make an assessment of severity for each SAR, and 
record this on the ACCORD SAE form according to one of the following 
categories: 
Mild: an event that is easily tolerated by the participant, causing minimal 
discomfort and not interfering with every day activities. 
Moderate: an event that is sufficiently discomforting to interfere with normal 
everyday activities. 
Severe: an event that prevents normal everyday activities. 

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Note: the term ‘severe’, used to describe the intensity, should not be confused 
with ‘serious’ which is a regulatory definition based on participant/event 
outcome or action criteria. For example, a headache may be severe but not 
serious, while a minor stroke is serious but may not be severe. 
 
11.4.4 Assessment of expectedness of SARs  
No SARs are considered expected by the sponsor for the purpose of 
expedited reporting of SUSARs based upon the reference safety 
information available in the most current version of the imaging contrast 
agents or study medications Investigator Brochure.  
 
11.5  Reporting of SARs/SUSARs 
As this trial is a non-CTIMP and involves procedures and interventions that 
are established in the medical community, with extensive information available 
regarding risks, only serious adverse reactions (SARs) and Suspected 
Unexpected Serious Adverse Reactions (SUSARs) will be reported to the 
Sponsor. 
 
Once the Investigator becomes aware that a study related SAR/SUSAR, has 
occurred in a study participant, the information will be reported to the 
ACCORD Research Governance & QA Office within 24 hours. If the 
Investigator does not have all information regarding an event, they should not 
wait for this additional information before notifying ACCORD. The ACCORD 
SAE report form will be used to submit the event report, and can be 
updated when the additional information is received.  
 
The SAE form will be sent via email to safety@accord.scot within 24 hours 
Only forms in a pdf format will be accepted by ACCORD via email. 
Where missing information has not been sent to ACCORD after an initial 
report, ACCORD will contact the investigator and request the missing 
information. 
 
All reports sent to ACCORD and any follow up information will be retained by 
the Investigator in the Investigator Site File. 
 
11.6 Reporting requirements 
The ACCORD Research Governance & QA Office is responsible for 
pharmacovigilance reporting on behalf of the co-sponsors (Edinburgh 
University and NHS Lothian). 
 
11.7 FOLLOW-UP PROCEDURES 

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After initially reporting a study related SAR/SUSAR, the Investigator will follow 
each participant until resolution or the completion of study follow-up. Follow-
up information will be reported to the ACCORD office. 
 
12 
OVERSIGHT ARRANGEMENTS 
12.1 INSPECTION OF RECORDS 
 
Investigators and institutions involved in the study will permit study related 
monitoring and audits on behalf of the Sponsor, REC review, and regulatory 
inspection(s). In the event of audit or monitoring, the Investigator agrees to 
allow the representatives of the Sponsor direct access to all study records and 
source documentation. In the event of regulatory inspection, the Investigator 
agrees to allow inspectors direct access to all study records and source 
documentation. 
 
12.2 STUDY MONITORING AND AUDIT 
The ACCORD Sponsor Representative will assess the study to determine if a 
study specific risk assessment is required.  
 
If required, a study specific risk assessment will be performed by 
representatives of the Sponsor(s), ACCORD monitors and the QA group, in 
accordance with ACCORD governance and sponsorship SOPs. Input will be 
sought from the Chief Investigator or designee. The outcomes of the risk 
assessment will form the basis of the monitoring plans and audit plans.  
 
If considered necessary, ACCORD clinical trial monitors, or designees, will 
perform monitoring activities in accordance with the study monitoring plan. 
This will involve on-site visits and remote monitoring activities as necessary. 
ACCORD QA personnel, or designees, will perform study audits in 
accordance with the study audit plan. This will involve investigator site audits, 
study management audits and facility (including 3rd parties) audits as 
necessary (delete where not required). 
 
13 
GOOD CLINICAL PRACTICE 
 
13.1 ETHICAL CONDUCT 
The study will be conducted in accordance with the principles of the 
International Conference on Harmonisation Tripartite Guideline for Good 
Clinical Practice (ICH GCP). 
 

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Before the study can commence, all necessary approvals will be obtained, 
and any conditions of approvals will be met. 
 
13.2 INVESTIGATOR RESPONSIBILITIES 
The Investigator is responsible for the overall conduct of the study at the site 
and compliance with the protocol and any protocol amendments.  
 
Responsibilities may be delegated to an appropriate member of study site 
staff. 
 
Delegated tasks must be documented on a Delegation Log and signed by all 
those named on the list prior to undertaking applicable study-related 
procedures. 
 
13.2.1 Informed Consent 
The Investigator is responsible for ensuring informed consent is obtained 
before any study specific procedures are carried out. The decision of a 
participant to participate in clinical research is voluntary and should be based 
on a clear understanding of what is involved.  
 
Participants must receive adequate oral and written information – appropriate 
Participant Information and Informed Consent Forms will be provided. The  
oral explanation to the participant will be performed by the Investigator or 
qualified delegated person, and must cover all the elements specified in the 
Participant Information Sheet and Consent Form. 
 
The participant must be given every opportunity to clarify any points they do 
not understand and, if necessary, ask for more information. The participant  
must be given sufficient time to consider the information provided. It should be 
emphasised that the participant may withdraw their consent to participate at 
any time without loss of benefits to which they otherwise would be entitled. 
 
The participant will be informed and agree to their medical records being 
inspected by regulatory authorities and representatives of the Sponsor(s). 
The Investigator or delegated member of the study team and the participant 
will sign and date the Informed Consent Form(s) to confirm that consent has 
been obtained. The participant will receive a copy of the signed consent form, 
and a copy will be filed in the participant’s medical notes. 
 
13.2.2 Study Site Staff 
The Investigator must be familiar with the protocol and the study 
requirements. It is the Investigator’s responsibility to ensure that all staff 
assisting with the study are adequately informed about the protocol and their 
study related duties. 
 
13.2.3 Data Recording 
The Principal Investigator is responsible for the quality of the data recorded in 
the CRF (study database) at each Investigator Site.  

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13.2.4 Investigator Documentation 
The Principal Investigator will ensure that the required documentation is 
available in local Investigator Site files.  
 
13.2.5 GCP Training 
For non-CTIMP (i.e. non-drug) studies all researchers are encouraged to 
undertake GCP training in order to understand the principles of GCP. This is 
not a mandatory requirement unless deemed so by the Sponsor. GCP training 
status for all investigators should be indicated in their respective CVs. 
 
13.2.6 Data Protection Training 
All University of Edinburgh employed researchers and study staff will 
complete the Data Protection Training through Learn.  
 
NHS Lothian employed researchers and study staff will comply with the NHS 
Lothian mandatory Information Governance Data Protection training through 
LearnPro.  
 
Non-NHS Lothian staff that have access to NHS Lothian systems will 
familiarise themselves and abide by all NHS Lothian IT policies, as well as 
employer policies.   
 
13.2.7 Information Security Training 
All University of Edinburgh employed researchers, students and study staff 
will complete the Information Security Essentials modules through Learn and  
will have read the minimum and required reading setting out ground rules to 
be complied with. 
 
NHS Lothian employed researchers and study staff will comply with NHS 
Lothian mandatory Information Governance IT Security training through 
LearnPro. 
 
Non-NHS Lothian staff that have access to NHS Lothian systems will 
familiarise themselves and abide by all NHS Lothian IT policies, as well as 
employer policies.  
 
13.2.8 Confidentiality 
All laboratory specimens, evaluation forms, reports, and other records must 
be identified in a manner designed to maintain participant confidentiality. All 
records must be kept in a secure storage area with limited access. Clinical 
information will not be released without the written permission of the 
participant. The Investigator and study site staff involved with this study may 
not disclose or use for any purpose other than performance of the study, any 
data, record, or other unpublished information, which is confidential or 
identifiable, and has been disclosed to those individuals for the purpose of the 
study. Prior written agreement from the Sponsor or its designee must be 
obtained for the disclosure of any said confidential information to other parties. 

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13.2.9 Data Protection 
All Investigators and study site staff involved with this study must comply with 
the requirements of the appropriate data protection legislation (including the 
General Data Protection Regulation and Data Protection Act) regarding the 
collection, storage, processing and disclosure of personal information. 
 
Computers used to collate the data will have limited access measures via 
usernames and passwords. 
 
Published results will not contain any personal data that could allow 
identification of individual participants. 
 
14 
STUDY CONDUCT RESPONSIBILITIES 
 
14.1 PROTOCOL AMENDMENTS 
Any changes in research activity, except those necessary to remove an 
apparent, immediate hazard to the participant in the case of an urgent safety 
measure, must be reviewed and approved by the Chief Investigator. 
 
Proposed amendments will be submitted to the Sponsor for classification, 
review and authorisation. 
 
Amendments to the protocol must be submitted in writing to the appropriate 
REC and local R&D for approval prior to implementation and prior to 
participants being enrolled into the amended protocol. 
 
14.2 MANAGEMENT OF PROTOCOL NON COMPLIANCE 
 
14.2.1 Protocol Waivers 
Prospective protocol deviations, i.e. protocol waivers, will not be approved by 
the Sponsors and therefore will not be implemented, except where necessary 
to eliminate an immediate hazard to study participants. If this necessitates a 
subsequent protocol amendment, this should be submitted to the REC and 
local R&D for review and approval if appropriate. 
 
14.2.2 Management of Deviations and Violations 
Deviations and violations are non-compliance events discovered after the 
event has occurred. Each protocol violation will be reported to the Sponsor 

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within 3 days of becoming aware of the violation. If the study becomes multi-
centre, deviation logs will be maintained for each centre.  
 
Deviation logs/violation forms will be transmitted via email to 
QA@accord.scot. Only forms in a pdf format will be accepted by ACCORD via 
email. Forms may also be submitted by hand to the office. Where missing 
information has not been sent to ACCORD after an initial report, ACCORD will 
contact the Investigator and request the missing information. The Investigator 
must respond to these requests in a timely manner. 
 
14.3 SERIOUS BREACH REQUIREMENTS 
 
A serious breach is a breach which is likely to effect to a significant degree: 
(a) the safety or physical or mental integrity of the participants of the study; or 
(b) the scientific value of the study. 
 
If a potential serious breach is identified by the Chief investigator, Principal 
Investigator or delegates, the Sponsor(s) (qa@accord.scot) must be notified 
within 24 hours. It is the responsibility of the Sponsor(s) to assess the impact 
of the breach on the scientific value of the study, to determine whether the 
incident constitutes a serious breach and report to research ethics committees 
as necessary. 
 
14.4 STUDY RECORD RETENTION 
All study documentation will be kept for a minimum of 5 years from the 
protocol defined end of study point. When the minimum retention period has 
elapsed, study documentation will be destroyed with permission from the 
Sponsor. 
 
14.5 END OF STUDY 
The end of study is defined as the last participant’s last visit. 
 
The Investigators and/or the Sponsor(s) have the right at any time to 
terminate the study for clinical or administrative reasons. 
 
The end of the study will be reported to the REC, and R&D Office(s) and 
Sponsor(s) within 90 days, or 15 days if the study is terminated prematurely. 
The Investigators will inform participants of the premature study closure and 
ensure that the appropriate follow up is arranged for all participants involved. 
End of study notification will be reported to the Sponsor(s) via email to 
researchgovernance@ed.ac.uk. 

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A summary report of the study will be provided to the REC within 1 year of the 
end of the study. 
 
14.6 INSURANCE AND INDEMNITY 
 
The Sponsor(s) are responsible for ensuring proper provision has been made 
for insurance or indemnity to cover their liability and the liability of the Chief 
Investigator and staff. 
 
The following arrangements are in place to fulfil the Sponsor(s)' 
responsibilities: 
• The Protocol has been designed by the Chief Investigator and researchers 
employed by the University and collaborators. The University has insurance in 
place (which includes no-fault compensation) for negligent harm caused by 
poor protocol design by the Chief Investigator and researchers employed by 
the University. 
• Sites participating in the study will be liable for clinical negligence and other 
negligent harm to individuals taking part in the study and covered by the duty 
of care owed to them by the sites concerned. The Sponsor(s) require 
individual sites participating in the study to arrange for their own insurance or 
indemnity in respect of these liabilities. 
• Sites which are part of the United Kingdom's National Health Service will 
have the benefit of NHS Indemnity. 
 
15 AUTHORSHIP POLICY 
Ownership of the data arising from this study resides with the study team. 
 
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