Conditions: Heart Failure, Diastolic Diastolic Heart Failure Hypertension
Keywords: magnetic resonance imaging positron-emission tomography echocardiography myocardial blood flow energy metabolism
Allocation: NON_RANDOMIZED | Intervention model: FACTORIAL
Enrollment: 55
Healthy volunteers: yes

Brief summary:
Unlike heart failure with reduced ejection fraction (HFrEF) where several medicines and devices have been demonstrated to reduce mortality, no such therapies have been identified in HFpEF. This may be in part due to incomplete understanding of the underlying mechanisms of HFpEF.

Recently, impaired myocardial blood flow, reduced myocardial energy utilization, and increased myocardial fibrosis have been postulated to play important pathophysiologic roles in HFpEF. The investigators and others have demonstrated that HFrEF may be associated with altered myocardial energy utilization and "energy starvation." However, there are limited data regarding "energy starvation" in HFpEF and the relationships between myocardial blood flow, energy utilization, and fibrosis in HFpEF are largely unknown. Therefore, the purposes of this study are to use non-invasive cardiac imaging techniques to describe cardiac structure, function, blood flow, energetics, and fibrosis, and the relationships between these in order to better understand underlying mechanisms in HFpEF.

Detailed description:
The investigators hypothesize that HFpEF is associated with reductions in myocardial blood flow and energy utilization and increased myocardial fibrosis as compared to age and gender matched hypertensive and healthy controls. The investigators will test their hypotheses by comparing measurements of myocardial blood flow, energy utilization, and fibrosis between three subject groups (HFpEF vs hypertension vs healthy). Myocardial blood flow will be quantitated from nitrogen (N)13-Ammonia positron emission tomography (PET) and gadolinium enhanced cardiac magnetic resonance (CMR) imaging, both at rest and stress following coronary vasodilation with regadenoson. Myocardial energy utilization will be quantified with 11C-acetate PET imaging and myocardial fibrosis will be assessed with gadolinium enhanced CMR and alterations in myocardial T1. Echocardiography will be utilized to quantify cardiac diastolic function.

It is anticipated that the results of this proposed study will provide a foundation that will inform future studies aimed at identifying novel preventive or therapeutic agents in HFpEF.

Arms:
arm 1: No cardiovascular abnormalities or diabetes. Estimated glomerular filtration rate (eGFR) \>60.

Studies: Echocardiography for left ventricular function and LV diastolic performance; cardiac magnetic resonance (CMR) imaging using gadolinium for LV fibrosis and regadenoson for myocardial blood flow (MBF); positron-emission tomography (PET) using regadenoson for MBF and 11C-acetate for oxidative metabolism. arm 2: No history of coronary artery disease or diabetes. Estimated glomerular filtration rate (eGFR) \>60.

Studies: Echocardiography for left ventricular function and LV diastolic performance; cardiac magnetic resonance (CMR) imaging using gadolinium for LV fibrosis and regadenoson for myocardial blood flow (MBF); positron-emission tomography (PET) using regadenoson for MBF and 11C-acetate for oxidative metabolism. arm 3: No history of coronary artery disease or diabetes. Estimated glomerular filtration rate (eGFR) \>60.

Studies: Echocardiography for left ventricular function and LV diastolic performance; cardiac magnetic resonance (CMR) imaging using gadolinium for LV fibrosis and regadenoson for myocardial blood flow (MBF); positron-emission tomography (PET) using regadenoson for MBF and 11C-acetate for oxidative metabolism.

Interventions: intervention 1: regadenoson
Intervention descriptions: intervention 1: evaluation of myocardial blood flow, interstitial fibrosis and oxidative metabolism in HFpEF, compared to hypertensive and normal participants

Protocol document:
Prot_SAP_000.pdf: 
Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 1 
 
 
 
 
 
Myocardial perfusion, oxidative metabolism, and fibrosis in HFpEF  
(HFpEF-PRoF) 
 
Principal Investigators: 
Marvin Kronenberg, MD 
Deepak Gupta, MD 
Vanderbilt Heart and Vascular Institute 
1215 21st Ave South, MCE 5414 
Nashville, TN 37232-8802 
phone (615)-936-2530  
marvin.w.kronenberg@vanderbilt.edu 
d.gupta@vanderbilt.edu 
 
Co-Investigators: 
Mark Lawson, MD 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 2 
 
 
Table of Contents 
 
1. 
Study Rationale ..................................................................................................................... 3 
2. 
Study Design.......................................................................................................................... 5 
2.1 Study Design and Hypotheses………………………………………..………….………….…………….……………………….………… 5 
2.2 Recruitment of Study Sample………………………………………………………..……………………………..…… 6 
2.2.1 Cohorts Defined………………………………………………………………………………………………….6 
 
 
2.2.2 Inclusion/Exclusion Criteria……………………………………………………………………….……….8 
2.3 Study Procedures…………………………………………………………………………………………………..………………..…..11 
3. 
Statistical Methods ............................................................................................................. 16 
4. 
Data Management .............................................................................................................. 22 
5. 
Safety Reporting ................................................................................................................. 22 
5.1. Serious Adverse Events ...................................................................................................................... 22 
References/Bibliography............................................................................................................. 23 
 
List of Tables 
 
Table 1 - Summary of Study Aims and Hypotheses 
Table 2 - Key Inclusion and Exclusion Criteria 
Table 3 - Protocol Visits 
Table 3a - Schedule of Events and Research Procedures for Study Participants 
Table 4 - Between group differences in myocardial blood flow measured by coronary flow 
reserve (CFR) for varying sample sizes and powers, at α=0.050 using ANOVA 
Table 5 - Between group differences in oxidative metabolism (Kmono) for varying sample sizes 
and powers, at α=0.050 using ANOVA 
Table 6 - Between group differences in myocardial fibrosis (T1 relaxation times, msec) for 
varying sample sizes and powers, at α=0.050 using ANOVA 
Table 7 - Margins of error for the Spearman correlation between myocardial blood flow, 
oxidative metabolism and myocardial fibrosis. 
 
 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 3 
 
 
1. Study Rationale 
 
Cardiovascular disease is the leading cause of death in the United States and in most countries 
around the world.1 Heart failure, in particular, is increasing in frequency and is associated with 
high morbidity, mortality, and economic cost.2 Heart failure with preserved ejection fraction 
(pump function) (HFpEF) accounts for approximately half of all cases of heart failure and is 
associated with 35-50% mortality over 5 years.2, 3 Unlike heart failure with reduced ejection 
fraction (HFrEF) where several medicines and devices have been demonstrated to reduce 
mortality, no such therapies have been identified in HFpEF.4  This may be in part due 
to incomplete understanding of the underlying mechanisms of HFpEF. 
 
Recently, impaired myocardial blood flow reserve, reduced myocardial energy utilization, and 
increased myocardial fibrosis have been postulated to play important pathophysiologic roles in 
HFpEF.5  We and others have demonstrated that HFrEF may be associated with altered 
myocardial energy utilization and “energy starvation.”6  However, there is limited data 
regarding “energy starvation” in HFpEF and the relationships between myocardial blood flow 
reserve, energy utilization, and fibrosis in HFpEF are largely unknown.7  Therefore, the purposes 
of this study are to use non-invasive cardiac imaging techniques to describe cardiac 
perfusion, energetics, and fibrosis, and their relationships, in order to better understand 
underlying mechanisms in HFpEF. 
 
Specifically, we hypothesize that HFpEF is associated with reductions in myocardial blood flow 
reserve and energy utilization and increased myocardial fibrosis as compared to  gender 
matched hypertensive and healthy controls. We will test our hypotheses by comparing 
measurements of myocardial blood flow reserve, energy utilization, and fibrosis between three 
subject groups (HFpEF vs hypertension vs healthy). Myocardial blood flow will be quantitated 
from 13N-Ammonia Positron Emission Tomography (PET) and gadolinium enhanced cardiac 
magnetic resonance imaging (cMRI) both at rest and stress following coronary vasodilation with 
regadenoson. Myocardial energy utilization will be quantified with 11C-Acetate PET imaging 
and myocardial fibrosis will be assessed with gadolinium enhanced MRI. Echocardiography will 
be utilized to quantify diastolic function and myocardial strain. 
 
It is anticipated that the results of this proposed study will provide a foundation that will inform 
future studies aimed at identifying novel preventive or therapeutic agents in HFpEF. In addition, 
the design of the study affords opportunities to compare myocardial perfusion between PET 
and cMRI across a spectrum of patient populations and obtain valuable normative perfusion 
data in an age group most likely to undergo stress testing. Importantly, the findings may also 
help establish a role for regadenoson stress myocardial perfusion 
imaging in the evaluation of coronary endothelial function and myocardial blood flow in 
hypertensive and HFpEF patients. 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 4 
 
 
 
Specific Aims 
 
Heart failure with preserved ejection fraction (HFpEF) is a major public health problem. HFpEF 
accounts for half of all new cases of heart failure (HF), is increasing in prevalence, and portends 
a poor prognosis. Unfortunately, medical therapies with proven mortality benefit in HFpEF are 
lacking. The absence of effective therapies is attributable in part to the poor understanding of 
the underlying mechanisms of HFpEF. 
 
Coronary endothelial dysfunction and impaired myocardial blood flow reserve are postulated 
to be important mechanisms in HFpEF. The delivery of oxygen and fuel sources needed for 
myocardial performance is dependent upon myocardial blood flow. Impaired myocardial blood 
flow at stress can occur even in the absence of flow limiting epicardial coronary artery disease, 
may be a marker of coronary endothelial dysfunction, and is associated with adverse 
cardiovascular outcomes.8  However, coronary endothelial function and myocardial blood flow 
reserve have not been well described in HFpEF. 
 
Experimental and clinical evidence suggests that myocardial oxidative metabolism is impaired 
in HF. Impaired oxidative metabolism has been associated with HF with reduced ejection 
fraction (HFrEF). However, few studies have investigated oxidative metabolism in HFpEF and in 
particular, whether differences in oxidative metabolism exist between hypertensive patients 
and those with HFpEF. 
 
Myocardial fibrosis is regarded as an important mechanism for HFpEF. Increased  extracellular 
fibrosis has been observed in animal models of hypertension, human left ventricular 
hypertrophy, and endomyocardial biopsies from patients with HFpEF. Hypertension is the major 
risk factor for HFpEF; however, it is unknown why some individuals with hypertension develop 
progressive myocardial fibrosis. 
 
Experimental evidence suggests that impaired oxidative metabolism contributes to 
myocardial fibrosis. In animal models of hypertension, therapies targeting oxidative 
metabolism have been associated with decreased LV fibrosis. Consequently, understanding 
oxidative metabolism and myocardial fibrosis in HFpEF is of biologic importance and may have 
therapeutic implications. 
 
In preliminary work, we have shown that we can quantitatively measure myocardial blood 
flow reserve, oxidative metabolism and myocardial fibrosis through non-invasive cardiac 
imaging. Myocardial blood flow reserve can be quantitatively measured with cardiac magnetic 
resonance imaging (cMRI) and positron emission tomography (PET). The mono-exponential 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 5 
 
decay rate (Kmono) of 11C-Acetate determined with PET is a validated measure of myocardial 
oxidative metabolism. Fibrotic myocardium can be quantified using cMRI T1 mapping. 
 
We postulate that reduced myocardial blood flow reserve contributes to impaired oxidative 
metabolism and increased myocardial fibrosis in HFpEF. We propose to test the following 
primary hypotheses: 1) Compared to healthy and hypertensive individuals, subjects with HFpEF 
have reduced myocardial blood flow reserve, 2) Compared to healthy and hypertensive 
individuals, subjects with HFpEF have reduced oxidative metabolism; 3) Compared to healthy 
and hypertensive individuals, subjects with HFpEF have increased myocardial fibrosis, and 4) 
Myocardial blood flow reserve is positively correlated with oxidative metabolism and inversely 
correlated with myocardial fibrosis. 
 
Aim 1. To compare myocardial blood flow reserve in individuals with HFpEF versus those with 
hypertension and healthy individuals. We will enroll 60 subjects (20 HFpEF, 20 hypertensive, 
and 20 healthy) who will undergo cMRI and 13N-Ammonia PET to quantify myocardial blood 
flow at rest and during pharmacologic stress with regadenoson. 
 
Aim 2. To compare myocardial oxidative metabolism in individuals with HFpEF versus those 
with hypertension and healthy individuals. Subjects enrolled in aim 1 will also undergo 11C-
Acetate PET imaging to quantify oxidative metabolism via the mono-exponential decay rate of 
11C-Acetate. 
 
Aim 3. To compare myocardial fibrosis in individuals with HFpEF versus those with 
hypertension and healthy individuals. Subjects enrolled in aim 1 will also undergo cMRI with 
T1 mapping to quantify myocardial fibrosis. 
 
Aim 4. To assess the relation between myocardial blood flow reserve, oxidative metabolism 
and fibrosis. The correlations between myocardial blood flow reserve, oxidative metabolism 
and fibrosis will be evaluated. 
 
2. Study Design 
 
2.1 Study Design and Hypotheses 
We propose a cross sectional study to investigate the hypotheses that HFpEF is associated with 
reduced myocardial blood flow reserve, reduced oxidative metabolism, and increased 
myocardial fibrosis, and that myocardial blood flow reserve is positively correlated with 
oxidative metabolism, while both are inversely correlated with myocardial fibrosis. We will 
quantify myocardial blood flow reserve, oxidative metabolism and fibrosis using non-invasive 
imaging in 60 subjects (20 healthy, 20 hypertensive, and 20 HFpEF). In addition, clinical 
information and circulating biomarkers will be collected during the protocol. All protocols will 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 6 
 
take place at Vanderbilt University Medical Center. We will specifically test the following 
primary hypotheses (Table 1): 
 
1) Compared to healthy and hypertensive individuals, subjects with HFpEF have reduced 
myocardial blood flow reserve, 
 
2) Compared to healthy and hypertensive individuals, subjects with HFpEF have reduced 
oxidative metabolism, 
 
3) Compared to healthy and hypertensive individuals, subjects with HFpEF have increased 
myocardial fibrosis, and 
 
4) Myocardial blood flow reserve is positively correlated with oxidative metabolism and both 
are inversely correlated with myocardial fibrosis. 
 
An important feature of the proposal is the inclusion of two control groups: healthy 
individuals and individuals with hypertension. This design not only maximizes contrast by 
evaluating healthy individuals and HFpEF patients, but also allows us to test whether a 
“gradient” of abnormal myocardial structure and function exists, by including a group with 
hypertension but no HF. This is of particular relevance because hypertension is the most 
common risk factor for the development of HFpEF and may promote reduced 
myocardial blood flow reserve and oxidative metabolism and increased fibrosis prior to 
the onset of HF. 
 
 
 
 
 
 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 7 
 
2.2 Recruitment of Study Sample 
 
2.2.1 Cohorts Defined 
 
Heart Failure Preserved Ejection Fraction (n=20). HFpEF patients will be aged 50 years or older, 
have a physician confirmed diagnosis of HF, be symptomatic (New York Heart Association class 
II or III), with a LVEF ≥50% within the prior 6 months, and no prior history of LVEF < 50%. HFpEF 
subjects will have evidence of elevated LV filling pressures by ≥ 1 of the following within the last 
6 months: invasive cardiac catheterization LV end diastolic pressure or pulmonary capillary 
wedge pressure > 12 mmHg, plasma B-type natriuretic peptide >100 pg/ml, or 
echocardiographic E/e’ > 15. HFpEF patients with known coronary artery disease (CAD) or 
angina) will be excluded to reduce potential confounding. Patients with significant valvular (≥ 
moderate stenosis or regurgitation), pericardial, or congenital heart disease, or symptoms due 
predominantly to right heart failure and pulmonary arterial hypertension will also be excluded, 
as will those with an estimated glomerular filtration rate (eGFR) < 45 ml/min/1.73m2. HFpEF 
patients in acute decompensated heart failure requiring intravenous diuretics, hospitalization, 
or use of intravenous vasoactive medications will be eligible for screening, but must be 
compensated at the time of the protocol visit. HFpEF patients will be recruited through the 
Vanderbilt Heart and Vascular Institute HFpEF program. 
 
Healthy (n=20): We will enroll volunteers matched on gender to the HFpEF group. Healthy 
subjects will be free of known cardiovascular disease (HF or cardiomyopathy, CAD, valvular 
heart disease, pericardial disease, arrhythmias, stroke, peripheral arterial disease), 
cardiovascular risk factors (hypertension, DM, tobacco use, dyslipidemia, family history of 
cardiovascular disease, CKD, autoimmune disorder), or use of medications for cardiovascular 
disease or its risk factors. In addition, healthy individuals must have normal cardiac structure 
and function, to be confirmed by conventional transthoracic echocardiography at the time of 
the screening visit. We will use the “Research Studies” email sent to accounts at Vanderbilt 
University Medical Center and the Research Study Volunteer Program, a registry that enables 
identification of healthy volunteers. 
 
Hypertensive (n=20). We will recruit subjects matched on gender to the HFpEF group. Subjects 
in the hypertensive group will have a physician confirmed diagnosis of hypertension, but 
without known cardiovascular disease (as defined above). Hypertension will be defined as a 
physician confirmed diagnosis of hypertension, history of blood pressure >140/90 mmHg and 
treatment with ≥ 1 anti-hypertensive medication. At the time of the protocol visit, the 
hypertensive subjects must have adequate blood pressure control, defined as < 160/100 
mmHg. Hypertensive subjects with other cardiovascular risk factors (as previously defined) will 
be excluded to reduce confounding. In addition, hypertensive individuals must have preserved 
LVEF, defined as ≥ 50%. We will recruit hypertensive patients through several means, including 
the “Research Studies” email sent to accounts at Vanderbilt University Medical Center, 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 8 
 
identification of patients from the Vanderbilt Heart and Vascular Institute clinics, and 
Vanderbilt general medicine and Hypertension clinics. 
 
2.2.2 Inclusion/Exclusion Criteria 
 
Subject Selection: Regardless of study group, any potential subjects with contraindications to 
cardiac imaging or adverse reactions to echocardiographic contrast, 11C Acetate, gadolinium, 
regadenoson or its reversal agent (aminophylline), or 13N-Ammonia will be excluded. Based 
upon the number of subjects needed for the study, we expect the number of subjects screened 
and eligible will exceed the number needed to enroll. An important aspect of the 
inclusion/exclusion criteria is the age requirement of ≥ 50 years old. The typical HFpEF patient is 
in the sixth decade of life or older,9, 10 and therefore, in order to capture a representative HFpEF 
population, we will specifically target this age range. Recruitment will be monitored on an 
ongoing basis by the PI. The investigators’ research group has substantial experience recruiting 
participants into clinical studies and trials using the mechanisms outlined above. 
 
Table 2.  Key Inclusion and Exclusion Criteria 
Study Group Inclusion 
Exclusion 
All 
• 
Age ≥ 50 years 
• 
Able to provide informed 
consent 
• 
eGFR ≥ 45 ml/min/1.73m2 
• 
Coronary artery disease 
• 
Contraindication to cMRI (e.g. 
claustrophobia, metal) 
• 
Weight > 350 lbs (PET & MRI limits) 
• 
Inability to lie flat for imaging 
• 
Pregnancy 
• 
Anemia, Hgb < 10 
• 
Contraindication to regadenoson or 
aminophylline 
Healthy 
• 
Normal cardiac structure & 
function 
• 
BP < 140/90 mmHg 
• 
Known cardiovascular disease (see text), 
risk factors (see text), or use of cardiac 
medications 
• 
Diabetes Mellitus  
Hypertensive 
• 
History of BP > 140/90 
mmHg 
• 
≥1 anti-hypertensive 
medication 
• 
LVEF ≥ 50% 
• 
Current BP < 160/100 mmHg 
• 
Known cardiovascular disease or risk 
factors (other than hypertension) or use 
of cardiac medications (other than anti-
hypertensives or statins) 
• 
Diabetes Mellitus 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 9 
 
HFpEF 
• 
Physician confirmed Dx of HF 
• 
Symptomatic HF (NYHA II or 
III) 
• 
LVEF ≥ 50% 
• 
↑LV filling pressures (see 
text) 
• 
Current BP < 160/100 mmHg 
• 
Prior history of LVEF < 50% 
• 
Acute decompensated HF 
• 
≥ Moderate valvular disease 
• 
Significant arrhythmias (AF, SVT, VT) 
• 
Pericardial disease 
• 
Congenital heart disease 
• 
1’ Pulmonary Arterial hypertension 
 
 
HFpEF subjects 
 
Detailed inclusion criteria 
1) Age ≥ 50 years old regardless of sex and race 
2) Physician confirmed diagnosis of HFpEF, with corroborating evidence of: 
a. Left ventricular ejection fraction ≥ 50%, and 
b. Symptomatic heart failure based upon Framingham criteria, and 
c. Recent heart failure hospitalization within the prior 6 months, or 
d. Evidence of elevated left ventricular filling pressures by any of the following (LVEDP or PCWP 
> 12 mmHg, BNP > 100 pg/ml, echocardiographic E/e’ > 15). 
 
Detailed exclusion criteria for HFpEF 
1) Age < 50 years old 
2) Inability to provide written informed consent 
3) Insufficient data to confirm the diagnosis of HFpEF 
4) Known coronary artery disease (prior myocardial infarction, coronary revascularization, or 
stenosis >50%), or subjects with symptoms or signs of acute myocardial ischemia, for example 
unstable angina or cardiovascular instability, who may be at greater risk 
5) HFpEF due to pericardial disease 
6) HFpEF due to right ventricular failure and/or pulmonary arterial hypertension alone. 
7) Inability to lie flat for non-invasive cardiac imaging procedures 
8) Decompensated and/or acute heart failure requiring active therapy with IV diuretics 
9) Estimated glomerular filtration rate < 45 ml/min/1.73m2 and/or on dialysis. 
10) Indwelling metal that is unsafe/incompatible with cardiac magnetic resonance imaging. 
11) Any history of adverse reactions or contraindications to cardiac imaging, including adverse 
reactions and/or contraindications to echocardiographic contrast, gadolinium, 11C-Acetate, or 
13N-Ammonia. 
12) Any history of adverse reactions or contraindications to regadenoson. 
13) Any history of adverse reactions or contraindications to aminophylline (the reversal agent 
for regadenoson). 
14) Uncontrolled hypertension (≥160/100mmHg) or symptomatic hypotension (systolic blood 
pressure <90mmHg) 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 10 
 
15) Obesity, with weight or waist circumference greater than the compatible limit of cMRI 
and/or PET scanners. 
16) Active pregnancy. 
17) Prior cardiac transplant. 
18) Inability to complete cardiac imaging procedures over 2 consecutive days. 
19) Inability to fast for 6 hours and abstain from caffeine intake for >24 hours 
20) Non-English speaking 
 
Hypertensive subjects without heart failure 
 
Detailed inclusion criteria for hypertensive controls 
1) Age ≥ 50 years old regardless of sex and race 
2) No known cardiovascular disease, including but not limited to coronary heart disease, heart 
failure, valvular stenosis or ≥ moderate valvular regurgitation, arrhythmias, pericardial disease, 
stroke, and/or peripheral arterial disease 
3) No known cardiovascular risk factors, other than hypertension. Specifically, no current or 
prior history of smoking, obesity, chronic kidney disease, or rheumatologic disorders. 
4) No known use of cardiovascular medications, other than anti-hypertensives, lipid lowering 
agents and/or aspirin for primary prevention. Specifically, no current therapy with digoxin 
and/or anti-arrhythmic medications. 
 
Detailed exclusion criteria for hypertensive controls 
1) Age < 50 years old 
2) Inability to provide written informed consent 
3) Known coronary artery disease (prior myocardial infarction, coronary revascularization, or 
stenosis >50%), or subjects with symptoms or signs of acute myocardial ischemia, for example 
unstable angina or cardiovascular instability, who may be at greater risk 
4) Diabetes mellitus  
5) Valvular heart disease (≥ moderate stenosis or regurgitation) 
6) Pericardial disease 
7) Right ventricular failure and/or pulmonary arterial hypertension. 
8) Inability to lie flat for non-invasive cardiac imaging procedures 
9) Estimated glomerular filtration rate < 45 ml/min/1.73m2 and/or on dialysis. 
10) Indwelling metal that is unsafe/incompatible with cardiac magnetic resonance imaging. 
11) Any history of adverse reactions or contraindications to cardiac imaging, including adverse 
reactions and/or contraindications to echocardiographic contrast, gadolinium, 11C-Acetate, or 
13N-Ammonia. 
12) Any history of adverse reactions or contraindications to regadenoson. 
13) Any history of adverse reactions or contraindications to aminophylline (the reversal agent 
for regadenoson). 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 11 
 
14) Uncontrolled hypertension (≥160/100mmHg) or symptomatic hypotension (systolic blood 
pressure <90mmHg) 
15) Obesity, with weight or waist circumference greater than the compatible limit of cMRI 
and/or PET scanners. 
16) Active pregnancy. 
17) Prior cardiac transplant. 
18) Inability to complete cardiac imaging procedures over 2 consecutive days. 
19) Inability to fast for 6 hours and abstain from caffeine intake for >24 hours 
20) Non-English speaking 
 
Healthy Controls 
 
Detailed inclusion criteria for healthy controls 
1) Age ≥ 50 years old regardless of sex and race 
2) No known cardiovascular disease, including but not limited to coronary heart disease, heart 
failure, valvular stenosis or ≥ moderate valvular regurgitation, arrhythmias, pericardial disease, 
stroke, and/or peripheral arterial disease 
3) No known cardiovascular risk factors, including but not limited to hypertension, diabetes 
mellitus, smoking, obesity, chronic kidney disease, or rheumatologic disorders. 
4) No known use of aspirin, statins, and cardiovascular medications, including but not limited to 
beta-blockers, inhibitors of the renal-angiotensin-aldosterone axis, diuretics and/or other anti-
hypertensive medications, digoxin, and/or anti-arrhythmic medications. 
 
Detailed exclusion criteria for healthy controls 
1) Age < 50 years old 
2) Inability to provide written informed consent 
3) Known coronary artery disease (prior myocardial infarction, coronary revascularization, or 
stenosis >50%), or subjects with symptoms or signs of acute myocardial ischemia, for example 
unstable angina or cardiovascular instability, who may be at greater risk 
4) Diabetes mellitus 
5) Valvular heart disease (≥ moderate stenosis or regurgitation) 
6) Pericardial disease 
7) Right ventricular failure and/or pulmonary arterial hypertension. 
8) Inability to lie flat for non-invasive cardiac imaging procedures 
9) Estimated glomerular filtration rate < 45 ml/min/1.73m2 and/or on dialysis. 
10) Indwelling metal that is unsafe/incompatible with cardiac magnetic resonance imaging. 
11) Any history of adverse reactions or contraindications to cardiac imaging, including adverse 
reactions and/or contraindications to echocardiographic contrast, gadolinium, 11C-Acetate, or 
13N-Ammonia. 
12) Any history of adverse reactions or contraindications to regadenoson. 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 12 
 
13) Any history of adverse reactions or contraindications to aminophylline (the reversal agent 
for regadenoson). 
14) Obesity, with weight or waist circumference greater than the compatible limit of cMRI 
and/or PET scanners. 
15) Active pregnancy. 
16) Prior cardiac transplant. 
17) Inability to complete cardiac imaging procedures over 2 consecutive days. 
18) Inability to fast for 6 hours and abstain from caffeine intake for >24 hours 
19) Non-English speaking 
 
2.3 Study Procedures 
 
Screening Visit: At the screening visit, which will take place at the Clinical Research Center, a 
medical history and physical examination will be administered with eligibility for the protocol 
determined (Table 2). A venous blood draw of will be performed for measurement of the 
complete blood count and creatinine to assess inclusion and exclusion criteria.  For healthy and 
hypertensive subjects, an echocardiogram will be performed at the screening visit to assess 
inclusion and exclusion related to cardiac structure and function, if an echocardiogram within 
the previous 6 months is not available. If the subject fulfills the inclusion criteria without 
exclusions to participation, a protocol visit will be scheduled through the Clinical Research 
Center (CRC). 
 
Protocol Visit: There will be one protocol visit during which the subject will undergo testing as 
outlined in Table 3. After arrival at the CRC, the subject will undergo a history and physical 
examination. If any intervening medical events between the screening and protocol visits 
occurred that would exclude the subject from participation, then the subject will be withdrawn 
from the study. A venous blood draw of will be performed for measurement of complete blood 
count, basic chemistry panel, B-type natriuretic peptide, and collection of plasma and serum for 
storage at -80⁰C for future assays of circulating biomarkers. If the subject is anemic 
(hemoglobin <10g/dL), or has an eGFR < 45ml/min/1.73m2 according to the Modified Diet and 
Renal Disease equation then the subject will be withdrawn from the study. Women of child 
bearing potential will also undergo a serum pregnancy test. If the serum pregnancy test is 
positive, the subject will be withdrawn from the study.  Eligible subjects will then undergo 
cardiac imaging as described below. For all imaging procedures, data will be deidentified with 
the exception of a randomly generated subject number, such that imaging data cannot be 
linked directly to subject data, particularly categorization as healthy, hypertensive, or HFpEF. 
This will ensure that the analyzing investigator will be blinded to study group.  
 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 13 
 
  
 
 
 
Echocardiography: At the start of echocardiography, heart rate and blood pressure will be 
recorded. A registered cardiac sonographer will perform comprehensive 2D, M-mode, and 
Doppler transthoracic echocardiography following a pre-specified protocol. A minimum of 3 
complete cardiac cycles will be recorded for each image. Images will be acquired using a Phillips 
IE33 (Phillips Corp, Andover, MD) machine and data will be stored digitally for off-line analysis 
using commercially available software (HeartLab, AGFA and Cardiac Performance Analysis, 
TomTec). Quantification of cardiac structure and function will be performed in accordance with 
guidelines from the American Society of Echocardiography.11-13  In particular, LV size and wall 
thicknesses will be measured using the 2D method, while LV volumes will be measured from 
the apical 4 and 2 chamber views via the Simpson’s Biplane method. LV ejection fraction will be 
calculated as LV stroke volume/end diastolic volume. To optimize assessment of left ventricular 
volumes and ejection fraction, intravenous echocardiographic contrast (Definity, Lantheus 
Medical Imaging, N Billerica, MA) will be given. LV mass will be calculated using the Devereux 
formula as recommended by the American Society of Echocardiography.14  Diastolic function 
will be assessed using mitral annular tissue Doppler e’ velocities, transmitral spectral Doppler E 
wave velocity and deceleration time, and the ratio of E and A wave velocities, as well as 
isovolumic relaxation time, and left atrial pressures (defined as E/e’). LV systolic function will be 
assessed by LVEF, mitral annular tissue Doppler S’ velocities, and speckle tracking derived global 
longitudinal  strain and strain rates, which are less angle and load dependent when compared 
to LVEF and tissue Doppler analyses. Our group has previous experience in quantitative 
echocardiography, including speckle tracking.15-18 
 
11C-Acetate Positron Emission Tomography for the assessment of oxidative metabolism. We 
will follow an established protocol for 11C-Acetate PET imaging previously used within our lab.6  
An intravenous line will be placed in an upper extremity by trained personnel. The subject will 
then lie supine on the PET/CT scanner and heart rate and blood pressure will be recorded at 
baseline and every 5 minutes. A limited transmission CT scan of the chest will be performed for 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 14 
 
attenuation correction. 11C-Acetate (0.286 mCi/Kg) will then be injected intravenously and PET 
imaging will begin. ECG gated images will be acquired in list mode for 30 minutes and 
reformatted into images of 12 frames of 10 seconds each, 8 frames of 30 seconds, 4 frames of 
60 seconds, 2 frames of 300 seconds, and 1 frame of 600 seconds. The 11C-Acetate images will 
be processed following CT attenuation correction. If the images are uninterpretable due to 
factors such as patient motion, attenuation artifacts, and/or inability to accurately gate images 
with ECG, then the subject will be withdrawn from the study with attempts to replace the 
subject to maintain sample size. Regions of interest over the LV blood pool and LV myocardium 
from the last image of the study will be applied to all preceding frames. Counts in the regions of 
interest will be determined and expressed as counts/pixel. The blood pool and LV myocardium 
activities will be plotted against time. The mono-exponential clearance rate, defined as Kmono, 
will be determined by least-squares fitting of the linear portion of the time-activity curve. 
Kmono has previously been demonstrated to correlate with myocardial oxygen consumption.19-
21  As the rate of cardiac metabolism may be influenced by cardiac demand, LV efficiency will be 
calculated as the work-metabolic index (WMI) using the equation WMI = (LVSVI x SBP x 
HR)/Kmono, where LVSVI is the LV stroke volume index (stroke volume/body surface area), SBP 
is systolic blood pressure, and HR is heart rate.22  In addition, oxidative metabolism may be 
influenced by blood supply, and therefore we will also assess myocardial blood flow through 
cMRI and PET as described below. 
 
Cardiac Magnetic Resonance Imaging of myocardial blood flow and myocardial fibrosis. cMRI 
will be performed on a 1.5T Siemens Magnetom Avanto scanner (Siemens, Erlangen, Germany). 
Subjects will be scanned with a phased array torso receiver coil. Following localizer images for 
identification of the heart within the thorax, cine imaging (“steady state free procession”) will 
be performed to generate conventional 4 and 2 chamber views, as well as short axis images. A 
short axis “stack” of cine images from the base to apex of the heart will be obtained, from 
which LV wall thickness, mass, volumes, and ejection fraction will be determined. Typical 
acquisition parameters will be used: field of view 300x340mm, matrix 156x192, slice thickness 8 
mm, flip angle 80⁰ with downward adjustment depending upon the specific absorption rate, 
echo time 1.1ms, bandwidth 930Hz/pixel, and at least 30 phases per cycle to maintain a 
repetition time < 50 ms. Parallel imaging will be used with generalized autocalibrating partially 
parallel acquisition (GRAPPA) using an acceleration factor of 2.6 
 
Following cine imaging, myocardial perfusion imaging will be performed at rest following dual 
bolus (pre bolus =0.005 mmol/kg; main bolus = 0.05 mmol/kg) intravenous injection of 
Gadobutrol (Gd-DO3A-butrol: Gadavist, Bayer Healthcare Pharmaceuticals, Wayne, IN), as 
previously described.23-27  First pass imaging of the wash in of Gd-DO3A-butrol through the LV 
myocardium will be obtained in 3 short axis imaging planes centered at the mid LV at the level 
of the papillary muscles, the basal LV below the mitral valve, and apical LV. T1 weighted 
saturation-recovery turbo fast low angle shot (FLASH) gradient echo sequences will be used for 
myocardial perfusion imaging. All short axis images for myocardial perfusion imaging will be 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 15 
 
acquired over 50-60 consecutive cardiac cycles beginning with injection of the Gd-DO3-butrolA 
contrast. Typical acquisition parameters for first pass imaging will be used: field of view 
320x400mm, matrix 116x192, slice thickness 8 mm, skip 8 mm, flip angle 12⁰, echo time 1.1 
msec, repetition time 160 msec, bandwidth 930Hz/pixel, and parallel imaging with an 
acceleration factor of 2. Stress myocardial perfusion imaging will be repeated 15 minutes 
following the resting gadolinium injection. Regadenoson (Astellas Pharma, Northbrook, IL) 400 
μg will be injected intravenously over 5-10 seconds, followed by 5-10 ml saline flush over 5-10 
seconds, as previously described.23, 28  Approximately 60 seconds following regadenoson 
injection, gadolinium will be injected intravenously using the dual bolus method with imaging 
performed as described above. Heart rate, rhythm, and blood pressure will be monitored 
throughout the imaging protocol. The myocardial perfusion index (MPI) will be calculated 
offline as the maximum slope of the time-intensity curve of myocardial enhancement according 
to the following equation: MPI = maximum slope (myocardium)/maximum slope (LV cavity).6  
MPI has previously been demonstrated to be a valid tool for quantification of myocardial blood 
flow.29, 30  Myocardial perfusion reserve index will then be calculated as the ratio of stress to 
rest myocardial perfusion indices. Similar analyses will be performed to evaluate myocardial 
blood flow (MPI and MPRI) in the subendocardial and subepicardial layers of the left ventricle. 
 
Myocardial fibrosis will be non-invasively quantified using cMRI.31  A short axis image at the mid 
LV will be obtained pre-contrast using the Modified Look-Locker Inversion recovery (MOLLI) 
sequence with imaging at the same level repeated at 10 and 15 minutes post intravenous 0.15 
mmol/kg Gd-DO3A-butrol contrast injection.32, 33  The MOLLI sequence consists of 3 consecutive 
inversion recovery prepared ECG gated Look-Locker trains. Each train begins with an inversion 
pulse at a specific inversion time (T1= 100, 200, and 350 msec), after which multiple single-shot 
steady state free procession images will be acquired over consecutive heart beats. All images 
will be acquired with the same trigger delay time in end diastole. Scanning parameters will be a 
field of view of 360x360mm, matrix 192x193, slice thickness 8 mm, flip angle 35, echo time 1.1 
ms, repetition time 2.2ms, GRAPPA factor of 2. Prior to the delivery of Gd-DO3A-butrol 
intravenous contrast, the “pre-contrast” T1 relaxation time will be determined. Similarly at 10 
and 15 minutes post contrast, T1 relaxation times will be determined. The T1 relaxation time 
has previously been demonstrated to correlate with the extracellular volume of fibrosis by 
histology.31, 34-36  The percent extracellular fibrosis will be quantified using the following 
previously validated equation: λ x (1 – hematocrit), where λ = (ΔRImyocardium/ΔRIblood pool) 
where RI=1/T1 and Δ corresponds to the change in RI pre and post Gd-DO3A-butrol contrast.37  
In addition to T1 mapping, we will also examine late gadolinium enhancement (LGE) as a 
measure of regional myocardial fibrosis.31  At 10-15 minutes post Gd-DO3A-butrol contrast 
injection, short axis myocardial LGE imaging will be performed using both single-shot inversion 
recovery and phase-sensitive inversion recovery true FISP imaging acquired every other cardiac 
cycle. In order to select the optimal inversion time (TI), a TI “scout” will be acquired prior to LGE 
imaging, using progressively longer TI times. The optimal TI time will be determined by visual 
estimation of when the myocardium appears the  darkest.6 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 16 
 
 
13N-Ammonia Positron Emission Tomography for the assessment of myocardial blood flow. 
13N-Ammonia studies will be performed at rest and stress on a whole body PET/CT scanner as 
previously described.38  An initial transmission scan will be obtained for the purpose of 
attenuation correction. Next, rest imaging will be initiated in list mode upon injection of 25mCi 
of intravenous 13N-Ammonia with serial images acquired over approximately 15-20 minutes. 
Approximately 50 minutes after the initial 13N-Ammonia injection (5 half-lives), imaging will be 
repeated following pharmacologic vasodilation with regadenoson (Astellas Pharma, 
Northbrook, IL). As with the cMRI protocol, 400 μg of regadenoson will be injected 
intravenously over 5-10 seconds, followed by a 5-10 ml saline flush. Approximately 60 seconds 
after the regadenoson injection, 25mCi of 13N-Ammonia will be injected with PET imaging in 
list mode. Heart rate, rhythm, and blood pressure will be monitored throughout the imaging 
protocol. Myocardial blood flow and coronary flow reserve will be quantified off-line using 
FlowQuant software (Ottawa Heart Institute, Ottawa, Canada). 
 
 
 
Reporting of clinical findings. As the imaging studies are being performed for research 
purposes, clinical reports will not be generated. However, if unexpected or previously unknown 
abnormalities of clinical importance are identified or urgent/emergent conditions are found, 
these will be clinically reported to both the patient and the patient’s physicians. 
 
3. Statistical Methods  
 
Aim 1. To compare myocardial blood flow reserve in individuals with HFpEF versus those with 
hypertension and healthy individuals.   
 
Primary Hypothesis:  Compared to healthy and hypertensive individuals, subjects with HFpEF 
have reduced myocardial blood flow reserve. 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 17 
 
 
Secondary Hypotheses: 
A) Myocardial blood flow is progressively impaired from healthy, to hypertensive, to HFpEF.  
B) Myocardial blood flow reserve by Gd enhanced cMRI (MPRI) is correlated with CFR by 13N-
Ammonia PET. 
C) Subendocardial MPRI is lower than subepicardial MPRI in HFpEF. 
D) Subendocardial MPRI is progressively impaired from healthy to hypertensive to HFpEF. 
E) Myocardial blood flow reserve is positively correlated with LV diastolic function.  
F) Myocardial blood flow reserve is positively correlated with LV systolic function. 
 
 
Analysis for aim 1.  Primary outcome: Myocardial blood flow reserve, as measured by MPRI 
from contrast enhanced cMRI first pass perfusion imaging and CFR from 13N-Ammonia PET 
perfusion imaging will be compared between the three study groups: healthy, hypertensives, 
and HFpEF.  The distributions of each within each study group will be assessed using 
histograms, Q norm plots, and the Shapiro Wilk test.  If normally distributed then analysis of 
variance will be used to test for differences in myocardial blood flow reserve between groups.  
If the p-value from the ANOVA is < 0.05, then pairwise comparisons using unpaired t-tests with 
unequal variances will be made between groups using a Bonferroni correction.  If MPRI or CFR 
is not normally distributed, then a Kruskal-Wallis test and Wilcoxon rank sum tests will be used 
in place of ANOVA and t-tests.  The null hypothesis is that myocardial blood flow reserve does 
not differ among HFpEF patients, healthy individuals, and hypertensive individuals.  We will also 
assess correlations (Spearman) between myocardial blood flow reserve, age, gender, blood 
pressure, heart rate, and body mass index.  To account for potential confounders, multivariable 
linear regression models will be constructed with myocardial blood flow reserve as the 
dependent variable, study group as the primary independent variable, with adjustments for 
age, gender, race/ethnicity, anti-hypertensive medications, systolic blood pressure, heart rate, 
body mass index, LVEF, LV end diastolic volume, and LV mass.  Prior to entry into models, 
variables will be log transformed, as appropriate.  We will check model fit by residual plots.  
Missing data will not be imputed as we will attempt to replace subjects with significant 
amounts of missing data due to uninterpretable images, as detailed above.  
  


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 18 
 
Statistical power for Aim 1:  Using a one-
way ANOVA at p = 0.05, we present 
between group differences in myocardial 
blood flow reserve as assessed by CFR 
under different sample size and power 
scenarios in Table 4.  We anticipate 
having 80% power to detect a between group difference (largest to smallest) in mean values of 
CFR of 0.87, using a conservative estimate for the standard deviation of 0.90 when compared to 
those reported for normal controls and patients with hypertension, 0.68 and 0.88, 
respectively.39  The between group difference in CFR is within the range of difference detected 
between normals (3.17 ± 0.68) and patients with hypertension (2.18 ± 0.88), previously 
reported.39  Of note even if sample size is reduced by 25%, we still anticipate having good 
power to detect a between group difference of 1.05.  Therefore, using conservative sample 
sizes that account for potential dropout or uninterpretable images, as well as larger estimates 
for standard deviation than previously reported, we should have good power to detect 
plausible differences in myocardial blood flow reserve between HFpEF, healthy, and 
hypertensive subjects. 
 
Secondary analyses:  The secondary hypotheses are presented above.  In addition to testing 
the primary hypothesis for between group differences in myocardial blood flow reserve, we will 
also evaluate for a significant trend across the three study groups using Spearman correlation.  
The relationships between myocardial blood flow reserve derived from cMRI (MPRI) and PET 
(CFR) will be compared by Spearman correlation as well as intraclass correlation in all subjects 
and by subject group.  Using ANOVA, we will also assess whether subendocardial MPRI is lower 
in HFpEF as compared to healthy and hypertensive controls.  Similarly we will assess whether 
subendocardial MPRI is lower than subepicardial MPRI among HFpEF subjects using paired t-
tests.  The relationships between myocardial blood flow reserve and cardiac function will also 
be evaluated.  Scatterplots and Spearman correlations will be performed with myocardial blood 
flow reserve as the independent variable and measures of diastolic function as the dependent 
variables (mitral annular tissue Doppler e’ velocities, isovolumic relaxation time, transmitral 
spectral Doppler E wave velocity, and E/e’).  A nominal p-value threshold of < 0.05 will be used 
for all correlations and results meeting significance at the nominal threshold will be considered 
for follow up study.  Similarly, we all also examine scatterplots and Spearman correlations 
between myocardial blood flow reserve and systolic function (LVEF, mitral annular tissue 
Doppler s’ velocities, and global longitudinal strain).  For both systolic and diastolic parameters, 
we will perform multivariable linear regression analyses adjusting for age, gender, 
race/ethnicity, systolic blood pressure, heart rate, body mass index, LVEF, LV end diastolic 
volume, and LV mass.  We will also test for effect modification on the relationships between 
myocardial blood flow reserve and diastolic and systolic function by including multiplicative 
interaction terms in the linear regression models.  Prior to entry into regression models non-
normally distributed variables will be appropriately transformed. 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 19 
 
 
Aim 2. To compare myocardial oxidative metabolism in individuals with HFpEF versus those 
with hypertension and healthy individuals.   
 
Primary Hypothesis:  Compared to healthy and hypertensive individuals, subjects with HFpEF 
have reduced oxidative metabolism. 
 
Secondary Hypotheses: 
A) Oxidative metabolism is progressively impaired from healthy, to hypertensive, to HFpEF.  
B) Oxidative metabolism is positively correlated with LV diastolic function.  
C) Oxidative metabolism is positively correlated with LV systolic function. 
 
Analysis for aim 2.  Primary outcome: Oxidative metabolism, quantified by Kmono from 11C-
Acetate PET imaging, will be compared between the three study groups: healthy, 
hypertensives, and HFpEF.  The distribution of Kmono within each study group will be assessed 
using histograms, Q norm plots, and the Shapiro Wilk test.  If normally distributed then analysis 
of variance will be used to test for differences in Kmono between groups.  If the p-value from the 
ANOVA is < 0.05, then pairwise comparisons using unpaired t-tests with unequal variances will 
be made between groups using a Bonferroni correction.  If Kmono is not normally distributed, 
then a Kruskal-Wallis test and Wilcoxon rank sum tests will be used in place of ANOVA and t-
tests.  The null hypothesis is that oxidative metabolism (Kmono), does not differ among HFpEF 
patients, healthy individuals, and hypertensive individuals.  We will also check for correlations 
(Spearman) between Kmono, age, gender, blood pressure, heart rate, body mass index, and 
resting myocardial blood flow.  To account for potential confounders, multivariable linear 
regression models will be constructed with oxidative metabolism (Kmono) as the dependent 
variable, study group as the primary independent variable, with adjustments for age, gender, 
race/ethnicity, anti-hypertensive medications, systolic blood pressure, heart rate, body mass 
index, LVEF, LV end diastolic volume, LV mass, and resting myocardial blood flow.  Prior to entry 
into models, variables will be log transformed, as appropriate.  We will check model fit by 
residual plots.  Missing data will not be imputed as we will attempt to replace subjects with 
significant amounts of missing data due to uninterpretable images, as detailed above.  
  
Statistical power for Aim 2:  Using a 
one-way ANOVA at p = 0.05, we 
present between group differences in 
Kmono under different sample size and 
power scenarios in Table 5.  We 
anticipate having 80% power to detect 
a between group difference (largest to smallest) in mean values of Kmono of 0.010, using a 
conservative estimate for the standard deviation of 0.010 when compared to those reported 
for normal controls and patients with HFrEF, 0.006 and 0.001, respectively.  The between group 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 20 
 
difference in Kmono is well within the range of difference detected between normals (0.060 ± 
0.006) and patients with HFrEF (0.041 ± 0.001), previously reported by our group.6, 40  Of note 
even if sample size is reduced by 25%, we still anticipate having good power to detect a 
between group difference of 0.012.  Therefore, using conservative sample sizes that account for 
potential dropout or uninterpretable images, as well as larger estimates for standard deviation 
than previously reported, we should have good power to detect plausible differences in 
oxidative metabolism between HFpEF, healthy, and hypertensive subjects.   
 
Secondary analyses:  The secondary hypotheses are presented above.  In addition to testing 
the primary hypothesis for between group differences in Kmono, we will also evaluate for a 
significant trend across the three study groups using Spearman correlation.  The relationships 
between oxidative metabolism and cardiac function will also be evaluated.  Scatterplots and 
Spearman correlations will be performed with oxidative metabolism as the independent 
variable and measures of diastolic function as the dependent variables (mitral annular tissue 
Doppler e’ velocities, isovolumic relaxation time, transmitral spectral Doppler E wave velocity, 
and E/e’).  A nominal p-value threshold of < 0.05 will be used for all correlations and results 
meeting significance at the nominal threshold will be considered for follow up study.  Similarly, 
we all also examine scatterplots and Spearman correlations between oxidative metabolism and 
systolic function (LVEF, mitral annular tissue Doppler s’ velocities, and global longitudinal 
strain).  For both systolic and diastolic parameters, we will perform multivariable linear 
regression analyses adjusting for age, gender, race/ethnicity, systolic blood pressure, heart rate, 
body mass index, LVEF, LV end diastolic volume, LV mass, and resting myocardial blood flow.  
We will also test for effect modification on the relationships between oxidative metabolism and 
diastolic and systolic function by including multiplicative interaction terms in the linear 
regression models.  Prior to entry into regression models non-normally distributed variables will 
be appropriately transformed. 
 
Aim 3.  To compare myocardial fibrosis in individuals with HFpEF versus healthy individuals 
and those with hypertension.   
 
Primary Hypothesis:  Compared with healthy and hypertensive individuals, subjects with HFpEF 
have increased myocardial fibrosis. 
 
Secondary Hypotheses:  
A)  Myocardial fibrosis progressively increases from healthy, to hypertensive, to HFpEF. 
B)  Myocardial fibrosis is inversely correlated with LV diastolic function.   
C)  Myocardial fibrosis is inversely correlated with LV systolic function. 
 
Analysis for aim 3.  Primary outcome: myocardial fibrosis assessed by T1 relaxation times by 
cMRI will be compared between the three study groups.  The analyses for aim 3 will be the 
same as for aim 2, except that myocardial fibrosis, instead of oxidative metabolism, will be 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 21 
 
compared. The distribution of T1 relaxation times, as a measure of myocardial fibrosis, within 
each study group will be assessed.  Based upon distributions, ANOVA or Kruskal-Wallis tests will 
be used to identify differences between study groups and if significant p < 0.05, then pairwise 
comparisons with T-tests or Wilcoxon rank sum tests will be performed with Bonferroni 
correction.  The null hypothesis is that T1 relaxation times do not differ among HFpEF, healthy, 
and hypertensive individuals.  We will also check for correlations (Spearman) between 
myocardial fibrosis, age, gender, blood pressure, heart rate, body mass index, and resting 
myocardial blood flow.  To account for potential confounders, multivariable linear regression 
models will be constructed with myocardial fibrosis as the dependent variable, with 
adjustments for the same covariates described for oxidative metabolism above. 
 
Statistical power for Aim 3:  Using an 
ANOVA at p = 0.05, we present between 
group differences in T1 relaxation times 
(msec) under different sample size and 
power scenarios in Table 6.  We anticipate 
having 80% power to detect a between 
groups difference (largest to smallest) in T1 relaxation times of 60 msec, using a conservative 
estimate for the standard deviation of 60 msec, which was based on a 51 msec SD observed in a 
HFpEF population (mean T1 time 381msec).41  The difference in T1 relaxation times is well 
within the range of expected difference based upon reported values for normals (459 ± 38) in 
the MESA study and patients with HFpEF (381 ± 51).32, 41  Of note, even if sample size is reduced 
by 25%, we still anticipate having good power to detect a between group difference of 70 msec, 
which is still less than the clinical relevant difference of ~80 msec.  Therefore, using 
conservative sample sizes, accounting for potential dropout or uninterpretable images, as well 
as a larger standard deviation estimate than previously reported, we should have good to 
excellent power to detect plausible differences in myocardial fibrosis between HFpEF, healthy, 
and hypertensive subjects.   
 
Secondary analyses:  The secondary hypotheses are detailed above and evaluate for 
progressive increases in myocardial fibrosis across study groups as well as the relationships 
between myocardial fibrosis and cardiac function.  The analyses for myocardial fibrosis will 
follow the same statistical methods to those described for oxidative metabolism.  Myocardial 
fibrosis, as assessed by T1 relaxation times, will be the independent variable and assessed in 
relation to features of diastolic and systolic function as previously defined above. 
   
 
Aim 4. To evaluate the relationships between myocardial blood flow reserve, oxidative 
metabolism and fibrosis. 
 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 22 
 
Primary Hypothesis: Myocardial blood flow reserve is positively correlated with oxidative 
metabolism and inversely correlated with myocardial fibrosis. 
 
Analysis for Aim 4: For all subjects completing gadolinium enhanced cMRI myocardial perfusion 
imaging, 13N-Ammonia PET perfusion imaging, 11C-Acetate PET imaging and cMRI fibrosis 
imaging, the relationships between myocardial blood flow reserve, oxidative metabolism, and 
fibrosis will be evaluated using Spearman correlation.  To account for potential confounders, 
multivariable linear regression models will be constructed with myocardial fibrosis or oxidative 
metabolism as the dependent variable, myocardial blood flow reserve as the primary 
independent variable with adjustments for age, gender, race/ethnicity, anti-hypertensive 
medications, systolic blood pressure, heart rate, body mass index, LVEF, LV end diastolic 
volume, LV mass, and study group.  We will check model fit by residual plots.  Prior to entry into 
regression models non-normally distributed variables will be appropriately transformed. 
 
Statistical power for Aim 4:  We present different margins of error for the Spearman 
correlation coefficient based upon 
different sample sizes and 
correlation coefficients in Table 7.  
Based upon a sample size of 60 
subjects, modest correlation 
coefficients of |0.40| to |0.50| 
would have 0.24 and 0.22 margins 
of error, respectively. 
 
 
4. Data Management 
 
Patient clinical data is stored in Vanderbilt’s electronic medical record. The hospital database is 
password protected and accessible only to the appropriate personnel.  Research data will be 
labeled with a patient study code and stored in a separate research database.  The code key 
connecting names to specific information will be available only to members of the research 
staff and will be kept in a separate, secure location. 
 
The research data will be stored in a HIPPA-compliant manner in a REDCap database accessible 
to only the PI and key study personnel.  Only those personnel that are directly involved in the 
study will have access to this database. 
 
5. Safety Reporting 
 
5.1 Serious Adverse Events 
 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 23 
 
Definitions of Serious Adverse Events  
A serious adverse event or reaction is any untoward medical occurrence that at any dose: 
 
• 
Results in death; 
• 
Is life-threatening 
(Defined as an event in which the patient 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.); 
• 
Requires inpatient hospitalization or prolongation of existing hospitalization; 
• 
Results in persistent or significant disability/incapacity; 
• 
Results in a congenital anomaly or birth defect; 
• 
Results in the development of drug dependency or drug abuse; 
• 
Is an important medical event- 
(Defined as a medical event(s) that may not be immediately life-threatening or result in 
death or hospitalization but, based upon appropriate medical and scientific judgment, 
may jeopardize the patient/subject or may require intervention (e.g., medical, surgical) 
to prevent one of the other serious events or reactions listed above.  Examples of such 
events include, but are not limited to, intensive treatment in an emergency room or at 
home for allergic bronchospasm and blood dyscrasias or convulsions that do not result 
in hospitalization. For reporting purposes, Lantheus also considers the occurrences of 
pregnancy, cancer or overdose (regardless of adverse outcome) as events which must 
be reported as important medical events.) 
 
SAE Reporting to Regulatory Bodies 
 
Regardless of causality, all SAEs will be documented and reported to regulatory bodies, 
including the IRB as required by the International Conference on Harmonization (ICH) 
Guidelines for Good Clinical Practice (GCP).  Follow-up information for SAEs and information for 
non-serious AEs that become serious will also be reported in the same manner. 
 
SAE’s will be reported to the Vanderbilt Human Research Protection Program according to 
institutional policies and procedures. 
 
Study Withdraw/Discontinuation 
Patients who wish to be withdrawn from the study will notify the PI who will have any of their 
data that has not been used or analyzed removed from the REDCap database.  
 
Privacy/Confidentiality Issues 
 
Patient privacy and confidentiality will be protected by the usual practices of the 
echocardiography laboratory.  The confidentiality of patient data will be protected by 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
Confidential 
 
Page 24 
 
maintaining a single master list linking Study ID (from the REDCap database) with StarPanel 
Medical Record Number, which will be stored in the REDCap file repository.   
 
 
 
 
 
 
 
 
 
REFERENCES 
 
1. 
Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Borden WB, Bravata DM, Dai 
S, Ford ES, Fox CS, Franco S, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, 
Huffman MD, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Magid D, 
Marcus GM, Marelli A, Matchar DB, McGuire DK, Mohler ER, Moy CS, Mussolino ME, Nichol 
G, Paynter NP, Schreiner PJ, Sorlie PD, Stein J, Turan TN, Virani SS, Wong ND, Woo D, Turner 
MB, American Heart Association Statistics C and Stroke Statistics S. Heart disease and 
stroke statistics--2013 update: a report from the American Heart Association. Circulation. 
2013;127:e6-e245. 
2. 
Roger VL. Epidemiology of heart failure. Circ Res. 2013;113:646-59. 
3. 
Meta-analysis Global Group in Chronic Heart F. The survival of patients with heart 
failure with preserved or reduced left ventricular ejection fraction: an individual patient 
data meta-analysis. Eur Heart J. 2012;33:1750-7. 
4. 
Borlaug BA and Redfield MM. Diastolic and systolic heart failure are distinct 
phenotypes within the heart failure spectrum. Circulation. 2011;123:2006-13; discussion 
2014. 
5. 
Paulus WJ and Tschope C. A novel paradigm for heart failure with preserved ejection 
fraction: comorbidities drive myocardial dysfunction and remodeling through coronary 
microvascular endothelial inflammation. J Am Coll Cardiol. 2013;62:263-71. 
6. 
Bell SP, Adkisson DW, Ooi H, Sawyer DB, Lawson MA and Kronenberg MW. 
Impairment of subendocardial perfusion reserve and oxidative metabolism in nonischemic 
dilated cardiomyopathy. J Card Fail. 2013;19:802-10. 
7. 
Doenst T, Nguyen TD and Abel ED. Cardiac metabolism in heart failure: implications 
beyond ATP production. Circ Res. 2013;113:709-24. 
8. 
Murthy VL, Naya M, Foster CR, Hainer J, Gaber M, Di Carli G, Blankstein R, Dorbala S, 
Sitek A, Pencina MJ and Di Carli MF. Improved cardiac risk assessment with noninvasive 
measures of coronary flow reserve. Circulation. 2011;124:2215-24. 
9. 
Owan TE, Hodge DO, Herges RM, Jacobsen SJ, Roger VL and Redfield MM. Trends in 
prevalence and outcome of heart failure with preserved ejection fraction. N Engl J Med. 
2006;355:251-9. 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
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10. 
Bhatia RS, Tu JV, Lee DS, Austin PC, Fang J, Haouzi A, Gong Y and Liu PP. Outcome of 
heart failure with preserved ejection fraction in a population-based study. N Engl J Med. 
2006;355:260-9. 
11. 
Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA, Picard MH, 
Roman MJ, Seward J, Shanewise JS, Solomon SD, Spencer KT, Sutton MS, Stewart WJ, 
Chamber Quantification Writing G, American Society of Echocardiography's G, Standards C 
and European Association of E. Recommendations for chamber quantification: a report 
from the American Society of Echocardiography's Guidelines and Standards Committee and 
the Chamber Quantification Writing Group, developed in conjunction with the European 
Association of Echocardiography, a branch of the European Society of Cardiology. J Am Soc 
Echocardiogr. 2005;18:1440-63. 
12. 
Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth OA, Waggoner AD, 
Flachskampf FA, Pellikka PA and Evangelista A. Recommendations for the evaluation of left 
ventricular diastolic function by echocardiography. J Am Soc Echocardiogr. 2009;22:107-33. 
13. 
Rudski LG, Lai WW, Afilalo J, Hua L, Handschumacher MD, Chandrasekaran K, 
Solomon SD, Louie EK and Schiller NB. Guidelines for the echocardiographic assessment of 
the right heart in adults: a report from the American Society of Echocardiography endorsed 
by the European Association of Echocardiography, a registered branch of the European 
Society of Cardiology, and the Canadian Society of Echocardiography. J Am Soc 
Echocardiogr. 2010;23:685-713; quiz 786-8. 
14. 
Devereux RB, Alonso DR, Lutas EM, Gottlieb GJ, Campo E, Sachs I and Reichek N. 
Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy 
findings. Am J Cardiol. 1986;57:450-8. 
15. 
Gupta DK, Shah AM, Castagno D, Takeuchi M, Loehr LR, Fox ER, Butler KR, Mosley 
TH, Kitzman DW and Solomon SD. Heart Failure with Preserved Ejection Fraction in 
African-Americans - The Atherosclerosis Risk in Communities (ARIC) Study. JACC Heart 
failure. 2013;1:156-163. 
16. 
Kraigher-Krainer E, Shah AM, Gupta DK, Santos A, Claggett B, Pieske B, Zile MR, 
Voors AA, Lefkowitz MP, Packer M, McMurray JJ, Solomon SD and Investigators P. Impaired 
systolic function by strain imaging in heart failure with preserved ejection fraction. J Am 
Coll Cardiol. 2014;63:447-56. 
17. 
Shah SJ, Aistrup GL, Gupta DK, O'Toole MJ, Nahhas AF, Schuster D, Chirayil N, Bassi 
N, Ramakrishna S, Beussink L, Misener S, Kane B, Wang D, Randolph B, Ito A, Wu M, Akintilo 
L, Mongkolrattanothai T, Reddy M, Kumar M, Arora R, Ng J and Wasserstrom JA. 
Ultrastructural and cellular basis for the development of abnormal myocardial mechanics 
during the transition from hypertension to heart failure. Am J Physiol Heart Circ Physiol. 
2014;306:H88-H100. 
18. 
Burke MA, Katz DH, Beussink L, Selvaraj S, Gupta DK, Fox J, Chakrabarti S, Sauer AJ, 
Rich JD, Freed BH and Shah SJ. Prognostic importance of pathophysiologic markers in 
patients with heart failure and preserved ejection fraction. Circ Heart Fail. 2014;7:288-99. 
19. 
Brown MA, Myears DW and Bergmann SR. Validity of estimates of myocardial 
oxidative metabolism with carbon-11 acetate and positron emission tomography despite 
altered patterns of substrate utilization. J Nucl Med. 1989;30:187-93. 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
MD 
Principal 
Investigator 
 
Protocol version:  04/10/2019 
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Page 26 
 
20. 
Henes CG, Bergmann SR, Walsh MN, Sobel BE and Geltman EM. Assessment of 
myocardial oxidative metabolic reserve with positron emission tomography and carbon-11 
acetate. J Nucl Med. 1989;30:1489-99. 
21. 
Brown M, Marshall DR, Sobel BE and Bergmann SR. Delineation of myocardial 
oxygen utilization with carbon-11-labeled acetate. Circulation. 1987;76:687-96. 
22. 
Knaapen P, Germans T, Knuuti J, Paulus WJ, Dijkmans PA, Allaart CP, Lammertsma 
AA and Visser FC. Myocardial energetics and efficiency: current status of the noninvasive 
approach. Circulation. 2007;115:918-27. 
23. 
Hsu LY, Groves DW, Aletras AH, Kellman P and Arai AE. A quantitative pixel-wise 
measurement of myocardial blood flow by contrast-enhanced first-pass CMR perfusion 
imaging: microsphere validation in dogs and feasibility study in humans. JACC Cardiovasc 
Imaging. 2012;5:154-66. 
24. 
Christian TF, Aletras AH and Arai AE. Estimation of absolute myocardial blood flow 
during first-pass MR perfusion imaging using a dual-bolus injection technique: comparison 
to single-bolus injection method. J Magn Reson Imaging. 2008;27:1271-7. 
25. 
Jerosch-Herold M. Quantification of myocardial perfusion by cardiovascular 
magnetic resonance. J Cardiovasc Magn Reson. 2010;12:57. 
26. 
Lee DC and Johnson NP. Quantification of absolute myocardial blood flow by 
magnetic resonance perfusion imaging. JACC Cardiovasc Imaging. 2009;2:761-70. 
27. 
Utz W, Greiser A, Niendorf T, Dietz R and Schulz-Menger J. Single- or dual-bolus 
approach for the assessment of myocardial perfusion reserve in quantitative MR perfusion 
imaging. Magn Reson Med. 2008;59:1373-7. 
28. 
Bhave NM, Freed BH, Yodwut C, Kolanczyk D, Dill K, Lang RM, Mor-Avi V and Patel 
AR. Considerations when measuring myocardial perfusion reserve by cardiovascular 
magnetic resonance using regadenoson. J Cardiovasc Magn Reson. 2012;14:89. 
29. 
Klocke FJ, Simonetti OP, Judd RM, Kim RJ, Harris KR, Hedjbeli S, Fieno DS, Miller S, 
Chen V and Parker MA. Limits of detection of regional differences in vasodilated flow in 
viable myocardium by first-pass magnetic resonance perfusion imaging. Circulation. 
2001;104:2412-6. 
30. 
Jerosch-Herold M, Vazquez G, Wang L, Jacobs DR, Jr. and Folsom AR. Variability of 
myocardial blood flow measurements by magnetic resonance imaging in the multi-ethnic 
study of atherosclerosis. Invest Radiol. 2008;43:155-61. 
31. 
Mewton N, Liu CY, Croisille P, Bluemke D and Lima JA. Assessment of myocardial 
fibrosis with cardiovascular magnetic resonance. J Am Coll Cardiol. 2011;57:891-903. 
32. 
Liu CY, Liu YC, Wu C, Armstrong A, Volpe GJ, van der Geest RJ, Liu Y, Hundley WG, 
Gomes AS, Liu S, Nacif M, Bluemke DA and Lima JA. Evaluation of age-related interstitial 
myocardial fibrosis with cardiac magnetic resonance contrast-enhanced T1 mapping: 
MESA (Multi-Ethnic Study of Atherosclerosis). J Am Coll Cardiol. 2013;62:1280-7. 
33. 
Bradham W, Bell SP, Smith H, Adkisson D, Lawson MA, Ooi H, Sawyer DB and 
Kronenberg MW. Myocardial T1 in Nonischemic Dilated Cardiomyopathy is Related to 
Circulating Procollagen Type 1 Carboxyterminal Peptide and Predicts Reverse Remodeling 
by Spironolactone Therapy. Circulation. 2013;128:A10179. 


Myocardial perfusion, oxidative metabolism, and fibrosis in 
HFpEF (HFpEF-PrOF) 
Marvin Kronenberg, 
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34. 
Sibley CT, Noureldin RA, Gai N, Nacif MS, Liu S, Turkbey EB, Mudd JO, van der Geest 
RJ, Lima JA, Halushka MK and Bluemke DA. T1 Mapping in cardiomyopathy at cardiac MR: 
comparison with endomyocardial biopsy. Radiology. 2012;265:724-32. 
35. 
White SK, Sado DM, Flett AS and Moon JC. Characterising the myocardial interstitial 
space: the clinical relevance of non-invasive imaging. Heart. 2012;98:773-9. 
36. 
Iles L, Pfluger H, Phrommintikul A, Cherayath J, Aksit P, Gupta SN, Kaye DM and 
Taylor AJ. Evaluation of diffuse myocardial fibrosis in heart failure with cardiac magnetic 
resonance contrast-enhanced T1 mapping. J Am Coll Cardiol. 2008;52:1574-80. 
37. 
Wong TC, Piehler K, Meier CG, Testa SM, Klock AM, Aneizi AA, Shakesprere J, 
Kellman P, Shroff SG, Schwartzman DS, Mulukutla SR, Simon MA and Schelbert EB. 
Association between extracellular matrix expansion quantified by cardiovascular magnetic 
resonance and short-term mortality. Circulation. 2012;126:1206-16. 
38. 
El Fakhri G, Kardan A, Sitek A, Dorbala S, Abi-Hatem N, Lahoud Y, Fischman A, 
Coughlan M, Yasuda T and Di Carli MF. Reproducibility and accuracy of quantitative 
myocardial blood flow assessment with (82)Rb PET: comparison with (13)N-ammonia 
PET. J Nucl Med. 2009;50:1062-71. 
39. 
Alexanderson E, Jacome R, Jimenez-Santos M, Ochoa JM, Romero E, Cabral MA, 
Ricalde A, Inarra F, Meave A and Alexanderson G. Evaluation of the endothelial function in 
hypertensive patients with 13N-ammonia PET. J Nucl Cardiol. 2012;19:979-86. 
40. 
Kronenberg MW, Cohen GI, Leonen MF, Mladsi TA and Di Carli MF. Myocardial 
oxidative metabolic supply-demand relationships in patients with nonischemic dilated 
cardiomyopathy. J Nucl Cardiol. 2006;13:544-53. 
41. 
Mascherbauer J, Marzluf BA, Tufaro C, Pfaffenberger S, Graf A, Wexberg P, 
Panzenbock A, Jakowitsch J, Bangert C, Laimer D, Schreiber C, Karakus G, Hulsmann M, 
Pacher R, Lang IM, Maurer G and Bonderman D. Cardiac magnetic resonance postcontrast 
t1 time is associated with outcome in patients with heart failure and preserved ejection 
fraction. Circ Cardiovasc Imaging. 2013;6:1056-65.