Effect of KNO3 Compared to KCl on Oxygen UpTake in Heart Failure With Preserved Ejection Fraction (KNO3CK OUT HFPEF)
Phase: PHASE2
Conditions: Heart Failure
Sponsor: University of Pennsylvania

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CLINICAL TRIAL PROTOCOL, R01 HL121510 
Effect of KNO3 Compared to KCl on Oxygen UpTake in Heart Failure with  
Preserved Ejection Fraction (KNO3CK OUT HFpEF) 
Version: V12 Date: 07-Oct-2021 
Sponsor: University of Pennsylvania 
Sponsor Representative: Emma A. Meagher, MD 
Sponsor email: psom-ind-ide@pobox.upenn.edu 
 
Funding Sponsor:  National Heart, Lung and Blood Institute (NHLBI) 
Study Chair / Grant Principal Investigator: 
Julio A. Chirinos, M.D., Ph.D. 
 
Site Principal Investigators (alphabetical order) 
Sanjiv Shah, M.D. (P.I., Northwestern Site) 
Sujith Kuruvilla (P.I., Corporal Michael J. Crescenz VAMC Site) 
Payman Zamani, M.D. (P.I., Penn Site) 
 
Sub-Investigators (alphabetical order) 
Thomas Cappola, M.D. 
Jesse Chittams, M.Sc. 
Alexandra Hanlon., Ph.D. 
Kenneth Margulies, M.D. 
Ravinder Reddy, MD, PhD 
Srinath Adusumalli, MD, M.Sc. 
 
Data Coordinating Center: 
Alexandra Hanlon, Ph.D. (Director) and Jesse Chittams, M.Sc. 
NIH Program Officer: 
Patrice Desvigne-Nickens, M.D. 
 

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Table of Contents 
1. SPECIFIC AIMS .................................................................................................................................. 5 
2. TRIAL SUMMARY TABLE (OBJECTIVES AND ENDPOINTS) ........................................................... 8 
3. BACKGROUND AND SIGNIFICANCE ................................................................................................ 9 
3.1. The nitrate–nitrite–NO activation pathway .................................................................................. 10 
3.2. Peripheral mechanisms of exercise intolerance in HFpEF .......................................................... 11 
exercise capacity ............................................................................................................................... 13 
3.4. Rationale behind the use of inorganic nitrate as a therapeutic approach in HFpEF .................... 14 
3.5. Preliminary data: effects of a single-dose of inorganic nitrate in HFpEF ..................................... 15 
3.6 Effect on Myocardial Perfusion Reserve in HFpEF (“Cardiac MRI sub-study”) ............................. 18 
4. STUDY DESIGN AND POPULATION ............................................................................................... 18 
4.1. Overview of study design ............................................................................................................ 18 
4.2 Study Sites .................................................................................................................................. 19 
4.3. Study Population ........................................................................................................................ 19 
4.4 Randomized intervention ............................................................................................................. 22 
5. STUDY VISITS AND PROCEDURES ............................................................................................... 22 
5.1. Initial Visit ................................................................................................................................... 23 
5.2. Randomization procedure ........................................................................................................... 25 
5.3. Intervention Phase 1 ................................................................................................................... 25 
5.4. 1-week washout period ............................................................................................................... 27 
5.5. Intervention Phase 2 ................................................................................................................... 27 
5.6 Adverse Event Reporting ............................................................................................................. 29 
5.7. Subject withdrawal / Early termination ........................................................................................ 29 
5.8. Concomitant Medication ............................................................................................................. 29 
6. DRUG DISPENSING, ACCOUNTABILITY, AND DESTRUCTION .................................................... 30 
6.1. Rules for un-blinding ................................................................................................................... 30 
7. ADVERSE EVENTS .......................................................................................................................... 30 
7.1. Key definitions ............................................................................................................................ 30 
7.2. Classification of AE/ADEs ........................................................................................................... 31 
7.3. Recording and Reporting of Adverse Events .............................................................................. 33 
7.4. Follow-up .................................................................................................................................... 34 
7.5. Management of Suspected Unexpected Serious Adverse Reaction ........................................... 34 
7.6. Pregnancy .................................................................................................................................. 34 
8. STUDY MEASUREMENTS AND DATA COLLECTION ..................................................................... 35 

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8.1. Assessment of exercise capacity ................................................................................................ 35 
8.2. Quality of life assessment ........................................................................................................... 35 
8.3. Assessment of the systemic vasodilatory response to exercise .................................................. 35 
8.4. Assessment of muscle perfusion and energetics ........................................................................ 36 
8.5. Doppler Echocardiography ......................................................................................................... 37 
8.6. Measurement of late systolic load and arterial wave reflections: ................................................. 37 
8.7. Blood nitrate/nitrite level measurements: .................................................................................... 38 
8.8 Cognitive Assessment ................................................................................................................. 38 
8.9. Other assessments ..................................................................................................................... 40 
9. MULTICENTER MANAGEMENT PLAN ............................................................................................ 41 
9.1. Study Chair/Grant Principal Investigator ..................................................................................... 41 
9.2. Site Principal Investigators ......................................................................................................... 41 
9.3. Investigators Committee ............................................................................................................. 41 
9.4. Operational Affairs Committee .................................................................................................... 42 
9.5. Data Management ...................................................................................................................... 42 
9.6. Quantification Core Laboratories ................................................................................................ 43 
9.7 Conflict of Interest Management .................................................................................................. 44 
10. STATISTICAL CONSIDERATIONS ................................................................................................. 44 
10.1. Power calculations .................................................................................................................... 44 
10.2. Data Analysis Plan ................................................................................................................... 45 
10.3. Subgroup analyses and considerations about sex .................................................................... 46 
10.4 Cardiac MRI sub-study analysis and power calculations ............................................................ 47 
11. PROTECTION OF HUMAN SUBJECTS .......................................................................................... 47 
11.1. Potential benefits of the proposed research, importance of the knowledge to be gained, and 
risk/benefit ratio ................................................................................................................................. 47 
11.2. Risks to study subjects ............................................................................................................. 48 
11.3. Adequacy of Protection Against Risks ...................................................................................... 53 
12. REGULATORY STANDARDS ......................................................................................................... 57 
12.1. Informed consent ...................................................................................................................... 57 
12.2. Institutional Review Board (IRB) ............................................................................................... 57 
12.3. Investigational New Drug (IND) application ............................................................................... 57 
12.4. Source document handling and archiving ................................................................................. 57 
13. DATA AND SAFETY MONITORING PLAN: .................................................................................... 57 
14. REFERENCES ................................................................................................................................ 58 
15. APPENDIX 1. SCHEDULE OF EVENTS ......................................................................................... 75 

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16. APPENDIX 2. ANTICIPATED STUDY TIMELINE AND KEY DATES .............................................. 76 
KEY DATES .......................................................................................................................................... 76 
17. APPENDIX 3 Instructions for wearing the Aurora Device (Pulse Watch) ......................................... 76 
18. APPENDIX 4. HISTORY OF CHANGES ......................................................................................... 77 
Version 2 (Feb 2016): Amended to specifically include ...................................................................... 77 
Version 3 (July 2016): ........................................................................................................................ 77 
Version 4 (July 2016) ......................................................................................................................... 78 
Version 5 (August 2016) .................................................................................................................... 78 
Version 6 (October 2016) .................................................................................................................. 78 
Version 7 (January 2017) .................................................................................................................. 78 
Version 8 (July 20177) ....................................................................................................................... 79 
Version 9 (November 2017) ............................................................................................................... 79 
Version 10 (October 2018) ................................................................................................................ 79 
Version 11 (March 2020) ................................................................................................................... 79 
Version 12 (07-Oct-2021) .................................................................................................................. 79 
 
 
 

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1. SPECIFIC AIMS 
Heart failure (HF) affects ~2% of the population and ~10% of adults aged >75 years. 
Approximately 50% of HF patients demonstrate a preserved left ventricular (LV) ejection fraction 
(EF), and thus have HF with preserved EF (HFpEF). Many therapeutic interventions are available 
for HF with reduced EF, but there are currently no proven effective pharmacologic interventions 
for HFpEF. Given its enormous public health, clinical and societal burden, finding effective 
therapies for HFpEF is a top priority. Accumulating evidence implicates various peripheral 
abnormalities in the pathophysiology of HFpEF. 
Exercise intolerance with reduced aerobic capacity is the hallmark of HFpEF and greatly impairs 
quality of life (QOL). Effective O2 extraction requires cardiac output redistribution towards and 
within exercising muscle, mediated by vasodilation in active muscle tissue. Dietary nitrate 
increases plasma nitrite, an endothelium-independent source of the potent vasodilator nitric oxide 
(NO). Importantly, the conversion of nitrite to NO occurs preferentially in the setting of hypoxia, as 
occurs in exercising muscle. Furthermore, nitrate-nitrite-derived NO has been shown to enhance 
mitochondrial function, representing an additional pathway to improve exercise tolerance. 
Therefore, nitrate supplementation represents a logical intervention to improve exercise tolerance 
in HFpEF. Our preliminary data from a double-blinded cross-over placebo-controlled trial 
demonstrates that a single dose of inorganic nitrate increases aerobic capacity (peak O2 
consumption during a symptom-limited maximal effort exercise test), enhances the peripheral 
vasodilator response to exercise, and improves skeletal muscle mitochondrial oxidative function. 
Late systolic LV load is an important determinant of LV dysfunction and remodeling. Wave 
reflections arise in peripheral arterial sites and travel back to the heart, arriving while the LV is still 
ejecting blood, increasing the late systolic workload of the LV. Available evidence causally link 
late systolic load with LV remodeling, diastolic dysfunction, and HF risk. Wave reflections are 
highly sensitive to NO. Our preliminary data demonstrates that a single oral dose of inorganic 
nitrate reduces late systolic load in HFpEF. Thus, in addition to an exercise-enhancing effects, 
augmentation of the nitrate-nitrite-NO pathway has the potential for chronic “disease modifying” 
benefits in HFpEF. 
Based on preliminary data demonstrating the effects of a single oral dose of inorganic nitrate in 
HFpEF, the current trial was designed to assess the efficacy of sustained oral administration of 
inorganic nitrate in a more diverse and representative sample of HFpEF patients and to further 
assess the mechanisms by which inorganic nitrate enhances oxygen uptake and exercise 
capacity. This is a randomized double-blind crossover clinical trial, in which 84 subjects with 
HFpEF will receive the following 2 interventions, in randomized order: (1) Potassium nitrate 
(KNO3), at a dose of 12-18 mmol/d by mouth for approximately 6 weeks, or; (2) Potassium chloride 
(KCl), at a dose of 12-18 mmol/d by mouth for approximately 6 weeks. A 1-week washout period 
will be introduced between the 2 interventions. The purpose of the trial is to test the efficacy of 
KNO3 on a number of clinical and physiologic endpoints in subjects with HFpEF. 
 
Our specific aims are: 

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Specific Aim 1: To assess whether KNO3 therapy improves endpoints with direct clinical 
relevance: aerobic capacity and quality of life (QOL). 
Hypothesis 1a: KNO3 will improve exercise capacity, quantified as: (1) Total work 
performed during a maximal-effort exercise test; (2) Peak oxygen consumption (VO2) 
during a symptom-limited maximal effort exercise test. These will be the co-primary 
endpoints of the trial. 
Hypothesis 1b: KNO3 will improve QOL, assessed using the Kansas City Cardiomyopathy 
Questionnaire. 
Specific Aim 2: To dissect the effects of KNO3 on specific physiologic adaptations to exercise 
Hypothesis 2a: KNO3 improves the systemic vasodilator response to exercise, measured 
as the change in systemic vascular resistance during a symptom-limited maximal effort 
exercise test. 
Hypothesis 2b: KNO3 improves muscle blood flow during exercise, measured with 
flowsensitive PC-MRI during a standardized plantar flexion exercise test. 
Hypothesis 2c: KNO3 improves muscle Cr recovery kinetics (a marker of oxidative 
capacity), measured with MRI. We will also assess the exercise-induced spatial matching 
(i.e., within muscle) of blood flow and oxidative capacity using a highly novel method 
(chemical exchange saturation transfer) that allows, for the first time, for highresolution 
spatial mapping of Cr in muscle. 
Hypotheses 2d-e: Since nitrates may exert venodilating effects and/or myocardial effects, 
we will test the hypotheses that KNO3 impacts LV diastolic filling parameters (measured 
with echocardiography at rest and after peak exercise) and improves myocardial strain 
(assessed with speckle-tracking echocardiography). 
Specific Aim 3: To assess whether KNO3 reduces late systolic LV load from wave reflections 
(assessed via comprehensive aortic pressure-flow relations, using arterial tonometry and Doppler 
echocardiography). 
We will explicitly ensure that a balanced sex distribution is present in our sample, and that African-
Americans are adequately represented. We will pre-specify stratified analyses of men vs. women 
and African American vs. non-African American subjects. We will also perform analyses to assess 
whether sex or race modify the effects of KNO3 on our endpoints. This is important, because sex 
differences have been demonstrated in responses to inorganic nitrate in other populations.1, 2 
Similarly, African Americans have been shown to have more pronounced wave reflections,3 and 
have been shown to demonstrate differential responses to other NO donors (organic nitrates) in 
the presence of HF with reduced ejection fraction (HFrEF).4, 5 
This trial has the potential to establish a novel, inexpensive, and readily implementable therapeutic 
paradigm for HFpEF and to characterize specific mechanisms involved, enhancing our 
understanding of this disease. 

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At 2 enrolling centers (Corporal Michael J. Crescenz Veterans Affairs Medical Center and 
University of Pennsylvania), we will perform an ancillary study (“Cardiac MRI sub-study”) to 
assess the effect of KNO3 on myocardial perfusion (MP) and myocardial perfusion reserve (MPR). 
This ancillary study will be funded by a grant from the Department of Veterans Affairs VISN-4 
Competitive Pilot Project Fund (CPPF) obtained by Dr. Sujith Kuruvilla (VA site PI and co-
Investigator in the ancillary studytrial). We will assess myocardial perfusion using novel MRI 
techniques. 
The sub-study will test the hypothesis that KNO3 administered over a period of ~4-6 weeks, will 
improve MP and MPR in subjects with HFpEF as compared to KCl. We will also explore whether 
MPR (measured during the placebo intervention, KCl) correlates with increased myocardial 
fibrosis (measured as the extracellular volume [ECV] ) using cardiac MRI), and with subclinical 
myocardial dysfunction (measured by impaired systolic/diastolic strain) in this population. 
We plan to enroll up to 20 subjects in this ancillary study. Subjects participating in the ancillary 
study will undergo all procedures per the parent trial, but in addition, will undergo a stress 
perfusion cardiac MRI study during the last 2 weeks of phase 1 and phase 2, during which MP, 
MPR values, myocardial fibrosis and myocardial strain measurements will be obtained. Of note, 
this may occur either before or after the exercise endpoint assessment, based on schedule 
availability. We anticipate that this substudy will contribute to a better characterization of the 
effects of inorganic nitrate therapy on the myocardium. 
 
 

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2. TRIAL SUMMARY TABLE (OBJECTIVES AND ENDPOINTS) 
Trial Title / Acronym 
Effects of KNO3 Compared to KCl on Oxygen UpTake in Heart Failure 
with Preserved Ejection Fraction (KNO3CK OUT HFpEFp) 
Funding Agency 
National Heart, Lung and Blood Institute, National Institutes of Health 
Phase 
Phase IIb Randomized Clinical Trial (RCT) 
Number of subjects 
84 
Study Sites 
University of Pennsylvania, Northwestern University, and the Corporal 
Michael J. Crescenz Veterans Affairs Medical Center 
Study Design 
Randomized, double-blinded, placebo-controlled crossover study 
Randomized 
Intervention 
Potassium Nitrate (KNO3) capsules, at a dose of 6 mEq (1 capsule) 
three times daily for approximately 6 weeks, versus, potassium chloride 
(KCl) capsules administered at a dose of 6 mEq (1 capsule) three times 
daily for  approximately 6 weeks  
Primary Aim 
To assess whether KNO3 therapy improves exercise capacity, 
quantified as: (1) Total work performed during a maximal-effort exercise 
test; (2) Peak oxygen consumption (VO2) during a symptomlimited 
maximal effort exercise test. These will be the co-primary endpoints of 
the trial. 
Secondary Aims 
1. To assess whether KNO3 improves quality of life 
2. To assess whether KNO3 improves the systemic vasodilator 
response to exercise 
3. To assess whether KNO3 improves muscle blood flow during 
exercise. 
4. To assess whether KNO3 improves muscle PCr recovery kinetics 
(a marker of oxidative capacity) 
5. To assess whether KNO3 improves LV diastolic function and 
myocardial systolic strain 
6. To assess whether KNO3 reduces late systolic LV load from wave 
reflections 
Exploratory Aims 
1. To assess whether KNO3 improves the exercise-induced spatial 
matching (i.e., within muscle) of blood flow and oxidative capacity 
(assessed by chemical exchange saturation transfer). 
2. To assess whether KNO3 has different effects in men vs. women and 
in African-American vs. non-African American subjects 
3. To assess whether KNO3 improves: 
•  Ventilatory threshold, VO2 kinetics  
• 
Natriuretic peptide levels 

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• 
Physical activity (measured via accelerometry) 
• 
24-hour late systolic pressure augmentation, measured with the 
Aurora device 
4. To assess the relationship between nitrate and nitrite levels achieved 
and the therapeutic response achieved in each subject  
(improvement in endpoints in response to KNO3 vs. placebo) 
 
Primary Endpoint 
Total work performed and peak oxygen uptake (VO2) during a 
symptom-limited maximal effort exercise test after  approximately 6 
weeks of KNO3 vs. KCl. 
Secondary Endpoints 
1. Quality 
of 
life 
score, 
assessed 
using 
the 
Kansas 
City 
Cardiomyopathy Questionnaire. 
2. The change in systemic vascular resistance reserve during exercise
during a symptom-limited maximal effort exercise test. 
3. Muscle blood flow during exercise, measured with flow sensitive PC-
MRI during a standardized plantar flexion exercise test. 
4. The time constant of Cr recovery after a plantar flexor exercise 
protocol. 
5. Measures of LV diastolic function (E/e’, left atrial volume index) and 
peak global systolic myocardial longitudinal and circumferential 
strain. 
6. Augmentation index, late systolic wall stress and aortic input
impedance. 
Ancillary Study 
Up to 20 subjects will be enrolled at the CMC VA Medical Center and 
the University of Pennsylvania into an ancillary study. The sub-study 
will test the hypothesis that KNO3 administered over a period of ~4-6 
weeks will improve myocardial perfusion and myocardial perfusion 
reserve in subjects with HFpEF as compared to KCl. Participation in the 
ancillary study will not change any of the main trial procedures. 
 
3. BACKGROUND AND SIGNIFICANCE 
Heart failure (HF) affects ~2% of the western population and 10% of adults age 75 years or older.6 
HF is the most common cause of hospitalization in adults >65 years of age.6 Approximately 46-
54% of patients with HF have HFpEF.6-18 The burden of HF has increased dramatically over the 
last several years.6, 19 The number of new HF cases increased from 348,000 in 2000 to 670,000 
in 2007, a 93% increase over this time period. Not only is HF already an epidemic, but with the 
aging of the population, a dramatic further increase in its prevalence is expected. Furthermore, 
the relative prevalence of HFpEF (as a proportion of the total burden of HF) appears to be 
increasing as the population ages.1, 14 Therefore, although already a huge epidemic, a further 
dramatic increase in the prevalence of HFpEF is expected.6 HFpEF is a malignant disease with a 
high mortality rate, high hospitalization risk and poor quality of life (QOL). Studies consistently 
demonstrate higher mortality rates in HFpEF patients compared with age- and sex-matched 
controls. Annual mortality rates ranged from ~3.5-6% in randomized trials20-22 to ~15% in 

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community-based studies.23 Approximately 50% of such deaths occur from cardiovascular 
causes.24 In addition to its high morbidity and mortality, HFpEF has been shown to be associated 
with an impaired QOL.25, 26 
Multiple therapies that provide substantial clinical benefit in HF with reduced ejection fraction are 
available. In contrast, there are currently no effective pharmacologic interventions that improve 
outcomes in patients with HFpEF.27 Thus, there is an urgent need to develop novel treatment 
strategies. Our trial will test a novel intervention (inorganic nitrate) to modify key physiologic 
abnormalities (arterial vasodilator reserve, muscle O2 delivery and utilization, mitochondrial 
function and late systolic LV load from arterial wave reflections), which have the potential for both 
immediate-term improvements in exercise tolerance and long-term “diseasemodifying” effects. 
The trial is designed to address the potential clinical benefit of this approach in HFpEF using 
endpoints with direct clinical relevance, and also to characterize the specific physiologic 
mechanisms involved. This would identify a new therapeutic paradigm and a readily 
implementable therapeutic intervention in HFpEF. 
 
3.1. The nitrate–nitrite–NO activation pathway 
NO formation occurs via 2 pathways in mammals (Figure 1): (1) NO synthases (NOS) catalyze 
the formation of NO from L-arginine and O2;28 (2) Circulating nitrate (previously considered an 
inert product of NO metabolism) can be converted to NO through the nitrate-nitrite-NO pathway, 
which is independent of NOS.28-33 
The nitrate-nitrite-NO pathway is dependent primarily on dietary inorganic nitrate intake. 
Vegetables (leafy green vegetables and beetroot, in particular)28, 33-36 are the main source of 
inorganic nitrate in the diet (>80%). Oral cavity commensal bacteria reduce nitrate to nitrite, which 
has a high oral bioavailability(>90%).33, 35, 36 Nitrite present in the blood stream is reduced directly 
to NO, a reaction catalyzed by several molecules, particularly deoxygenated myoglobin,37-40 and 
deoxygenated hemoglobin,41 but also xanthine oxidoreductase,42 mitochondrial respiratory chain 
enzymes43, aldehyde oxidase44, carbonic anhydrase,45 vitamin C46, polyphenols47, 48 and even 
endothelial NO synthase.49, 50 Nitrate is also directly absorbed in the gastrointestinal tract (without 
requiring conversion to nitrite), with high bioavailability (>90%)51 and circulates in plasma with a 
half-life of ~6-8 hours, in contrast to nitrite, which has a very short half-life (~30-40 minutes).52-54 
Circulating nitrate is taken up by the salivary glands and secreted in the saliva (thus being 
susceptible to both direct reabsorption as nitrate or to conversion to nitrite followed by absorption). 
This entero-salivary cycle provides a natural, “slow” and sustained source of nitrite from ingested 
nitrate, thus allowing for favorable pharmacokinetics and for safe oral administration of 
“pharmacologic” doses in HFpEF (as discussed further in this application). Most (65%) inorganic 
nitrate eventually undergoes renal excretion. 

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NO production by the nitrate-nitrite-NO pathway is enhanced under hypoxic conditions: exercising 
muscle is characterized by a low pO2,33 which favors the formation of NO from circulating nitrite.  
Deoxyhemoglobin supports the reduction of nitrite to NO and is thought to play a key role in 
modulating small resistance vessels (particularly of skeletal muscle), where O2 extraction from the 
circulation to the tissues is most marked. Here, the O2 saturation of hemoglobin approaches the 
P50 (the O2 concentration at which half the heme is saturated), an optimum balance point between 
the greater reductive potential of heme in the R (oxy) state tetramer and the number of un-ligated 
deoxy-heme sites necessary for nitrite binding (which are more plentiful in the T-state tetramer). 
This results in near-maximal conversion rates of nitrite to NO and hence vasodilatation.55, 56 
Similarly, NO by deoxy-myoglobin enhances blood flow to skeletal muscle and matches O2 supply 
to increased metabolic demands under hypoxic conditions.37 Xanthine oxido-reductase also 
converts nitrite to NO when O2 levels are low.42 In sharp contrast to NO production from the nitrate-
nitrite-NO pathway, which is enhanced in the presence of hypoxia, NO production via the classic 
L-arginine pathway is strongly inhibited by hypoxia50 (Figure 1). In summary, the endogenous 
nitrate-nitrite pathway is a physiological effector of hypoxic vasodilation via NO release, which is 
independent of the L-arginine pathway and largely independent of NO synthase. 
 
3.2. Peripheral mechanisms of exercise intolerance in HFpEF 
Exercise intolerance is the hallmark of HFpEF and determines a poor quality of life.25, 26, 57, 58 
Therefore, enhancing exercise capacity in this population is a key objective with immediate clinical 
relevance. We believe that new attempts to achieve this unmet goal should be based on careful 
considerations regarding the pathophysiology of exercise intolerance in HFpEF. The early 
pathophysiologic paradigm was that increases in LV filling pressure during exercise were not 
accompanied by increases in end-diastolic volume, failing to recruit the Frank-Starling mechanism 
and to augment stroke volume.59 Studies also reported the presence of chronotropic 
incompetence, leading to an abnormal cardiac output reserve.60-62 However, various studies have 
not found abnormal end-diastolic LV volume during exercise,60, 63 stroke volume reserve (i.e., 

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increase during exercise),60-62 chronotropic incompetence63 or cardiac output reserve.61 Thus, 
rather than resulting exclusively from cardiac abnormalities, HFpEF is now seen a complex 
disease and there is a need to address not only cardiac, but also peripheral abnormalities in this 
condition.64, 65 
Reduced O2 utilization is a key abnormality in the pathophysiology of exercise intolerance in 
HFpEF. Peak O2 uptake (VO2) during exercise, the most widely accepted index of aerobic 
capacity, is consistently reduced in HFpEF.59-62, 66 Peripheral O2 utilization requires not only an 
adequate cardiac output during exercise, but also an adequate flow distribution, characterized by 
blood flow being preferentially directed to exercising skeletal muscle, in order to achieve an 
adequate matching of perfusion with metabolic demands. This preferential flow distribution is 
dependent on the exercise-induced reduction in the local resistance of skeletal muscle arterioles 
(i.e., exercise-induced vasodilation). Therefore, as discussed below, the normal exercise induced 
vasodilation in exercising muscle serves 2 important roles: (1) To reduce the “total” (i.e., systemic) 
vascular resistance during exercise, which reduces LV afterload, promoting a greater cardiac 
output for any given contractile state; (2) To preferentially divert flow to highly oxidative muscle, 
promoting matching between metabolic needs and oxygenated blood delivery, thus allowing the 
“periphery” to optimally “utilize” the cardiac output to maximize O2 consumption. 
Exercise arterial vasodilatory reserve is abnormal in HFpEF, leading to excessive LV afterload. 
During exercise, LV afterload must decrease to accommodate increases in cardiac output without 
excessive rise in arterial pressure. In several studies, compared to age-matched hypertensive 
subjects   during exercise allows the local vasculature to overcome humoral and reflex-mediated 
vasoconstriction.67 NO bioavailability and release is a key mechanism mediating this response.67, 
68 Importantly, impaired vascular responses within skeletal muscle can have dramatic 
consequences for O2 extraction, creating a marked imbalance between O2 delivery and 
requirement in muscle which results in a large O2 deficit, accentuated intracellular metabolic 
perturbations and enhanced glycogenolysis even at low levels of activity (reviewed in 67). The 
vasodilator response in exercising muscle is dependent on both endothelium and 
endotheliumindependent pathways.67 As will be discussed below, nitrite-derived NO is a potent 
endotheliumindependent mediator of hypoxic vasodilation.37, 68, 69 and increase muscle blood flow 
during exercise.29, 70 Of note, HFpEF patients exhibit endothelial dysfunction71, 72 and may be 
particularly dependent on endothelium-independent NO to enhance skeletal muscle flow during 
exercise. 
Importance of flow distribution within muscle is also important. Although blood flow toward 
locomotive muscle groups is clearly important during exercise, blood flow (Q) within active 
muscles is not homogeneous, being greater in highly oxidative muscles, which normally 
demonstrate greater vasodilatation.73, 74 Hypoxic vasodilation favors preferential vasodilation in 
tissues with high oxidative capacity, which optimizes supply-demand matching (reviewed in 67, 68). 
Dysregulation of these control processes provides an excess flow and therefore O2 delivery to 
muscles with less oxidative capacity, reducing muscle and whole-body fractional O2 extraction.74 
Nitrite-derived NO-mediated hypoxic vasodilation37, 68, 69 thus may not only enhance overall 
muscle bulk flow, but also optimize flow distribution within muscle. Therefore, we believe that it is 
important for a study that targets muscle hypoxic vasodilation to not only address “overall” bulk 
muscle flow as a mechanistic endpoint, but also flow distribution within muscle. This important 

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and elusive goal, although well characterized in animal models,67 has been limited in human 
studies by the lack of techniques to spatially map (i.e., image) oxidative capacity. Our group has 
recently developed and validated an ad hoc technique75, 76 which, for the first time, allows for non-
invasive high-resolution spatial mapping (i.e., producing an anatomic image) of phosphocreatine 
kinetics (a marker of oxidative capacity). Therefore, our study will not only assess the effects of 
our intervention on exercise-induced bulk muscle flow and muscle oxidative capacity , but also on 
the matching of blood flow distribution and oxidative capacity within locomotive muscle. 
 
3.3. Importance of wave reflections and late systolic load: beyond improvements in  
exercise capacity 
While addressing exercise intolerance in HFpEF is a key therapeutic goal, there is also a need to 
address long-term underlying abnormalities that contribute to chronic LV remodeling and the long-
term course of the disease. As discussed below, a large body of evidence now indicates that late 
systolic pulsatile load from wave reflections (which are increased in HFpEF)77, have adverse long-
term consequences on LV remodeling and function. Our preliminary data suggests that enhancing 
nitrate-nitrite-pathway-derived NO production favorably impacts these systemic arterial 
abnormalities. Therefore, in addition to their exercise-enhancing mechanistic effects, interventions 
that enhance the nitrate-nitrite-NO pathway exert peripheral arterial effects with a potential for 
chronic “disease-modifying” benefits in HFpEF. 
The pulse wave generated by the LV travels forward in arteries and is partially reflected at sites 
of impedance mismatch (i.e., bifurcations, points of change in arterial size or wall stiffness, 
predominantly in middle-sized conduit arteries).78-80 Wave reflections travel back to the heart, 
merging into a discrete reflected wave and arrive while the LV is still ejecting blood in mid-to-late 
systole.80, 81 Wave reflections increase the late systolic workload of the LV and profoundly impact 
the LV loading sequence (late relative to early systolic load).78, 79, 82-84 
Late systolic load from wave reflections leads to LV hypertrophy. For any given level of systolic 
pressure, late-systolic load exerts deleterious effects on the LV.80, 85, 86 Kobayashi et al85 used a 
Wistar rat model and performed constriction of either the ascending aorta (which increased LV 
early systolic load) or suprarenal abdominal aorta (which caused prominent late systolic loading 
from a reflected wave at the distal aortic constriction site arriving at the heart in mid-to-late 
systole).85 Despite an identical peak LV pressure in rats that underwent ascending vs. descending 
aortic constriction, rats that underwent descending aortic banding (and were thus exposed to 
greater late systolic load) demonstrated much greater LV hypertrophy and fibrosis than those 
undergoing ascending aortic banding (which were exposed to increased early systolic load).85 
These animal causal findings are supported by our observational human data demonstrating an 
association between reflected wave amplitude an LV hypertrophy in the general population87 and 
by Hashimoto’s study demonstrating that changes in reflection magnitude during antihypertensive 
therapy strongly predicted regression of LV mass independent of blood pressure reduction.88 Of 
note, effects of standard antihypertensive therapy on wave reflections are unpredictable, with 
reflections actually increasing in some patients.88 

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Late systolic load promotes diastolic dysfunction. Gillebert et al studied the effect of the timing of 
systolic load on LV relaxation in dogs by inflating balloons in the ascending aorta during either 
early or late systole. They found that late systolic inflation of an aortic balloon increases the LV 
relaxation constant tau much more than early systolic inflation, demonstrating a cause-effect 
relationship between late systolic load and diastolic dysfunction.89 In support of these causal 
findings, wave reflections have been shown to be independently associated with diastolic 
dysfunction in clinical cohorts.90, 91 We have developed a novel technique to assess timeresolved 
LV wall stress during ejection,92 and more recently showed in the population-based Asklepios 
study 83, 92-95 that wave reflections selectively increase late LV myocardial wall stress83, 96 and that 
late systolic wall stress was associated with lower mitral annular relaxation velocities, in sharp 
contrast to early systolic wall stress which was associated with greater relaxation velocities.95 
Therefore, available data implicates the loading sequence as an independent correlate of 
myocardial relaxation in humans. Wave reflection magnitude was a strong predictor of incident 
heart failure among 5,934 participants in the Multiethnic Study of Atherosclerosis, who were free 
of clinically apparent cardiovascular disease84, 97 during 7.61 years of follow-up (and after 
adjustment for multiple confounders, including blood pressure), reflection magnitude strongly 
predicted incident HF (Hazard ratio per 10%-increase=2.69; 95%CI=1.79-4.04; P<0.0001), and 
was a stronger predictor of HF than blood pressure and any other standard modifiable risk factor. 
Similarly, for any given level of systolic and diastolic blood pressure, a greater area under the 
pressure curve in late systole (relative to early systole) is strongly predictive of incident HF.98 Our 
findings implicate late systolic load from arterial wave reflections as a novel strong risk factor for 
HF, strongly supporting animal and human mechanistic findings from previous studies and 
demonstrating the relevance of late systolic load in humans. 
 
3.4. Rationale behind the use of inorganic nitrate as a therapeutic approach in HFpEF 
The arterial hemodynamic characteristics of HFpEF patients (stiff arteries with wide pulse 
pressure,77 reduced exercise-induced vasodilation62, 63, 66, enhanced wave reflections77 dictate a 
set of “ideal” characteristics for a vasoactive intervention in this condition, namely, one that: (1) 
Does not significantly reduce mean arterial pressure at rest, avoiding hypotension; (2) Enhances 
exercise-induced vasodilation to reduce LV afterload during exercise; (3) Has selectivity for 
enhancing local vasodilation in hypoxic/acidotic environments, in order to match blood flow to 
metabolic demands (i.e., directing blood flow to exercising muscle); (4) Reduces wave reflections 
and late systolic load, which likely contribute to LV diastolic dysfunction and maladaptive 
remodeling.80, 85, 86, 88-91, 95 Inorganic nitrate precisely satisfies these characteristics. Inorganic 
nitrate exerts weak vasodilator effects in the absence of hypoxia but acts as a potent arterial 
vasodilator in the presence of local hypoxemia. Concentrations of nitrite (for which nitrate is a 
precursor) as high as 100-1000 µmol/L are typically required to induce relaxation of arterial rings 
in vitro.99 Similarly, a lack of vasodilator activity was seen with a concentration of 200 µmol/L of 
nitrite in the forearms of healthy volunteers.100 This situation is, however, different under hypoxic 
conditions. Maher et al studied 40 volunteers and demonstrated that, during normoxia, forearm 
arterial blood flow during local infusions of nitrite at incremental doses (from 40 nmol/min to 7.84 
µmol/min) increased by a maximum of 64% during normoxia, but it increased by as much as 121% 
during hypoxia.101 The nitrate-nitrite pathway thus seems ideal for enhancing vasodilation, O2 
extraction and utilization in HFpEF. By virtue of enhancing hypoxia-mediated vasodilation, the 

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vasodilatory reserve would be increased, reducing LV afterload during exercise and directing 
blood flow “where and when it is needed”, enhancing O2 delivery and this utilization. Indeed, 
inorganic nitrate improves muscle blood flow during exercise.29, 70 Our preliminary data support 
the promise of inorganic nitrate in HFpEF, via promotion of exercise-induced vasodilation, 
ultimately leading to enhanced aerobic capacity, in the absence of blood pressure reductions at 
rest, while at the same time reducing LV late systolic load. This represents an optimal “tailored” 
approach to abnormal hemodynamics in HFpEF. 
 
3.5. Preliminary data: effects of a single-dose of inorganic nitrate in HFpEF 
We performed a randomized double-blind cross-over trial (clinicaltrials.gov NCT01919177) of a 
single dose of 12.9 mmol of inorganic nitrate given as 140 mL of concentrated nitrate-rich beetroot 
juice (NR-BR; Beet it; Sport, James White Drinks Ltd., Ipswich, UK) versus an otherwise identical 
beetroot juice which had been nitrate-depleted (used as the control or “placebo” juice, PB), 
allowing for a genuine double-blinded design.102 The intervention was given before a maximal-
effort cardiopulmonary exercise test, with both experiments separated by a wash out period of at 
least 5 days (mean=11.8 days). We administered NR-BR 3 hours prior to testing because this 
period of time has been shown to correspond to peak circulating nitrite levels after oral nitrate 
administration. We tested the hypothesis that a single dose of inorganic nitrate would increase 
exercise capacity in HFpEF and assessed key physiologic adaptations to exercise. Specifically, 
we assessed: (1) Peak oxygen consumption (VO2); (2) Total work performed; (3) Exercise 
efficiency, defined as the ratio of total work performed to total O2 consumed (efficiency has been 
improved by inorganic nitrate in young athletes)70, 103-107; (4) Exercise vasodilatory reserve (the 
change in peripheral vascular resistance from rest to peak exercise, normalized to the resting 
value); (5) Skeletal muscle mitochondrial oxidative function; (6) Aortic augmentation index, a 
metric of late systolic load. 
We included subjects with symptomatic HF who had a preserved LV EF (>50%) and who were 
required to have a ratio of the early mitral inflow velocity (E) to septal tissue Doppler velocity (e’) 
>8 and at least one other sign of chronically-elevated filling pressures including: (1) left atrial 
volume index >34 mL/m2; (2) elevated NT-pro-BNP level; or (3) elevated filling pressures 
(pulmonary capillary wedge pressure > 12 mmHg) on prior cardiac catheterization. Subjects had 
to be on stable medical therapy. Exclusion criteria included non-cardiac conditions limiting 
exercise tolerance (i.e. orthopedic issues, peripheral arterial disease with claudication, 
neuromuscular disorders); gait instability; rhythm other than sinus; infiltrative or hypertrophic 
cardiomyopathy; pericardial disease; primary pulmonary arteriopathy; coronary revascularization 
within 60 days; clinically significant valvular disease; clinically significant lung disease felt to 
contribute to exercise intolerance; significant ischemia seen on stress testing within the past year 
and not revascularized. 
After each intervention, we performed a resting echocardiogram and arterial tonometry, followed 
by a maximal-effort supine-bicycle cardiopulmonary stress test using a graded exercise protocol, 
beginning at 15 Watts (W) for 3 minutes, increasing to 25 W for 3 minutes, then increasing by 25 
W every 3 minutes thereafter.108 Measurements during exercise included expired gas analyses 
(ParvoMedics True One 2400; Parvomedics, Utah, USA), echocardiographic assessments of 
stroke volume and continuous interrogation of muscle oxygenation of the left gastrocnemius 

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muscle with near-infrared spectroscopy (Portamon MkII device, Artinis medical systems; The 
Netherlands). We also assessed mitochondrial oxidative function measured via the recovery of 
oxygen consumption (measured with near-infrared spectroscopy) in forearm skeletal muscle after 
a bout of handgrip exercise, which corresponds to the phosphocreatine recovery kinetics, as 
previously described and validated against 31PMRI.109, 110 Details of the procedure may be found 
in previous publications. 108,109-111 In brief, the procedure measures the rate of oxyhemoglobin 
decline during brief high pressure inflations (at 200 mHg using a rapid inflation system), during 
which the decline in local muscle O2 is due exclusively to consumption, as the arterial occlusion 
removes the confounding impact of arterial inflow. After a brief bout of forearm exercise, several 
of such brief occlusions are performed, allowing for assessments of the rate of O2 consumption 
at various time points after exercise. Exercise induces increases in the rate of O2 consumption, 
which upon cessation of exercise, declines towards baseline in a mono-exponential fashion. This 
is characterized by a time constant (τ, tau) that corresponds to the time constant of PCr recovery 
kinetics.109, 110 Although we used this relatively inexpensive near-infrared spectroscopy procedure 
in our pilot trial, we will use more informative MRI techniques in this proposal. 
Participants’ mean age was 66±9 years. The population was enrolled from the Philadelphia VA 
Medical Center and was 88% male and 82% African-American (we will explicitly assure adequate 
representation of women in the KNO3CK OUT HFpEF trial). Subjects were obese (BMI 35.4±5.4), 
had a high prevalence of hypertension (100%). Median NT-pro-BNP was 326 (IQR=290-352) 
pg/mL. Mean LA volume index was 35.7±10.9 mL/m2. Serum NO metabolites were markedly 
greater after NO3- supplementation (median 326.0 [IQR 290.0-352.0] versus 10.0 [IQR 9.0-13.0] 
µM, P=0.0003). 
3.5.1. Changes in exercise capacity and oxygen uptake: Nitrate supplementation resulted in 
a greater peak VO2 (12.6±3.7 versus 11.6±3.1 mL O2/kg/min; P=0.005) and total work performed 
(55.6±35.3 vs. 49.2±28.9 kJ; P=0.04). Ventilatory threshold (a marker of the cellular anaerobic 
threshold) was greater following NO3- supplementation (7.6±1.8 versus 7.0±1.4 mL O2/kg/min, 
P=0.03). Because total work performed and O2 consumption increased in tandem, exercise 
efficiency was no different. This is interestingly, different than the effect seen in athletes and 
younger subjects, among whom exercise efficiency increases70, 103-107, demonstrating the 
importance of characterizing specific adaptations to exercise in this particular patient population. 
3.5.2. Exercise vasodilatory reserve: NR-BRJ led to a significant fall in systemic vascular 
resistance (SVR) at peak exercise (-42±17%) compared to PB (-32±20%; P=0.03). This was 
accompanied by an increase in cardiac output (% change in NR-BRJ 122±60 vs PB: 89±53%, 
P=0.006), increased heart rate (78.0±24.1 versus 65.6±21.0%, P=0.001) and a trend towards 
greater stroke volume (22.6±22.4 versus 12.7±25.4%, P=0.13). 
3.5.3. Effects on filling parameters: Interestingly, we found no evidence of reduced preload, 
since end-diastolic volume (EDV) significantly increased with beetroot supplementation (112±12 
mL vs 93±6 mL; P=0.04), whereas the E/E’ ratio did not change (NR-BRJ: 12.1±0.9 vs. PB: 
11.5±0.8; P=0.90). The increased LV EDV may suggest improvements in diastolic function, which 
we plan to further assess in this proposal using measurements of intraventricular pressure 
gradients. 

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3.5.4. Augmentation index: The aortic AIx (derived from radial tonometry) was significantly 
decreased by NO3- supplementation (NO3- 132.2±16.7 versus PB 141.4±21.9%, P=0.03). 
3.5.5. Changes in resting blood pressure: There was no significant change in resting BP 
 
(mean arterial pressure change=0.17 mmHg; systolic BP change=0.7 mmHg; both P>0.85) with 
NO3- supplementation. This is in contrast to BP-lowering effect in healthy volunteers and drug 
naïve hypertensive subjects112, 113 and likely explained by recent data indicating that the 
BPlowering effect of inorganic nitrate is mediated by its renal effects in pathways downstream of 
the AT1-receptor.114 These effects are likely attenuated in patients who are treated with ACE 
inhibitors/angiotensin receptor blockers for BP control (such as occurs in HFpEF patients). 
In summary, a single dose of inorganic nitrate increased peak VO2 and total work during a 
symptom-limited maximal effort exercise test. It enhanced the exercise vasodilatory reserve, 
without affecting SVR or blood pressure at rest. It also reduced late systolic LV load, which is 
implicated in diastolic dysfunction and LV remodeling. Nitrate also led to increased cardiac output 
during exercise, likely from a reduced afterload, but potentially also mediated by increased EDV 
(suggesting effects on diastolic function). Finally, in accordance with other studies, our pilot data 
suggest an improvement in mitochondrial oxidative function, suggesting improved ATP 
production.115, 116 However, nitrate did not affect the O2 cost of exercise, as has been 
demonstrated in younger and/or healthier populations.70, 105, 106, 117, 118 This demonstrates the 
importance of having disease-specific and agent-specific data to design our phase IIb trial. Our 
pilot trial was limited by its small sample size, inclusion of mostly men (we will explicitly assure 

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adequate representation of women in this trial). However, it does support the need for a larger 
phase IIb randomized trial of prolonged nitrate supplementation in HFpEFp. 
3.6 Effect on Myocardial Perfusion Reserve in HFpEF (“Cardiac MRI sub-study”) 
Myocardial biopsy studies in HFpEF patients have revealed cardiomyocyte hypertrophy, interstitial 
myocardial fibrosis, incomplete myocardial strip relaxation,  increased cardiomyocyte stiffness, 
increased production of reactive oxygen species at the coronary endothelial cell level, along with 
increased expression of adhesion molecules and production of pro-inflammatory cytokines.119-122 
In view of these findings, a new paradigm has been suggested in the pathophysiology of HFpEF 
(Figure 3). The paradigm proposes that the co-morbidities seen in HFpEF patients induce a 
proinflammatory state which activates coronary microvascular endothelial cells to produce 
reactive oxygen species and induces cardiomyocyte hypertrophy resulting in concentric LV 
remodeling and increased interstitial collagen deposition proceeding to diastolic dysfunction. 
 
Figure 3: Adapted from Paulus et al. Novel paradigm in HFpEF patients. JACC Volume 62, Issue4 (236-271) 
MRI is an ideal tool to assess these pathophysiological phenotypes and assess the effect of KNO3 
on microvascular myocardial function. Coronary microvascular dysfunction which is proposed to 
underlay this disease process can be estimated by measuring MPR by means of quantitative 
vasodilator stress cardiac MRI. MPR is calculated by a ratio of stress coronary perfusion over rest 
perfusion. Myocardial fibrosis  can be quantitatively assessed by the measurement of ECV by 
myocardial T1 mapping. 123 
The ancillary study of the KNO3CK OUT HFpEF trial represents a unique opportunity to assess 
the effect of inorganic nitrates on MPR values in this patient population. 
4. STUDY DESIGN AND POPULATION 
4.1. Overview of study design 
In this phase IIb, double-blind, cross-over placebo-controlled trial, we will assign 84 subjects with 
HFpEF to: (A) Potassium nitrate administered by mouth at a dose of 6 mEq three times daily for 
approximately 6 weeks, or (B) Potassium chloride (KCl) at identical doses. The order of the 
interventions (AB-BA design) will be randomized, with a 1-week washout period separating each 

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intervention. A crossover design will enable each subject to receive both treatments, reducing 
inter-individual variability and maximizing our power to detect effects of potassium nitrate vs. 
placebo  (KCl). The active drug (KNO3-) and control (KCl) will be prepared at the Investigational 
Drug Pharmacy at the University of Pennsylvania and dispensed by an investigational drug 
pharmacist, blinded to both the subjects and the investigators. 
4.2 Study Sites 
The three sites for this trial will be the Hospital of the University of Pennsylvania, Northwestern 
University and the Corporal Michael J. Crescenz VA Medical Center. 
4.3. Study Population 
We will enroll 84 subjects meeting the following criteria.  Eligibility will be determined by the site 
PI according to the criteria outlined below. 
4.3.1. Inclusion Criteria: 
1. Adults aged 18-90 years of age 
2. A diagnosis of heart failure with NYHA Class II-III symptoms 
3. LV ejection fraction >50% during baseline echocardiography 
4. Stable medical therapy: no addition/removal/changes in antihypertensive medications, or beta-
blockers in the preceding 30 days 
5. Elevated filling pressures as evidenced by at least 1 of the following: 
a. Mitral E/e’ ratio > 8 (either lateral or septal), with low e’ velocity (septal e’<7 cm/sec or 
lateral e’< 10 cm/sec), in addition to one of the following:    
i 
  Enlarged left atrium (LA volume index >34 ml/m2)    
ii   Chronic loop diuretic use for control of symptoms 
iii  Elevated natriuretic peptides (BNP levels >100 ng/L or NT-proBNP levels >300 
ng/L) 
b. Mitral E/e’ ratio > 14 (either lateral or septal) 
c. Elevated invasively-determined filling pressures previously (resting LVEDP>16 mmHg 
or mean pulmonary capillary wedge pressure [PCWP] > 12 mmHg; or 
PCWP/LVEDP≥25 mmHg with exercise) 
d. Acute heart failure decompensation requiring IV diuretics 
 
4.3.2. Exclusion Criteria: 
1. Supine systolic blood pressure <100 mm Hg 
2. Pregnancy: Women of childbearing potential will undergo a pregnancy test during the 
screening visit 
3. Orthostatic hypotension defined as >20 mm Hg decrease in systolic blood pressure 3-5 
minutes following the transition from the supine to standing position 
4. Uncontrolled atrial fibrillation, as defined by a resting heart rate>100 beats per minute 
5. Hemoglobin < 10 g/dL 
6. Inability/unwillingness to exercise 
7. Moderate or greater left sided valvular disease (mitral regurgitation, aortic stenosis, aortic 
regurgitation), any degree of mitral stenosis, severe right-sided valvular disease, or presence 
of a prosthetic valve in the mitral position 

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8. Hypertrophic, infiltrative, or inflammatory cardiomyopathy 
9. Clinically significant pericardial disease, as per investigator judgement. 
10. Current angina 
11. Acute coronary syndrome or coronary intervention within the past 2 months 
12. Primary pulmonary arteriopathy 
13. Clinically significant lung disease as defined by: Chronic Obstructive Pulmonary Disease 
meeting Stage III or greater GOLD criteria, treatment with oral steroids within the past 6 
months for an exacerbation of obstructive lung disease, or the use of daytime supplemental 
oxygen 
14. Ischemia on stress testing without either (1) subsequent revascularization, or; (2) a 
subsequent angiogram demonstrating the absence of clinically significant epicardial coronary 
artery disease, as per investigator judgement. 
15. Left ventricular ejection fraction <45% in any prior echocardiogram or cardiac MRI, unless this 
was in the setting of uncontrolled atrial fibrillation. 
16. Treatment with phosphodiesterase inhibitors that cannot be withheld 
17. Treatment with organic nitrates 
18. Significant liver disease impacting synthetic function or volume control (ALT/AST > 3x ULN, 
Albumin <3.0 g/dL) 
19. eGFR < 30 mL/min/1.73m2 
20. G6PD deficiency. In males of African, Asian or Mediterranean decent, this will be formally 
evaluated by enzyme testing prior to drug administration. A negative screening test for G6PD 
will be required in these subjects for inclusion in the study. If a quantitative test is being 
performed, a clinically significant reduction in G6PD activity (<60% of normal) will exclude 
subjects. 
21. Methemoglobinemia – baseline methemoglobin level >5% 
22. Serum K>5.0 mEq/L 
23. Severe right ventricular dysfunction 
24. Any medical condition that, in the opinion of the investigator, will interfere with the safe 
completion of the study. 
25. Contraindications to MRI (except as noted below), including the presence of a pacemaker, 
metal implants, claustrophobia, or that have known medical conditions which can be 
exacerbated by stress such as anxiety or panic attacks. Inability to lie flat in the MRI scanner 
for 90 minutes is also an exclusion criterion. 
Participants who have contraindications for MRI but are otherwise good candidates for the study, 
may be enrolled. In this case, participants will undergo all study procedures except for the MRI 
scans.** (See paragraph below for more detail) 
In addition, specific exclusion criteria for undergoing scanning in the 7-Telsa MRI scanning system 
include (these will only apply when the 7T scanner is used. In addition, a 3T scanner is also 
available at Penn, allowing the choice of the scanner depending on availability (to maximize 
safety, exclusion criteria for 7T will be applied at Penn for all participants, even if they ultimately 
are scanned at 3T):** 
– ANY intra-luminal implant, filter, stent, or valve replacement 
– ANY type of life assist device, pump, or prosthetic 
– ANY vascular clip or clamp 

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– ANY surgically placed clips or clamps or bands on visceral organs 
– ANY intracranial implants of any type other than dental fillings 
– ANY non-removable piercings, jewelry, or medicinal patch 
– ANY personal history of intraocular injury or fragment in or around the orbit that cannot 
be cleared through radiologic examination 
– ANY personal history of bullet, shrapnel, or stabbing wounds that cannot be cleared 
through radiologic evaluation. 
 
** If any of the above apply, we may investigate the issue further to ensure subject safety. This 
may include obtaining X-rays or obtaining reports from prior radiographic studies. We may also 
discuss the case with our radiologists and MRI technicians. In these circumstances, an MRI will 
only be performed if deemed safe by an attending radiologist on a case-by-case basis. 
Subjects that qualify for a 3T or 7T scanner (for the leg MRI study) also qualify for scanning in a 
1.5T scanner (for the cardiac MRI substudy). However, the opposite is not necessarily true. 
Subjects who are determined eligible to undergo scanning in a 3T or 7T scanner (in the primary 
study) are eligible to undergo scanning in the 1.5T scanner (for the cardiac MRI study). Some 
situations (such as many intraluminal stents and prosthetic valves) which would exclude scanning 
at high field (7T or 3T) are considered safe for 1.5T scanning. Subjects who are deemed ineligible 
to undergo 7T or 3T scanning in the parent trial, may still be enrolled in the ancillary myocardial 
perfusion study and undergo a cardiac MRI study (at 1.5T). In all cases, participation in the 
myocardial perfusion / cardiac MRI ancillary study will be approved by Dr. Kuruvilla, who has 
appropriate clinical expertise to assess the safety of this test, according to current clinical 
standards. Dr. Kuruvilla is the Director of Advanced Cardiac Imaging at the CMJC Philadelphia 
VA Medical Center and Assistant Professor at the University of Pennsylvania. 
4.3.3. Criteria that will prompt exclusion from the trial at the 6 week visit 
Subjects will be discontinued from study participation if they meet any of the following criteria at 
the 6-week study visit: 
1. Acute coronary syndrome or coronary intervention for unstable coronary disease after 
enrollment 
2. New onset ischemia on stress testing since the time of enrollment, including clinically 
significant reversible ischemia in a coronary distribution identified during the cardiac MRI 
substudy. 
3. New treatment with organic nitrates or phosphodiesterase inhibitors that cannot be withheld 
4. Serum K>5.5 mEq/L 
5. Any medical condition that, in the opinion of the investigator, will interfere with the safe 
completion of the study, or the validity of endpoint assessments. Although the ultimate 
authority/decision making will rely on each site PI, these ad-hoc discontinuations will be 
discussed with the other site PIs, the chair of the trial and the medical director of the IND 
for input. 
Subjects who meet the criteria below will not be immediately discontinued, but will be scheduled 
for an ad hoc additional visit for reassessment of values prior to drug initiation in phase 2: 

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1. Supine systolic blood pressure <90 mm Hg 
2. Acute kidney injury with eGFR < 30 mL/min/1.73m2 
3. Methemoglobinemia – baseline methemoglobin level >5% 
 
If these criteria persist during the ad-hoc visit, the subject will be discontinued from the study. 
4.3.4  Conditions that will preclude participation in the myocardial perfusion ancillary study 
include: 
1. Prior allergic reactions to gadolinium-based contrast agents. 
2. Allergy, or prior severe adverse reactions to regadenoson or aminophylline. 
3. 2nd degree AV block, 3rd degree AV block or high-grade AV block. 
 
4.4 Randomized intervention 
The randomized interventions will be the following (in random order, separated by a 1-week 
washout period) 
(A) Potassium nitrate: Potassium nitrate crystals will be purchased and placed into oral gelatin 
capsules with an inert filler (lactose monohydrate). Capsules will be prepared at the University of 
Pennsylvania Investigational Drug Service [3600 Spruce Street, Ground floor Maloney Building, 
Philadelphia, PA 19104]. Each capsule will contain 610 mg KNO3-, corresponding to 6.03 mmoles 
of NO3-, plus 190mg of lactose monohydrate, spray dried, NF. The dose for this trial will be 18 
mmoles of NO3-per day, given as one capsule (6 mmoles) three times a day. 
(B) Control drug: For the purpose of conducting this clinical trial, a control capsule will be made 
consisting of potassium chloride, granular, USP (450mg) plus lactose monohydrate, spray dried, 
NF (300mg) combined and packed into an identical capsule shell. We chose potassium chloride 
in order to isolate the effects of nitrate from any potential effect of potassium supplementation. 
The dose for this trial will be 18 mmoles of KCl  per day, given as one capsule (6 mmoles) three 
times a day. 
5. STUDY VISITS AND PROCEDURES 
A letter may be sent to potential subjects prior to initial contact. An overview of the study design 
and flow of study visits is presented in Figure 4. 
 
 
Figure 4. Overview of the study design and procedures. 

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The following investigational devices will be used during the study: 
• 
Aurora device: wearable pulse collection device (watch) 
• 
Portalite: continuous wave near infrared spectroscopy (NIRS) system to measure 
oxygenation of muscle tissue 
• 
Portamon: continuous wave near infrared spectroscopy (NIRS) system to measure 
oxygenation of muscle tissue 
5.1. Initial Visit 
During the baseline study visit, inclusion and exclusion criteria will be reviewed to ensure subject 
suitability. After  subject eligibility is confirmed, written informed consent will be obtained from the 
subject using Institutional Review Board (IRB)-approved documents. Informed consent will be 
obtained before any study procedures. Subjects will be given the opportunity to have all questions 
regarding their participation answered in detail in a private setting before entering the study. 
Informed consent may be obtained in-person or virtually. 
Following informed consent, a physical examination, with measurement of orthostatic blood 
pressures, will be performed. A urine pregnancy test will be performed in women with childbearing 

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potential. Blood will be collected for measurement of (a) comprehensive metabolic panel; (b) 
complete blood count; (c) NT-pro-BNP; and (d) methemoglobin. A pregnancy test (women of 
childbearing potential) and Glucose-6-phosphate dehydrogenase (G6PD) deficiency screening 
(males of males of African, Asian or Mediterranean decent) will be performed as needed. Blood, 
urine, and saliva samples will be collected and frozen for nitrate/nitrite level determination. 
Following the collection of baseline labs and biomarker samples, subjects will be provided with a 
standard low nitrate breakfast.The Kansas City Cardiomyopathy Questionnaire (KCCQ) will be 
administered. Subjects will also be given an informational brochure regarding foods that are high 
in nitrate/nitrite and will be instructed to avoid such foods while participating in the study. 
Computerized cognitive assessment testing will be performed using cogstate software (Cogstate; 
New Haven, CT). The assessment includes a customized battery of seven tests that measure 
various cognitive domains. The battery includes the following tests: 
• 
International Shopping List Test 
• 
Gorton Maze Learning Test 
• 
Detection Test 
• 
Identification Test 
• 
One Card Learning Test 
• 
One Back Test 
• 
International Shopping List Test- Delayed Recall (memory) 
The system works with an internet server administered and maintained by Cogstate, which 
quantifies the subject’s performance/responses and calculates cognitive scores. No personal 
identifiable information will be entered/uploaded into the server. 
Echocardiography will then be performed using a standardized protocol. Images will be obtained 
from the parasternal long axis, short axis, apical 5-, 4-, 3-, and 2-chamber, subcostal, and 
suprasternal views for offline analysis of myocardial strain. Dedicated ventricular chamber images 
will be obtained in the 4- and 2-chamber apical positions for determination of left ventricular 
volumes. Mitral inflow velocities, including color M-mode interrogation, will be assessed in the 4-
chamber view. Tissue Doppler imaging will be performed at the mitral septal position, 
approximately 1-cm apical to the mitral valve plane. Additional images will be obtained in the 
parasternal short axis at the level of the papillary muscles, 2-chamber, and 4-chamber apical 
views for assessment of myocardial strain. Pulse-wave Doppler interrogation of the left ventricular 
outflow tract (LVOT) will be performed in the apical 5-chamber view. 
Concurrent arterial tonometry will be performed using a high-fidelity tonometer at the carotid, 
femoral, and radial arteries, using a Sphygmocor device. Waveforms will be calibrated using the 
brachial artery blood pressures, obtained using a validated oscillometric device. Waveforms will 
be digitally-stored for off-line analysis. Where available, we will also measure blood pressure using 
a BP+ device (Uscom, Sydney, Australia), which is an FDA-approved device that measures 
brachial and central blood pressure using a standard brachial blood pressure cuff. Body surface 
measurements will be made to determine distance between the suprasternal notch to the carotid, 
radial, and femoral arteries. A 6-minute walk test will also be performed during this visit. Patients 
who demonstrate significant desaturation during exercise (<=94% or a fall in Spo2 >= 5%) will be 
excluded from the study 

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Initial Visit Procedures 
 
• Informed Consent a 
• Eligibility assessment 
• Medical History, Concomitant Medications 
• Dietary counseling 
• KCCQ 
•Physical Exam (including orthostatic vital signs and anthropometric measurements) 
•ECG, Laboratory tests (CBC, comprehensive metabolic panel, NTproBNP or proBNP, 
methemoglobin; pregnancy test and G6PD deficiency screening as needed) 
• Blood, urine and saliva collection 
• Cognitive assessment 
• 2D echocardiogram, arterial tonometry 
• Device (Aurora and Actigraph) education 
• 6 minute walk test with O2 saturation and Aurora device 
 
a Informed consent will be obtained before any study procedures 
 
  
5.2. Randomization procedure 
A 
stratified 
blocked 
randomization 
will 
be 
performed 
based 
on 
enrollment 
site 
(UPenn/Northwestern/ VAMC), sex, and ethnicity. A sufficient number of complete blocks will be 
generated for each stratum so that randomized assignments are available for every eligible 
subject. Strata-specific treatment order assignments and will be provided by a member of the data 
management team. Each block will contain an equal number of allocations to KNO3 followed by 
KCl versus KCL followed by KNO3. The order of treatment sequences within each block will be 
randomized. 
To preserve blinding, the strata-specific treatment order assignments and the associated 
studyspecific patient identification number will be provided to each study IDS representative site 
in electronic format (excel spreadsheet) pre-sealed envelopes. 
Upon determination of subject eligibility into the trial, the IDS representative will break the seal of 
the next envelope for the appropriate stratum to determine the treatment group assignment based 
on the appropriate stratum-specific scheme and will record the patient identification number for 
that study subject. 
5.3. Intervention Phase 1 
Subjects will be randomized to either active drug (KNO3) or placebo (KCl) as the “first” drug for 
phase A. The initial dose will be one 6 mmol capsule administered twice daily, to be taken with 
meals. We will provide subjects with a 14-day supply of study medication to account for potential 
delays in the shipment of the 2nd batch of medication (i.e., after the 1-week call). 

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1-week call: Subjects will be called by telephone ~1 week later for side-effect assessment. 
Subjects will receive reinforcement of the dietary restrictions imposed during the study. Subjects 
will be interrogated about headaches and dizziness. If these symptoms are not present, or are not 
worse compared to the subject’s baseline, the drug dose will be up titrated to 18 mmol daily (One 
6-mmol capsule taken 3 times per day). The presence of orthostatic symptoms (i.e. sustained 
lightheadedness upon standing) will prompt a visit to assess for orthostatic vital signs. The 
presence of symptomatic orthostatic hypotension (20 mmHg reduction in systolic blood pressure 
with associated symptoms such as dizziness) will prompt exclusion of patients from the trial. 
Subjects may experience symptoms upon dose uptitration that do not prompt exclusion from the 
trial, as per the criteria noted above (for example, but not limited to, mild dizziness, gastrointestinal 
symptoms, fatigue). In these instances, the Site Principal Investigator can reduce the dose back 
to 6-mmol capsule taken twice per day, for the remainder of the treatment phase. For subjects 
who have a baseline serum K between 4.7 and 5.0 mEq/L in the presence of either: (a) potassium-
sparing diuretic use, or (b) estimated glomerular filtration rate of 30-39 mL/min/1.73m2, we will 
check a serum potassium 1 week after the implementation of the 18 mmol/d dose as a safety 
measure. A serum potassium >5.5 mEq/L will prompt exclusion at this point. 
At approximately 5 weeks into each interventional phase, the Aurora and Actigraph devices will 
be given to the subjects. The goal will be for the subjects to wear these devices during the final 
week of each interventional phase to look for differences with each therapy. The devices will be 
brought back by the subject at his/her next visit. 
6-week endpoint assessment: Following ~6-weeks of therapy with KNO3/KCl, subjects will 
return for endpoint assessments. Physical examination with measurement of orthostatic blood 
pressure, pill count, KCCQ, dietary counseling, cognitive assessment, and side effect 
assessments will be performed. A urine pregnancy test will be performed in women with 
childbearing potential. An intravenous catheter will be placed in the antecubital vein and securely 
fixed in place. Blood, urine, and saliva samples will be collected prior to, and approximately 2 
hours after the morning dose administration, given with a standardized low-nitrate breakfast. 
Blood RNA samples will be collected prior to the morning dose administration using Paxgene 
tubes (BD Biosciences, Franklin Lakes, NJ). Subjects will then undergo repeat echocardiography, 
arterial tonometry, and blood pressure measurements (including BP+ central blood pressure 
measurements). Subjects will then perform a maximal-effort peak oxygen consumption (VO2) test 
using a supine bicycle exercise test with expired gas analysis. Cardiac output at rest will be 
measured immediately prior to exercise using pulsed wave Doppler echocardiography. Exercise 
cardiac outputs will also be obtained using the same technique. An additional blood sample will 
be obtained at peak exercise. The Aurora watch will be worn during exercise when possible. 
Subjects enrolled at Penn or the VA will undergo an assessment of skeletal muscle mitochondrial 
oxidative capacity using MRI either before, or at least 3 hours after, peak VO2 testing. Based on 
scheduling and availability, the MRI may be performed on a different day than the exercise study. 
The Kansas City Cardiomyopathy Questionnaire (KCCQ) will be administered. 
 
Phase 1: 6-week Visit Procedures 
 
• Interim medical history, concomitant medications 

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• Physical exam (including orthostatic vital signs) 
• Pill count 
• Blood, urine and sputum collection, blinded drug administration 
• ECG, labs (CBC, comprehensive metabolic panel, methemoglobin, pregnancy test as needed, 
BD PAXgene RNA samples) 
• KCCQ 
• Blood, urine, and saliva collection approximately 2 hours post-administration 
• Cognitive assessment 
• 2D echocardiogram, arterial tonometry 
•Cardiopulmonary test with resting and exercise cardiac output measurements, blood draw at 
peak exercise 
• If feasible, plantar flexor exercise / MRI (fat/water imaging / Creatine CEST / PC-MRI); these 
measurements are not performed at the Northwestern site 
• Data download of the Aurora device and Actigraph 
 
Additional visit for subjects participating in the Cardiac MRI sub-study: Subjects who 
participate in the MPR ancillary study will undergo a cardiac MRI with regadenoson administration. 
This will be scheduled during the last 2 weeks of the study intervention (4-6 weeks after initiation 
of drug administration in Phase 1), such that it will not interfere with the scheduling of the main 
trial visits. The cardiac MRI may take place on a different day than the exercise study or the lower 
extremity MRI scan and may occur either before or after the exercise endpoint study visit. 
5.4. 1-week washout period 
Following the endpoint assessment, subjects will enter a 1-week washout period during which 
they will not receive any study medications. 
5.5. Intervention Phase 2 
Following the washout period, subjects will receive either active drug (KNO3) or placebo (KCl) 
during phase 2 of the trial. Subjects will receive the intervention (KNO3 or KCl) that was not 
administered in phase 1, such that each subject will receive both study interventions in this cross-
over design. The ~1-week call and the ~6-week study visit will be repeated as above. We will 
provide subjects with a 14-day supply of study medication to account for potential delays in the 
shipment of the 2nd batch of medication (i.e., after the 1-week call). 
1-week call: Subjects will be called by telephone ~1 week later for side-effect assessment. 
Subjects will receive reinforcement of the dietary restrictions imposed during the study. Subjects 
will be asked about headaches and dizziness. If these symptoms are not present, or are not 
different than the subject’s baseline, the drug dose will be up-titrated to 18 mmol daily (One 6mmol 
capsule taken 3 times per day). The presence of orthostatic symptoms will prompt a visit to assess 
for orthostatic vital signs. The presence of symptomatic orthostatic hypotension (20 mmHg 
reduction in systolic blood pressure with associated symptoms such as dizziness) will prompt 
exclusion of patients from the trial. 
Subjects may experience symptoms upon dose uptitration that do not prompt exclusion from the 
trial, as per the criteria noted above (for example, but not limited to, mild dizziness, gastrointestinal 

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symptoms, fatigue). In these instances, the Site Principal Investigator can reduce the dose back 
to 6-mmol capsule taken twice per day, for the remainder of the treatment phase. 
At approximately 5 weeks into each interventional phase, the Aurora and Actigraph devices will 
be given to the subjects. The goal will be for the subjects to wear these devices during the final 
week of each interventional phase to look for differences with each therapy. The devices will be 
brought back by the subject at his/her next visit. 
6-week endpoint assessment: Following ~6-weeks of therapy with KNO3/KCl, subjects will 
return for a repeat endpoint assessment. Physical examination with measurement of orthostatic 
blood pressure, pill count, KCCQ, dietary counseling, cognitive assessment, and side effect 
assessments will be performed. A urine pregnancy test will be performed in women with 
childbearing potential. An intravenous catheter will be placed in the antecubital vein and securely 
fixed in place. Blood, urine, and saliva samples will be collected prior to, and approximately 2 
hours after, morning dose administration, given with a standardized low-nitrate breakfast. Blood 
RNA samples will be collected prior to the morning dose administration using Paxgene tubes (BD 
Biosciences, Franklin Lakes, NJ). Subjects will then undergo repeat echocardiography, arterial 
tonometry, and blood pressure measurements (including BP+ central blood pressure 
measurements). Subjects will then perform a maximal-effort peak oxygen consumption (VO2) test 
using a supine bicycle exercise test with expired gas analysis. Cardiac output at rest will be 
measured immediately prior to exercise using pulsed wave Doppler echocardiography. Exercise 
cardiac outputs will also be obtained using the same technique. An additional blood sample will 
be obtained at peak exercise. The Aurora watch will be worn during exercise when possible. 
Subjects enrolled at Penn or the VA will undergo repeat assessment of skeletal muscle 
mitochondrial oxidative capacity using MRI either before, or at least 3 hours after, peak VO2 
testing. Based on scheduling and availability, the MRI may be performed on a different day than 
the exercise study. The Kansas City Cardiomyopathy Questionnaire (KCCQ) will be administered. 
Following the final endpoint assessment, subjects’ participation in the study will be over. 
 
Phase 2: 6-Week Visit (final visit) Procedures 
 
• Interim medical history, concomitant medications 
• Physical exam (including orthostatic vital signs) 
• Pill count and side effect assessment 
• Blood, urine, and sputum measurements, followed by blinded drug administration  
• ECG, labs (CBC, comprehensive metabolic panel, methemoglobin, pregnancy test as needed, 
BD PAXgene RNA samples) 
• KCCQ 
• Blood, urine and, saliva collection approximately 2 hours post-administration  
• Cognitive assessment 
• 2D echocardiogram, arterial tonometry 
• Cardiopulmonary test with resting and exercise cardiac output measurements, blood draw at 
peak exercise 
• If feasible, plantar flexor exercise / MRI (fat/water imaging / Creatine CEST / PC- 
MRI); these measurements are not performed at the Northwestern site 
• Data download of the Aurora device and Actigraph 

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Additional visit for subjects participating in the Cardiac MRI Sub-study: Subjects who are to 
participate in cardiac MRI sub-study will undergo a Stress Cardiac MRI with regadenoson 
administration. This will be scheduled during the last 2 weeks of the study intervention (4-6 weeks 
after initiation of drug administration in phase 2), such that it will not interfere with the scheduling 
of the main trial visits. The cardiac MRI may take place on a different day than the exercise study 
or the lower extremity MRI scan and may be performed either after or before the exercise 
assessment. 
5.6 Adverse Event Reporting 
All adverse events will be reported following FDA guidelines. The research team will keep a log 
of all adverse events that occur in the trial, and any reportable events will be reported per Section 
7 in this protocol. The study team in charge of the conduct of the trial is up to date on all trainings 
pertaining to safety guidelines and adverse event reporting. Adverse Events will be reported to 
the site IRB, the Data Safety Monitoring Board, the Sponsor andand the NIH Program Office in a 
timely fashion, as specified in Section 7. 
5.7. Subject withdrawal / Early termination 
Subjects may voluntarily withdraw from the study at any time and for any reason, or this may be 
at the investigator’s discretion. The investigator may withdraw a patient from the study due to 
protocol non-compliance, incorrect enrollment or randomization, or for any other reasons related 
to subject safety. The reason for study discontinuation will be recorded on the source documents 
and all such subjects will be asked to complete an early termination visit. 
During this visit, we will document: (1) vital signs; (2) compliance with the medications, including 
pill count; (3) adverse effects, (4) specific reason for withdrawal. 
Early Termination Visit Procedures 
• Vital signs and physical exam 
• Pill count and medication adherence log 
• Safety labs 
• Adverse event assessment 
• Documentation of reason for withdrawal 
• Retrieval of Aurora and Actigraph devices and data download 
 
5.8. Concomitant Medication 
Subjects should be treated with standard of care medications for HFpEF or associated 
comorbidities. As per inclusion criteria, subjects should be on a stable medical regimen for HFpEF 
prior to entry. Further adjustment of diuretics or blood pressure medications during the study 
period is discouraged and should only be performed according to new and clinically compelling 
worsening of clinical status. Therapy with organic nitrates or phosphodiesterase-5 inhibitors is 
contra-indicated during the study period. 

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6. DRUG DISPENSING, ACCOUNTABILITY, AND DESTRUCTION 
Drug dispensing will be managed by the Penn Investigational Drug Pharmacy, in collaboration 
with the Northwestern Investigational Drug Pharmacy. KNO3 and KCl capsules will be 
compounded at the Penn IDS. Penn IDS will bottle, label, and dispense product for the Penn and 
VA sites. Penn IDS will ship product to Northwestern IDS for bottling, labeling, and dispensing of 
product at the Northwestern site. Prior to initiation of each phase, participants will receive a 
sufficient supply of KNO3 or placebo capsules to last until the end of each treatment phase, 
allowing for 100% compliance with the regimen for the entire treatment phase, including a window 
of ±7 days for the 1-week up-titration (to ensure an adequate supply of medication) and ±1 week 
for the 6-week endpoint assessment visit. 
Subjects will be instructed to take the medication as required by the protocol, and compliance will 
be assessed via pill count performed by IDS. 
Subjects will be instructed to return unused capsules at the end of each treatment phase. A pill 
count will be performed prior to destruction of the drug. Returned trial capsules will be stored 
separately from the non-allocated trial capsules until returned trial capsules are disposed of. The 
IDS personnel will keep track of all received, used, partly used and unused trial products. 
Used and unused study drug will be destroyed at the instruction of the sponsor. A copy of the drug 
destruction SOP should be maintained in the pharmacy section of the Regulatory Binder. Study 
drug should not be destroyed until drug accountability has been completed and the Sponsor has 
authorized it. 
 
6.1. Rules for un-blinding 
Randomization data will be kept strictly confidential, accessible only to authorized IDS personnel, 
until the time of un-blinding. The investigators will be given access to the treatment code for their 
patients for emergency un-blinding by calling the respective IDS. This is considered to be a very 
unlikely occurrence. Any suspected study drug-related events will be treated as though the patient 
received active (KNO3). Nevertheless, in the rare event ofemergency un-blinding, the site PI and 
the Sponsor must be notified. 
7. ADVERSE EVENTS 
7.1. Key definitions 
Adverse Event: An adverse event (AE) is any untoward medical occurrence associated with the 
use of a drug in a subject whether or not considered drug or biologic related. An AE can therefore 
be any unfavorable and unintended sign, symptom, or disease temporally associated with the use 
of the pharmaceutical product. 
Suspected Adverse Reaction: A suspected adverse reaction (SAR) is any adverse event for 
which there is a reasonable possibility that the drug caused the event. “Reasonable possibility” 
suggests there is a causal relationship between the drug and the adverse event. “Suspected 
adverse reaction” implies a lesser degree of certainty about causality than adverse reaction, which 
means any adverse event caused by a drug. 

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Serious Adverse Events (SAE): An adverse event or suspected adverse reaction is considered 
serious if the investigator or sponsor believes any of the following outcomes may occur: 
• 
Death 
• 
Life-threatening AE: Places the subject at immediate risk of death at the time of the event 
as it occurred. It does not include an AE that, had it occurred in a more severe form, might 
have caused death. 
• 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions. 
• 
Inpatient hospitalization or prolongation of hospitalization. 
• 
Congenital anomaly or birth defect. 
• 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered a serious adverse event when, based upon appropriate 
medical judgment, they may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition above. 
This determination is based on the opinion of either the investigator, or sponsor (i.e., if any one of 
these believes it is serious, it must be considered serious). 
Unanticipated Adverse Device Effect (ADE): Any serious adverse effect on health or safety, 
any life-threatening problem or death caused by, or associated with a device, if that effect, 
problem, or death was not previously identified in nature, severity, or degree of incidence in the 
application; or any other unanticipated serious problem associated with a device that relates to 
the rights, safety, or welfare of subjects. 
7.2. Classification of AE/ADEs 
A medically-qualified investigator must assess all AEs in terms of causal relationship to 
intervention, severity, and “expectedness” using the following guidelines. 
Classification of Adverse Events for Causal Relationship to Study Interventions 
Not related 
There is not a reasonable causal relationship to the investigational 
product and the adverse event. 
Unlikely related 
No temporal association or the cause of the event has been identified, or 
the drug or device is unlikely to be implicated, but there is a low  
likelihood that a causal relationship exists. 
Possibly related 
There is reasonable evidence to suggest a causal relationship between 
the drug and adverse event. 
Related 
There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely. 
 
 
 
 

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1 
Mild AE: Awareness of sign, symptom, or event, but easily tolerated; no 
treatment required 
2 
Moderate AE: Discomfort enough to cause interference with usual activity 
and may warrant intervention. In the latter scenario, AE responds to 
treatment 
3 
Severe AE: Incapacitating, limiting usual/normal activities or significantly 
affects clinical status requiring hospitalization or prolongation of 
hospitalization. 
4 
Life-threatening or disabling 
5 
Fatal AE 
 
 
Expectedness: The expectedness of an AE/ADE or SAR shall be determined according to the 
specified reference document containing safety information (e.g., most current investigator’s 
brochure or product label). Any AE/ADE that is not identified in nature, severity, or specificity in 
the current study reference document(s) (e.g. protocol or investigator’s brochure) is considered 
unexpected. Events that are mentioned in the investigator's brochure as occurring with a class of 
drugs or as anticipated from the pharmacological properties of the drug, but not specifically 
mentioned as occurring with the particular drug under investigation, are considered unexpected. 
The following AEs are expected, disease-related events in patients with HF with preserved 
ejection fraction (HFpEF). 
1. Unplanned hospitalization, ER visit or clinic visit for worsening HF 
2. Arrhythmias, particularly atrial fibrillation 
3. Sudden cardiac death 
4. Acute coronary syndrome 
5. Cerebrovascular event 
6. Venous thromboembolism 
7. Lightheadedness 
8. Worsening renal function 
9. Shortness of breath at rest or during/after exercise  
10. Fatigue at rest or during/after exercise 
The following are potential expected side effects of KNO3: 
1. Stomach discomfort 
2. Slight headache 
3. Dizziness 
Classification of Adverse Events Regarding Severity Scale 

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4. New onset or worsening lightheadedness 
5. Low blood pressure 
6. Stomach ache, diarrhea, nausea, or vomiting 
7. Worsening shortness of breath 
8. Worsening fatigue 
9. Flushing 
10. Rash 
11. Mild orthostatic hypotension 
Clinically significant methemoglobinemia or significant symptomatic orthostatic hypotension (20 
mmHg or greater reduction in systolic blood pressure with associated symptoms such as dizziness 
upon standing) are potential expected side effects to KNO3, although we will consider them 
unexpected given prior data in this patient population with this particular drug and dosing scheme. 
The following are potential expected side effects of regadenoson administration (for subjects 
participating in the cardiac MRI sub-study): 
1. Headache 
2. Dizziness 
3. Nausea or stomach discomfort 
4. Decreased sense of taste 
5. Mild chest discomfort 
6. Shortness of breath 
7. Flushing (warmth, redness, or tingly feeling under the skin) 
These side effects are transient and generally resolve upon clearance of the drug and/or 
reversal of drug effects with dypiridamole. 
The following are potential expected side effects of gadolinium-based contrast administration (for 
subjects participating in the cardiac MRI sub-study): 
1. Injection site pain (from the needle) 
2. Burning at injection site (if the dye contacts the skin) 
3. Headache 
4. Dizziness or faintness 
5. A decrease in blood pressure 
6. Nausea 
7. Vomiting 
8. Sweating 
9. Paresthesias 
 
These side effects occur in a small minority of patients, but if they do occur they will be noticed 
within minutes of the injection and are transient in nature. 
7.3. Recording and Reporting of Adverse Events 
The site PIs will continuously supervise all aspects of the trial and review the records of the study 
subjects following each visit and at the end of their participation. The site PIs will be responsible 

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for ensuring that all adverse events are noted, followed and reported to the  Sponsor database 
and IRB as per local requirements. 
SAEs occurring from the time of signed informed consent to the Week 15 phone visit will be 
captured on a CIOMS form. AEs will be classified according to the guidelines/definitions specified 
in section 7 of the protocol. Any AE rated >=3 in severity and all SAEs must be reported by the 
site investigator or qualified designee within 1 working day of first becoming aware of the event, 
to the Sponsor via email (at psom-ind-ide@pobox.upenn.edu), and the medical director (Dr. 
Townsend), with CC to the study chair (Dr. Chirinos). The IRB should be notified as per 
institutional guidelines at each respective site. The medical director  will make an immediate 
determination about the necessity to modify the protocol, include additional information in the 
consent form, inform previous participants, temporarily hold enrollment of patients, or terminate 
the study.  
The investigator or qualified designee will enter the required information regarding the AE into the 
appropriate module of the eCRF, All study procedures and cumulative adverse events are subject 
to full IRB review at least yearly and DSMB review every 6 months. 
Events significant enough to necessitate modification of study drug dosing will be captured on an 
appropriate eCRF module (“Study Drug Dosing” page). Should the PI determine that a 
modification to study drug is appropriate, the PI must obtain written approval from the Sponsor to 
proceed with this exception to the protocol. 
7.4. Follow-up 
The Investigator will record follow-up safety information according to the same process used for 
reporting the initial event as described above. The Investigator will follow all safety events until 
resolution, stabilization or the event is otherwise explained. 
The DSMB will review detailed safety data approximately every 6 months throughout the study, 
as detailed in the Data Safety Monitoring Section of this protocol and in the Data Safety Monitoring 
Charter. 
7.5. Management of Suspected Unexpected Serious Adverse Reaction 
AEs that meet the criteria of serious, related to study intervention, and unexpected for the study 
intervention, qualify for expedited reporting by the Sponsor to the regulatory authorities. The Site 
Investigator will assess all SAEs occurring at his/her site and evaluate for “unexpectedness” and 
relationship to study drug. The Site Investigator is required to complete a report for any event 
identified as serious, study drug related and unexpected, using the CIOMS format, and to submit 
it to the Sponsor within 1 working day of the PI or site personnel being aware of it. The sponsor 
will submit a safety report according to regulations. 
A copy of the report sent by the PI to the Sponsor should be kept at the site Regulatory Binder 
7.6. Pregnancy 
Pregnancy is a contraindication to enrollment in the study. Pregnancy occurring during the study 
period, although not considered an SAE, must be reported to the Sponsor within the same 

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timelines as an SAE, and to the  IRB per local requirements . The pregnancy will be recorded on 
the appropriate note to file. The drug will be discontinued immediately and subject terminated from 
the trial, but the pregnancy will be followed until final outcome. Any associated AEs or SAEs that 
occur to the mother or fetus/child will be recorded in the AE or SAE case report form. 
8. STUDY MEASUREMENTS AND DATA COLLECTION 
8.1. Assessment of exercise capacity 
We will use a supine bicycle exercise protocol in conjunction with expired gas analysis to assess 
oxygen consumption (VO2) during exercise. Subjects will perform a maximal exertion limited 
exercise test using a graded-exercise protocol. We will use a supine cycle ergometer designed 
for stress echocardiography (Stress Echo Ergometer 1505, Medical Positioning, Inc, Kansas City, 
MO). Subjects will undergo expired gas analysis with a Parvo Medics True One 2400 device 
(Parvo Medics, Sandy, UT), an Innocor device (Innovision Inc) or equivalent. Resistance began 
at 15 W for 3 minutes, increasing to 25 W for 3 minutes, and then increasing by 25 W every 3 
minutes thereafter. Breath-by-breath information will be recorded. We will use custom-designed 
software already developed in Matlab (MathWorks, Natick, MA) at our lab for offline processing 
and quantification of all exercise data.111 All data quantification will be blinded to treatment. Total 
work performed will be computed and exercise efficiency will be defined as (total work/total oxygen 
consumed). Exercise efficiency is not a formal endpoint of the trial, but will be used to interpret 
changes in the co-primary outcomes. 
 
8.2. Quality of life assessment 
This will be assessed with the Kansas City Cardiomyopathy Questionnaire (KCCQ).124 We will 
administer the KCCQ to subjects prior to randomization and at the end of each intervention phase 
(6-week time point in both phases). This is a validated 23-item questionnaire that assess physical 
function, symptoms, social function, self-efficacy and knowledge, and quality of life. It has been 
used extensively in heart failure studies (http://cvoutcomes.org/pages/3214). 
8.3. Assessment of the systemic vasodilatory response to exercise 
Blood pressure will be measured with a validated oscillometric device at rest, at each stage of 
exercise and after exercise. Cardiac output will be measured at rest and at each stage of exercise 
using echocardiography. Systemic vascular resistance (SVR) will be calculated at rest and at peak 
exercise as mean arterial pressure / cardiac output. Systemic vasodilatory reserve will be 
measured as the reduction in SVR during exercise, relative to SVR at rest ([rest SVR– peak 
exercise SVR] / rest SVR).111 Depending on equipment availability at each site, we may also 
monitor and record muscle oxygenation in the calf (lateral aspect of left gastrocnemius) and 
forearm (flexor digitorum superficialis) during exercise non-invasively using a Portamon or 
Portalite near-infrared spectroscopy device (Artinis Medical Systems; The Netherlands). Both the 
Portamon and Portalite are investigational devices and not currently approved medical devices in 
the U.S. 

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8.4. Assessment of muscle perfusion and energetics 
If available equipment and personnel are available at the time of enrollment, MRI studies will be 
performed at rest and immediately after a standardized plantar flexion exercise test using a 3T or 
a 7T scanner equipped with a 15-channel transmit/receive radiofrequency coil and an MR 
compatible pneumatically controlled foot pedal. Phase contrast MRI using a fast 1D projection 
technique125 will be used to image blood flow with high temporal resolution (Figure 5a-c) .  
 
 
The primary measure of recovery kinetics will be measured with creatine chemical exchange 
saturation transfer (CrCEST), except for subjects enrolled at the Northwestern site. Sample 
CrCEST recovery images of the calf are shown in figure 5d. The first 2 acquisitions are pre-
exercise (red box), with the remainder representing sequential acquisition immediately after 
cessation of exercise. Recovery kinetics of creatine levels will be quantified. Phosphorus 
spectroscopy is limited by the lack of anatomic information, being able only to assess PCr levels 
in a large voxel of tissue. In contrast, the chemical exchange saturation transfer (CEST) technique, 
a novel sensitivity enhancement method developed by our group,75, 126-128 can detect metabolite 
content based on exchangerelated properties at either 7T127, 128 or 3T,126 utilizing hydrogen signal 
 
 

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(thus not depending on the much weaker phosphorus signal). Creatinine exhibits a concentration 
dependent CEST effect between its amine (-NH2) and bulk water protons (CrCEST)75 which allows 
for the monitoring of Cr concentration changes with high spatial and temporal resolution. In 
contrast to phosphorus spectroscopy, this technique allows for spatial mapping (creation of an 
highresolution anatomic image) of PCr levels (figure 3D). This cannot be accomplished with 
phosphorus spectroscopy. This novel technique can be used to assess PCr recovery kinetics 
following exercise, allowing for accurate selection of muscle tissue (rather than fat or bone). More 
importantly, the serial anatomically mapped PCr information can be used to assess oxidative 
capacity of muscle tissue from specific pixels in the image. We will also assess muscle fat using 
a 3-point Dixon separation technique and hydrogen spectroscopy. We will exclude fat from 
CrCEST computations. 
 
8.5. Doppler Echocardiography 
This procedure is similar to clinical cardiac echocardiograms. An ultrasound probe will be placed 
on the surface of the skin to obtain images of the heart. This takes approximately 60 minutes. This 
will occur at all three visits. 
Measurement of LV filling and myocardial strain: Given that inorganic nitrate may also induce 
venodilation and myocardial effects, we will also assess the effects of potassium nitrate on (1) 
Early mitral annular early diastolic tissue velocity; (2) The ratio of early diastolic mitral inflow 
velocity to mitral annular tissue velocity (a surrogate of LV filling pressures); (3) Peak early 
diastolic intraventricular pressure gradient, a marker of ventricular relaxation, assessed with color 
M-mode interrogations of mitral inflow.129, 130 We note that the latter is not simply based on the 
inflow propagation velocity, but rather on solving Euler momentum equation.129 This method is 
able to accurately assess LV relaxation.129 Systolic function will be assessed via systolic 
myocardial strain (using speckle tracking echocardiography), which we have successfully applied 
in previous studies.92, 95, 131 
 
8.6. Measurement of late systolic load and arterial wave reflections: 
We will use a high-fidelity Millar applanation tonometer80 to record carotid pressure waveforms, 
which will be calibrated using brachial artery pressures. Central arterial tonometric recordings and 
Doppler flow velocity files will be processed off-line using custom-designed software written in 
Matlab (The Mathworks, Natick, MA) as previously described.92, 132 We have successfully 
implemented this method in multiple previous studies79, 83, 92, 93, 133 and have published a tutorial 
detailing our analysis methods.78, 79 After signal-averaging of pressure and flow, time alignment of 
carotid pressure and LV outflow curves will be performed to maximize concordance of the rapid 
systolic upstroke of pressure and flow, concordance of the dicrotic notch and cessation of flow, 
zero value of the phase angle of higher-frequency harmonics (7th to 10th) of input impedance, and 
linearity of the early systolic pressure-flow relationship.132 After computation of aortic input 
impedance, proximal aortic characteristic impedance (Zc) will be computed in the frequency 
domain as previously described.132 Pressure and flow harmonics were separated into forward and 
backward components using standard wave separation analysis.78, 79, 132, 134 The sum of forward 
and backward pressure harmonics yields the forward and backward waves, respectively. We will 

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assess the reflection coefficient in the first 3 harmonics. As reflection coefficient is derived from 
the ratio of two sine waves, it is a complex number with an amplitude and phase-angle, which can 
correspond to different degrees of destructive or constructive interference between forward and 
backward waves. Therefore, the net-effect of reflections will be expressed as the real part of Γ, 
which becomes increasingly positive as pressure from wave reflections increases (constructive 
interference), and negative when destructive interference leads to a net decrease in pressure by 
wave reflections at a given harmonic.135 Since all harmonics of wave reflection contribute variably 
to systolic LV load, our primary measure of late systolic load will be the net pressure related to 
wave reflections during ejection in the time domain, which better represents the impact of 
reflections on LV afterload. We will first compute the product of flow and aortic Zc (QZc product), 
which represents the pressure resulting from the interaction of blood flow with aortic root Zc.79, 80 
The relation between QZc and measured pressure reveals the direct effect of wave reflections on 
the arterial system.136 We will therefore quantify the additional ejection-phase pressure load from 
wave reflections arising distal to the root (i.e., reflection-related pressure time integral during 
ejection, as the difference between measured pressure and the QZc product). Carotid-femoral 
pulse wave velocity (PWV), an index of large artery stiffness,137, 138 will also be measured80, 139 
using a Sphygmocor device (Atcor Medical).137 We will also measure carotid-radial PWV using 
the Sphygmocor device. PWV is not a formal endpoint of this trial, since our pilot trial did not 
demonstrate an effect of nitrate-rich beetroot juice on this endpoint (unpublished data). However, 
it may aid in interpreting changes in pulsatile hemodynamics in the trial. Similarly, when available, 
arterial tonometry may be performed during and immediately after exercise for exploratory 
purposes. 
8.7. Blood nitrate/nitrite level measurements: 
We will document an intervention-related change in blood nitrate and nitrite. Nitrate and nitrite 
levels will be sampled before and approximately 2 hours after the last oral dose on the morning 
of the exercise test, after the patient has been on each intervention for approximately 6 weeks. 
Measurements will be performed at Dr. Harry Ischiropoulos’ lab at the University of Pennsylvania. 
Venous blood samples will be drawn into lithium-heparin tubes (which have very low levels of 
nitrate/nitrite) and centrifuged at 4,000 rpm for 10 min within 3 min of collection and frozen at -
80°C for later analysis. Blood will also be obtained at peak exercise. After thawing at room 
temperature, blood samples will be deproteinized using cold ethanol precipitation as previously 
described.118 The nitrate and nitrite content of deproteinized blood will be determined using a 
modified detection chemiluminescence technique using a Ionics/Sievers nitric oxide analyzer 
(NOA 280), as first described by Dr. Ischiropoulos’ lab140 and later adapted by Allen et al for human 
blood.141 
8.8 Cognitive Assessment 
Computerized cognitive assessment testing will be performed using cogstate software (Cogstate; 
New Haven, CT). These tests will be administered to subjects prior to randomization and at the 
end of each intervention phase (6-week time point in both phases).The assessment includes a 
customized battery of seven tests that measure various cognitive domains. The battery includes 
the following tests: 
 

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COGNITIVE ASSESSMENT TESTS 
 
 
TEST NAME AND 
INSTRUCTIONS 
COGNITIVE DOMAIN 
PRIMARY OUTCOME 
INTERPRETATION 
International Shopping 
List Test 
Tell me the items on the 
shopping list. 
Verbal learning 
Number of correct 
responses remembering 
the word list on three 
consecutive trials 
Higher score = Better 
performance 
Gorton Maze Learning 
Test 
Find the hidden pathway. 
Executive function 
Number of errors 
learning the same 
hidden pathway across 
the consecutive learning 
trials 
Lower score = Better 
performance 
Detection Test 
Has the card turned over? 
Psychomotor function 
Speed of performance; 
mean of the log10 
transformed reaction 
times for correct 
responses 
Lower score = Better 
performance 
Identification Test 
 
Is the card red?  
Attention 
Speed of performance; 
transformed reaction 
times for correct 
responses 
Lower score = Better 
performance 
One Card Learning Test 
Have you seen this card 
before in this test? 
Visual learning 
Accuracy of 
performance 
Higher score = Better 
performance 
One Back Test 
Is the card the same as 
the previous card? 
Working memory 
Speed of performance; 
transformed reaction 
times for correct 
responses 
Lower score = Better 
performance 
International Shopping 
List Test - Delayed Recall 
 
Tell me the items on the 
shopping list that you 
learned earlier. 
Memory 
Number of correct 
responses 
Higher score = Better 
performance 
 

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8.9. Other assessments 
Weight and Height: Weight will be measured to the nearest 0.1 kg with the use of calibrated 
scales, while the subjects are wearing light clothing and no shoes. Height will be measured at 
baseline to the nearest 0.1 cm using a wall-mounted stadiometer. 
Physical Activity: We will measure activity level in all subjects with the Actigraph device, which 
has been previously validated. This device can be worn on the wrist, and measures acceleration 
each minute in the anterior-posterior, mediolateral, and vertical axis, and summarizes that 
information as a vector magnitude. The software will use this data to calculate physical activity in 
METS and steps. The accelerometer will be provided during both phase 1 and phase 2. We will 
give the devices (Aurora [see below] and Actigraph) to the subjects during approximately the final 
week of each interventional phase. We will ask the subjects to put the devices on and will provide 
informational materials to assist with device placement. We will also discuss device placement 
with the subjects at the study visits as well as over the phone as needed. Subjects will bring the 
devices to the endpoint assessment visits. Subjects will be fully oriented in the use of this device. 
The device may be removed for showering or swimming but otherwise should be worn 
continuously. The specific details in the use of this device will be provided to study subjects. We 
will use the average METS of activity over this time period as an estimate of physical activity. We 
will also records steps taken. 
Ambulatory Pulse Wave Analysis: Ambulatory pulse wave analysis will be performed, when 
feasible, with a novel watch research device (Microsoft Aurora device). This device is not 
commercially available or FDA approved and is investigational. It continuously measures the 
radial pulse via a watch-mounted tonometer. The device also measures transit time from the heart 
to the wrist in an ambulatory fashion. The device will be worn outside of the exercise lab, as well 
as during the exercise studies themselves. The specific details in the use of this device will be 
provided to study subjects. 
Genetic Expression Analysis: Blood RNA samples will be collected using BD PAXgene blood 
RNA tubes (BD Biosciences) in order to explore changes in genetic expression in response to the 
study medication (potassium nitrate) and whether patterns of genetic expression predict response 
to the study medication. Two 2.5 mL samples (total of 5 mL of blood) will be collected at each 6-
week endpoint assessment visit. 
Myocardial Perfusion Reserve: Subjects who agree to participate in the cardiac MRI sub-study 
will undergo a Stress Cardiac MRI during the last 2 weeks of each cross-over treatment phase. 
Study parameters assessed during this MRI scan will include: assessment of myocardial perfusion, 
assessment of myocardial fibrosis, and myocardial strain. 
Myocardial perfusion: Stress perfusion images will be obtained following IV injection of 
regadenoson. Three to four short-axis perfusion images will be acquired each heart beat during 
the injection of the gadolinium-based contrast agent at a rate of 3 to 4 mL/s via a power injector 
during first pass of the contrast agent. Forty to sixty image frames will be obtained. Following 
reversal of regadenoson with intravenous aminophylline, cine images and delayed enhancement 
images (for focal fibrosis) will be obtained. At the end of the study, perfusion images will be 
obtained at rest using the same imaging protocol. Myocardial perfusion will be assessed from the 
time-intensity curves of the myocardium and blood pool. Overall MPR will be obtained as the ratio 

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of myocardial perfusion after vasodilator administration divided by the myocardial perfusion after 
reversal of the vasodilator (“rest” images). 
Myocardial fibrosis: We will use cardiac MRI to assess diffuse myocardial fibrosis by computing 
the extracellular volume fraction from pre- and post-gadolinium normalized myocardial 1/T1 and 
blood 1/T1 changes, after correction for hematocrit. T1 mapping pre- and post- administration of 
a macrocycic gadolinium-based contrast agent (i.e., Gadobutrol 0.1 mmol/kg or an equivalent 
dose of Dotarem) will be performed using a modified Look-Locker inversion recovery (MOLLI) 
sequence with image analysis using CMR42 software (Circle CVI, Calgary, AB, Canada). 
Myocardial strain: We will assess myocardial longitudinal and circumferential strain using Cine 
Displacement ENcoding with Stimulated Echoes (DENSE) sequences. 
 
9. MULTICENTER MANAGEMENT PLAN 
9.1. Study Chair/Grant Principal Investigator 
Dr. Chirinos will serve as the Study Chair and grant Principal Investigator for this protocol. In this 
role, he will administer the grant, and communicate with the NIH about study progress. He will 
communicate with site PIs regarding the science and any operational issues that arise during the 
trial, establish subcommittees and working groups to complete specific activities, monitor study 
implementation across sites (Penn, Northwestern, and Corporal Michael J. Crescenz VAMC), and 
hold regular investigator calls/meetings. As DSMB coordinator, he will convene an independent 
DSMB, communicate with the DSMB, and participate in open DSMB meetings. Dr. Chirinos will 
supervise the investigative team to develop and coordinate procedures and generate reports and 
presentations. He will maintain communication with the DSMB and will provide central fiscal 
management and reporting. Other roles of Dr. Chirinos will include directing the echocardiography 
and arterial hemodynamics core laboratory for this trial. 
9.2. Site Principal Investigators 
A principal investigator will lead operations at each enrollment site (Penn, Northwestern, and 
Corporal Michael J. Crescenz VAMC) and will be responsible for various aspects of the study, 
including data collection, adherence to all policies and procedures, and maintenance of study 
protocol to ensure that the specific objectives are being met. In conjunction with the Sponsor, they 
will oversee the safety of study participants. They will interact with the Sponsor team, including 
the Medical Director, the data management team, the study chair and the IND sponsor regulatory 
lead, as needed. 
9.3. Investigators Committee 
An Investigators Committee will hold a monthly call and discuss aspects of the study, including 
enrollment, retention, data management, quality assurance, and other issues as they arise. Dr. 
Chirinos will chair the committee, which will include the following members: Julio Chirinos, 
Payman Zamani, Sanjiv Shah, Sujith Kuruvilla, and Jesse Chittams. Minutes of these meetings 
will be distributed and kept on file. The study chair and the site PIs will have authority to make 

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decisions that require immediate attention when the committee is unavailable to meet. The study 
chair will adjudicate disagreements within the committee. The sponsor has the final responsibility 
for all study decisions. 
9.4. Operational Affairs Committee 
This committee will be responsible for oversight and coordination of the day-to-day activities of 
the project. Committee members will include the site PIs, the study coordinators at both enrollment 
sites, the post-doctoral researcher in charge of processing the MRI fat/water imaging, creatine-
CEST and PC-MRI data, the echocardiography/tonometry quantification technician and staff 
members of the biostatistical team. This group, along with all of the members of the Investigators 
Committee, will hold an in-person meeting at the beginning of the project to review the study 
protocol and the policies and procedures for its implementation. Members of the Operational 
Affairs Committee will participate in the Investigators Committee, but may hold additional 
meetings/calls throughout the project to review subject recruitment, retention, data transfer, 
quality of data, etc. Minutes of these meetings will be distributed and kept on file. 
9.5. Data Management 
The UPENN site will serve as the data coordinating center (DCC) and will play a fundamental role 
in overseeing randomization, data entry, data coordination, data transfer, and DSMB meetings. 
Dr. Alexandra Hanlon will be director of the DCC. Within the UPENN School of Nursing, DCC will 
have access to state-of-the-art computing resources capable of supporting studies ranging in size 
from one to more than 100,000 subjects. The equipment is fully supported by the Office of 
Technology and Information Systems (OTIS), which has complete business continuity and 
disaster recovery via redundant, distributed data centers and backup processes, in addition to 
traditional office and project management software. 
The data for this trial will be collected in ad hoc source documents by the research staff during 
each study visit, using trial-specific and visit-specific data collection forms. All source documents 
collected in this trial will be housed inside of a locked cabinet in the offices of the research staff, 
located at each study site. Data capture and storage will be accomplished within the framework 
of the Research Electronic Data Capture (REDCap) project. REDCap is a secure, web-based 
application designed exclusively to support data capture for research studies. It provides an 
intuitive interface for data entry with data validation, audit trails for tracking data manipulation and 
export procedures, automated export procedures for seamless data downloads to common 
statistical packages, including SAS, and procedures for importing data from external sources. As 
of March 2015, REDCap was in production use or prototype build-status for more than 99,000 
studies spanning numerous research focus areas across a consortium of 940 active institutional 
partners, including the University of Pennsylvania. Once the DCC launches the REDCap 
database into production mode, the study coordinators will be responsible for transmitting the 
participant coded electronic data using standardized data collection instruments to the database 
manager who will check the data for accuracy and completeness and follow-up with the research 
assistants as needed. The research coordinators will perform manual data entry from source 
documents and self-administered participant questionnaires (such as quality of life 
questionnaires), and the DCC will perform comprehensive data quality checks. 

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9.6. Quantification Core Laboratories 
Various physiologic core laboratory units will be in charge of analyzing physiologic data from the 
trial. 
9.6.1 Echocardiography Core Laboratory: The echocardiography core lab, directed by Dr. 
Chirinos, has extensive experience in analyzing echocardiographic data for multicenter trials. The 
lab functions as the core lab for 2 ongoing international multicenter trials in heart failure (CVRx 
HOPE4HF and CVRx NeoHF) and one US-based multicenter study focused on HFpEF and 
HFrEF (BMS CV002004). The core lab has all the capabilities required for echocardiographic data 
management and quantification in this study. The core lab has well established standard 
procedures for receiving and logging imaging data, quantitative analyses, quality control, audit 
trails, and communication with study sites (queries about individual issues, feedback for quality 
control). The core lab will be responsible for generating and updating manuals for 
echocardiographic data acquisition, which will standardize procedures across the sites. The core 
lab will certify individual technicians performing measurements for the study after reviewing of 
sample scans (from normal volunteers) across the 2 sites, and will review and update certification 
as needed. The core lab will have the capacity to veto individual technicians from performing 
studies for the trial, based on data quality. The echocardiographic core lab will hold monthly calls 
with a representative of the data management core to discuss issues regarding core lab data 
management. 
 
9.6.2 Arterial hemodynamics and exercise physiology core laboratory: The arterial 
hemodynamics core lab, directed by Dr. Chirinos, has experience in analyzing hemodynamic data 
for various large cohort studies (MESA, Asklepios)83, 93, 95, 97, 142-145 and currently functions as the 
core lab for physiologic data for several ongoing multicenter studies (ACRIN 4008, BMS 
CV002004, iCAP study, CREST study). The core lab has specific experience in assessing specific 
exercise-related phenotypes relevant to inorganic nitrate effects102 and has adequate capabilities 
required for physiologic data management and quantification in this study. The core lab will have 
ad hoc teleconference calls with Dr. David Poole (consultant in this grant). Dr. Poole will provide 
input about specific datasets in which there may be questions about physiologic phenomena. In 
addition to enhancing the interpretation of our human datasets, this will facilitate scientific 
interactions and will spark new ideas that will facilitate the translation of human observations to 
animal models and vice-versa. The physiology core lab will hold monthly calls with a 
representative of the data management core to discuss issues regarding core lab data 
management. 
 
9.6.3  MRI core laboratory: The MRI quantification core lab will be directed by Dr. Ravinder 
Reddy and will include a designated post-doctoral researcher (35% effort throughout the grant 
duration) who will be in charge of implementing the scans, performing QC of data as the study 
progresses, and performing standardized quantification of MRI images. Dr. Reddy’s laboratory 
has extensive experience in MRI quantification, with >100 publications in quantitative MRI and 14 
completed studies using CEST imaging in the last 5 years.75, 126, 128, 146-155 The MRI core lab will 
hold monthly calls with a representative of the data management core to discuss issues regarding 
core lab data management. 
 

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9.6.4 Imaging data management: All imaging electronic data will be stored within a secure 
HIPAA-compliant network-attached storage server, and is accessible only to those who are 
specifically given access by the core lab director. The secure server has mechanisms for 
redundant storage and data backup at different physical locations, to prevent data loss from 
disasters (such as a fire). 
 
9.6.5 Data Safety Monitoring Board: The DSMB will provide independent oversight of the 
project. The DSMB will be responsible for assessing: 1) baseline comparability between groups; 
2) participant accrual rate and retention; 3) resource availability; 4) data quality with special 
emphasis on eligibility data; and 5) patient safety. The board will make recommendations 
regarding: study continuation, protocol modification, and review of additional data. The conference 
call meetings and progress reports will be set by the DSMB. 
9.6.6 Cardiac MRI core laboratory: Cardiac MRI quantification will be supervised by Dr. Kuruvilla, 
with support from Dr. Chirinos. Dr. Kuruvilla will have access to software available in Dr. Chirinos’ 
core laboratory, including CMR 42 software and Matlab software. 
9.7 Conflict of Interest Management 
Dr. Chirinos is named as inventor on a patent application which has been assigned to the 
University of Pennsylvania. This constitutes Clinical trial intellectual property (as defined under 
University of Pennsylvania policy) relevant to this trial. Therefore, the Offices of Research 
Services and Vice Provost for Research have formulated a management plan for Dr. Chirinos’ 
participation in the trial as Study Chair and Grant PI. Notification of thist management plan will be 
distributed to DSMB members, site PIs and Sub-Investigators and the operations of the trial will 
adhere to this plan. 
 
10. STATISTICAL CONSIDERATIONS 
10.1. Power calculations 
We will randomize 84 subjects to one of 2 sequences (i), each of which consists of 2 periods 
(AB/BA design). We now have pilot data regarding the distribution of the changes in peak VO2 
(the primary endpoint of the study) as well as various secondary endpoints, among patients with 
HFpEF. We considered an increase in peak VO2 of ~0.6 ml/kg/min to be the minimum clinically 
significant change. This was based on the fact that even “modest” changes of comparable 
magnitude in peak VO2 have been associated with improved outcomes.156 The standard deviation 
of the change in peak VO2 in our pilot trial was 1.23 ml/kg/min. Therefore, the standardized effect 
size is 0.49 or greater. Assuming a 90% retention rate, enrolling 84 subjects in this cross-over trial 
will have 80% power to detect such an effect size in the intervention induced change of our study 
endpoints, with a two-sided α=0.05. Based on the distribution of the changes in study endpoints 
in our pilot trial or from repeated measurements of the Kansas City Cardiomyopathy Questionnaire 
in HFpEF patients, we estimated that this trial study is powered (80%) to detect the following 
changes in other endpoints: 4.6 points for the Kansas City Cardiomyopathy Questionnaire, 8.2% 
for exercise vasodilatory reserve, 19% for cardiac output reserve, 5 seconds for PCr/Cr recovery 
kinetics, and 4.2% change in reflection magnitude. We note that these differences are well below 
what is considered a clinically significant change in KCCQ (10 points or more, 
http://cvoutcomes.org/pages/3217) or the effect size that has been observed for these endpoints 

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in our pilot  trial.111 Therefore, we are confident that we will achieve adequate power not only for 
our clinical endpoints, but also for our physiologic measures. PASS11157 was used to perform 
power analyses. 
10.2. Data Analysis Plan 
The co-primary outcome variables will be peak VO2 and work performed during a symptom-limited 
maximal effort exercise test. All secondary outcome measures are continuous variables. The 
predictor of interest for all aims will be intervention (KNO3 therapy vs. placebo), with analyses 
based upon the total number of subjects randomized. Initial descriptive estimates of all measures 
will be generated for study participants at each time point by treatment group. Statistics will include 
estimates of central tendency, measures of variability, and derived moments of skewness and 
kurtosis. Analyses of distributional properties will be performed to determine if variance stabilizing 
or normalizing transformations should be applied. Outliers will be assessed via visual inspection 
of distributions and checked for accuracy. Aim 1 will assess the effects of potassium nitrate 
therapy on peak O2 / work performed (co-primary outcomes) and quality of life score (secondary 
outcome). An initial assessment of the treatment effect will be performed using the paired t-test 
and the non-parametric Wilcoxon sign-rank test on the difference between the paired within 
subject outcome measures. This will be followed by a more comprehensive linear mixed-effects 
model analysis158 allowing for assessments of the treatment effect on each continuous outcome 
of interest while controlling for effects of other covariates such as period, sequence, and a random 
subject effect nested within sequence. For non-normal distributed outcomes, we will utilize non-
parametric methods or consider distribution-stabilizing transformations. The intervention groups 
will initially be compared within each period (time invariant covariates will only be compared for 
period 1) according to continuous covariates described above using parametric or non-parametric 
one-way ANOVA models, depending upon whether or not normality appears to be in question. 
Levine’s tests will be used to assess homogeneity of variance. Additionally, the intervention 
groups will be compared within period according to categorical covariates using Fisher’s Exact 
tests. Significant differences between groups on these variables will result in their use as control 
variables in the modeling of outcome. Separate models will be generated for each of the two 
outcome measures, with each outcome measure regressed on intervention group assignment, 
along with baseline outcome and any other covariates deemed prognostic in preliminary analyses. 
The linear mixed-effects models will incorporate adjustments for any period effect and include 
data from dropouts.159-161 The model will include subject-specific intercepts as random effects, and 
assumes independent and identically distributed random errors within subject. Restricted 
maximum likelihood estimation will be used, and an appropriate covariance matrix will be 
specified. Model assumptions will be examined (eg,QQ plots to assess  normally distributed 
residuals for valid Wald tests). All mixed-effects models will be analyzed using the SAS PROC 
MIXED procedure162 in SAS version 9.3 (SAS Institute, Inc., Cary, NC). We will make every 
possible effort to minimize missing data and ensure final assessments for participants opting to 
discontinue study participation. Missing data, however, is an inevitable problem in a longitudinal 
study. The mechanism for missingness-missing completely at random (MCAR), missing at 
random (MAR), nonignorable or not missing at random (NMAR)-will be evaluated prior to 
implementing methodology intended to minimize bias from missing data.163 We anticipate that 
~10% of randomized subjects will not complete the study. 

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The intent-to-treat principle of including all randomized participants in the outcome models will be 
followed. SAS software procedures will be used for creating and analyzing multiple imputed data 
sets for incomplete multivariate data. Instead of filling in a single value for each missing value, 
Rubin's164 multiple imputation procedure replaces each missing value with a set of plausible 
values that represent the uncertainty about the correct value to impute. These multiple imputed 
data sets are then analyzed using standard procedures for complete data and combining the 
results from this analysis. Assuming monotone missing data patterns emerge, either a parametric 
regression method that assumes multivariate normality or a nonparametric method that uses 
propensity scores will be implemented, depending upon distributional patterns. 
Analytic methods described above will take advantage of all available data. The possibility of 
systematic bias in the outcomes for those who withdraw exists. Baseline characteristics will be 
compared among subjects with and without complete follow-up, recognizing that statistical power 
associated with finding true statistical differences may be limited. To assess potential biases, a 
comparison of withdrawal rates and or/time to withdrawal will be included. If the number of 
subjects lost to follow-up is small and the missing observations can be documented as being MAR 
or MCAR, then the primary hypotheses will be tested using the complete observed data. If missing 
observations cannot be assumed to be MAR or MCAR, more complex approaches will be 
considered. Sensitivity analyses for these models will be performed should they be 
implemented.165 
Our secondary aims involve exploratory analyses investigating mechanisms of action. In addition 
to assessing the effect of our randomized intervention on each mechanistic endpoint, exploratory 
structural equation modeling will be used to evaluate associations between outcomes and biologic 
mechanistic pathways. The modeling will be carried out in three sequential steps: (1) exploratory 
factor analysis, (2) confirmatory factor analysis, and (3) structural equation modeling. The 
exploratory factor analysis will be based on principal axis factoring to decrease the number of 
variables. For both theoretical and empirical reasons, it will be assumed that retained factors are 
correlated and thus an oblique rotation method will be used. Measured and latent variables will 
be examined for co-linearity, and related variables will be combined into single factors with 
individual factor loadings. To determine the number of factors, the model will be evaluated against 
the following four rules: (1) eigenvalues greater than 1.0166; (2) Glorfeld’s167 extension of parallel 
analysis, where a large number of random correlation matrices are generated  to compare the 
number of eigenvalues that are significant by chance168; (3) high internal consistency (an alpha 
coefficient of > .70) for unit-weighted factors,169 and (4) interpretability.170 The heaviest weight will 
be placed on the Minimum Average Partial and parallel analysis methods, with the scree test as 
a visual adjunct.171 The next step involves incorporating the factors into a model using 
confirmatory factor analysis. The model will be tested using goodness of fit tests to assess the 
overall fit of the model to the data.  Various models will be tested and compared prior to arriving 
at the best fitting model. And finally, the best fitting model obtained from confirmatory factor 
analysis will incorporate a structural equation model designed to examine the links between the 
randomized intervention, the mechanistic variables and the clinical variables assessed in the trial. 
10.3. Subgroup analyses and considerations about sex 
Although women represent 60% of patients with HFpEF, our sample will include 50% women and 
50% men. This is due to the fact that we explicitly wish to perform sex-stratified analyses, in 

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addition to testing for a potential modifying effect of sex on the effects of KNO3. Having an equal 
balance in terms of the number of males and females maximizes the statistical power to test for 
significant interaction effects between sex and other variables of interest. Furthermore, the slight 
increase in the proportion of males sampled will help to maximize the statistical power for sub-
analyses including only males. Balancing the sample on sex will also reduce the chances of 
having confounding between the race effect and other variables in the model. Lastly, since the 
over sampling of males is only slightly above the estimated actual population of 40%, the results 
of the analysis of the sample data should still be generalizable to the overall HFpEF population. 
Additional pre-specified subgroup analyses will include diabetes vs. no diabetes, hemoglobin 
value above and below median, and use vs. non-use of calcium channel blockers. 
10.4 Cardiac MRI sub-study analysis and power calculations 
The aim of this ancillary study is to assess the paired difference in myocardial blood flow and 
perfusion reserve in response to KNO3 or the control intervention (KCl) in a cross-over design. 
We will therefore utilize paired-t tests to estimate the difference. The non-parametric Wilcoxon 
sign-rank test will be considered, as appropriate. For exploration of the relationship between MPR 
and myocardial fibrosis or myocardial function, we will compute Pearson’s correlation coefficients 
for linear associations, or the Spearman’s Rank Correlation coefficient for monotonic non-linear 
associations. A p-value less than 0.05 will be considered significant. 
Power calculations: The standard deviation of the change in MPR in repeated measures in prior 
studies is 0.5. Assuming a dropout rate of 10% (effective n=18), the ancillary study will have 85% 
power to detect a standardized effect size of 0.75 or greater. Therefore, our study will be powered 
to detect an improvement of 0.375 in MPR with KNO3 vs. KCl. 
 
11. PROTECTION OF HUMAN SUBJECTS 
11.1. Potential benefits of the proposed research, importance of the knowledge to be gained, and 
risk/benefit ratio 
Potential benefits: There are no anticipated direct benefits to the subjects as a result of their 
participation in this study nor will this be implied when obtaining consent. However, if our 
hypothesis is correct, subjects in the active medication groups may experience improvements in 
their functional class and quality of life, although this will not be implied in any way during informed 
consent or enrollment. 
Importance of the knowledge to be gained: If our hypothesis is correct, this study may identify an 
effective specific intervention to treat peripheral mechanistic abnormalities in HFpEF patients. 
Furthermore, if improving these peripheral abnormalities leads to an enhanced exercise capacity 
in this patient population, this would lead to a new paradigm in the field and a better understanding 
of the mechanisms that lead or contribute to HFpEF, which will accelerate the discovery of new 
treatments for this condition. In addition, if potassium nitrate proves effective in enhancing 
exercise capacity and/or quality of life in this trial, this would identify a readily implementable, 
inexpensive therapy for this condition. 

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Risk/benefit ratio: the results of this study may ultimately lead to an effective treatment for HFpEF, 
which is urgently needed to help millions of patients. Since there is minimal risk and potential 
benefits to medical knowledge and society, the risk / benefit ratio is acceptable. 
11.2. Risks to study subjects 
The conduct of these studies will involve 84 human subjects, each exposed to both the active 
drug (potassium nitrate) and control (potassium chloride) intervention (cross-over design), and 
therefore studied as their own controls. All subjects will be adults able to give informed consent. 
Subjects will be enrolled with explicit assurance for adequate representation of women and African 
Americans. Assuring representation of women is key for the scientific aspects of this disease (high 
incidence in women). Assuring adequate representation of African-Americans is key for this 
intervention with a nitric oxide donor (since African Americans with HF and reduced EF have 
demonstrated differential responses to NO donors compared to Caucasians).172-175 Children will 
not be enrolled. Participants will be recruited from the Hospital of the University of Pennsylvania, 
Northwestern University Medical Center, and the Corporal Michael J. Crescenz VAMC. Assuring 
this gender and ethnic distribution will not be a problem given the clinical populations in the 
enrolling medical centers. 
The study involves various tests (arterial tonometry, Doppler echocardiography, a 
cardiopulmonary exercise stress, a lower extremity MRI without gadolinium administration with a 
standardized plantar flexor exercise protocol, blood draws) and the administration of randomized 
therapy (potassium nitrate vs. placebo). The ancillary study involes a cardiac MRI with gadolinium 
and regadenoson administration. 
11.2.1 Potential Risks of study intervention (potassium nitrate):  
The main potential risks of nitrate administration are related to its potential effect on (1) blood 
pressure; and (2) methemoglobin levels. 
Effects on blood pressure: Regarding the blood pressure reduction, as described above, many 
studies have demonstrated a reduction in blood pressure in both hypertensive and normal 
subjects following nitrate supplementation. This was summarized in a recent meta-analysis, 
demonstrating an approximate 4 mm Hg reduction in systolic blood pressure and 1 mm Hg 
reduction in diastolic blood pressure.51 Importantly, in our study of a single dose of inorganic 
nitrate in this specific population (HFpEF), we did not observe any change in blood pressure 
following nitrate ingestion. This suggests that subjects with HFpEF may have a different response 
to nitrate than other populations and thus hypotension may be a less pronounced side effect. 
Importantly, the lack of a blood pressure response to nitrate has been shown in both elderly 176 
and diabetic individuals,177 further suggesting that there may be important differences in nitrate 
effect by subgroups. In our pharmacokinetics study, we found a mild (~12 mm Hg) asymptomatic 
reduction in blood pressure recorded at the time of our study visits during 2-week drug 
administration, which occurred in the absence of any hypotensive symptoms.178 We did not find a 
reduction in ambulatory blood pressure. Therefore, although we don’t anticipate major changes 
in BP or significant hypotension with this dose, we will implement exclusion criteria and safety 

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blood pressure checks throughout the conduct of the study to ensure subject safety (see next 
section). 
Impact of NO3- on methemoglobin levels: Nitrate supplementation increases serum nitrite levels 
through the actions of an enterosalivary circuit and reduction of nitrate to nitrite by anaerobic 
bacteria in the oral cavity. 28, 33-36 Methemoglobin is generated by the reaction of nitrite with 
hemoglobin. According to Toxnet, doses between 2-9 grams of NO3- have been associated with 
methemoglobinemia. The dose we will use in our study (18 mmol KNO3-) will provide only 1.11 
grams (18 mmol NO3- x 62 grams/mol NO3- x 1mol/1000 mmol), which is well below this threshold. 
Moreover, this level of nitrate supplementation provides less inorganic nitrate than that found in a 
traditional Japanese diet, which is rich in vegetables (traditional Japanese diet provides 18.8 
mg/kg of nitrate per day x 70 kg individual = 1.3 grams of nitrate).179 Moreover, not a single cases 
of methemoglobinemia was observed in our pharmacokinetics trial , at the same doses used in 
this trial and in the same patient population. In the pharmacokinetics study we measured 
methemoglobin every half hour after the first administration as well as during steady-state 
continued oral administration at doses of both 12 mmol/d and 18 mmol/d (same dosing scheme 
used in the KNO3CK OUT HFpEFp trial) and did not find any clinically-significant elevations in 
methemoglobin. We don’t anticipate any significant issues with methemoglobinemia in the 
KNO3CK OUT HFpEFp study since this is a problem that has been reported with nitrite, but not 
with nitrate at the doses used in this study. 
Pilot safety data: In our double-blind randomized controlled trial in HFPEF, in which 12.9 mmol 
of inorganic nitrate were administered to patients with HFpEF, efficacy was demonstrated for the 
study endpoint (please refer to research plan). In this pilot study, no side effects were noted in 
any subject, except for pink urine (which was not different between the nitrate-rich and nitrate-
poor beetroot juice, because this is a consequence of other components of beetroot juice). As the 
body is able to reduce oxidized Fe3+ in methemoglobin back to Fe2+ at a rate of approximately 
15% per hour,180 the gradual increase in nitrite that occurs after nitrate administration (and the 
consequent formation of methemoglobin), should occur slowly enough to prevent significant rises 
in methemoglobin. To the best of our knowledge, methemoglobinemia has not been reported with 
oral nitrate administration. In our pharmacokinetic study, we did not observe any clinically-relevant 
elevations in methemoglobin levels.178 
Other potential safety considerations: There has been concern regarding whether a high 
nitrate diet may predispose to gastric cancer in humans through conversion to nitrite in the 
stomach or through increased urinary excretion of nitrate metabolites. This is a controversial issue 
derived from animal studies181, although the relationship between nitrate intake and cancer has 
not been demonstrated in humans.31, 106, 182 In fact, many studies show either no relationship or 
even an inverse relationship between a high intake of nitrate and the occurrence of gastric 
cancer.183, 184185, 186 The Joint FAO/WHO Expert Committee on Food reviewed all the available 
evidence, but failed to establish a definite link between nitrate intake and risk of developing 
cancer.189, 190 Furthermore, The World Cancer Research Fund/American Institute of Cancer 
Research found no evidence linking ingestion of vegetables which are known to be high in nitrate 
with the development of cancer.191 We note that the highly controversial concern regarding cancer 
risk associated with nitrate intake is related to long-term intake and not a significant issue with our 
6-week administration scheme of potassium nitrate. 

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11.2.2. Potential Risks of study procedures:  
Potential risks are associated with the study tests, the study interventions (potassium nitrate) and 
potential breaches in confidentiality. 
Lower extremity MRI: The non-invasive nature and the lack of ionizing radiation make the risks 
associated with MRI small. A MRI scan requires the participant to be in a partially enclosed space 
inside the scanner. Some people may find this to be uncomfortable. Claustrophobia is an 
exclusion criterion for the study. The MRI scanner produces different types of noises during a 
scan. Participants are given special earplugs to reduce the noise. A MRI scanner has a strong 
magnet which attracts certain metals. If anyone has these types of metal in their body, the MRI’s 
strong magnetic field can cause them to move which may cause injury. Subjects with be 
thoroughly screened for the presence of body metal and excluded from the study if they are unable 
to safely undergo the MRI study. If body metal/device may be present, we may investigate the 
issue further by obtaining X-rays or prior radiographic reports. We may also discuss the case with 
our radiologists and MRI technicians. An MRI will only be performed if deemed safe by an 
attending radiologist. 
The greatest risk of MRI scanning is a metallic object flying through the air toward the magnet and 
hitting the subject. To reduce this risk we require that all people involved with the study remove 
all metal from their clothing and all metal objects from their pockets. No metal objects are allowed 
to be brought into the magnet room anytime. In addition, once the subject is in the magnet, the 
door to the room will be closed so that no one inadvertently walks into the magnet. Some of the 
imaging sequences and/or RF coils are not FDA approved but are considered nonsignificant risk 
investigational devices. 
Risks of 7.0T MRI: To date no persistent adverse effects have been reported by facilities with 
magnetic field strengths at 7.0T. However, there has been an increase in the number of people 
reporting transient dizziness or nausea upon being moved into the field. This dizziness lasts less 
than 10 minutes and can be reduced by reducing the speed at which the subject is placed into the 
magnet. Risks are not cumulative and effects are transient. In addition, there has also been an 
increase in the number of subjects reporting a metallic taste in their mouth over that of 1.5 and 
3.0T systems. The FDA has approved the use of 7.0T magnets for research imaging in humans. 
All subjects will be informed of the possibility of having these sensations during the informed 
consent process. 
If an X-ray is deemed to be necessary to investigate any potential metal in the body, the subject 
will be exposed to minor doses of radiation: 
 

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(data from http://www.radiologyinfo.org/en/info.cfm?pg=safety-xray, accessed 10/5/2016; this website is maintained by the 
Radiological Society of North America) 
Plain film X-rays expose subjects to very low doses of radiation. From this table, it can be seen 
that a bone X-ray of an extremity would expose subjects 0.001 mSv, which is less radiation than 
an intraoral X-ray performed at the dentist’s office (0.005 mSv) and less radiation than a chest X-
ray (0.1 mSv). 
Cardiopulmonary stress test: This test is used extensively for research purposes with minimal 
risk to subjects. The most significant risks of the test are dysrhythmias or other cardiovascular 
complications, which are extremely rare. These procedures will be performed by qualified 
personnel 
according 
to 
established 
American 
Heart 
Association 
Guidelines.192, 
193 
Nonrevascularized myocardial ischemia, which may increase the risk of complications during 
exercise testing, is an exclusion criterion for the study. We note that prior studies in HFpEF also 
enrolled subjects with atrial fibrillation194 and that rate-controlled atrial fibrillation is not a 
contraindication to exercise testing. 

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Subjects may feel uncomfortable as a result of pushing themselves during the maximal effort 
exercise test. Subjects will likely feel short of breath and fatigued as a result of the exercise test. 
Various other complaints, such as nausea, lightheadedness, and other aches and pains are also 
possible as a result of the maximal effort exercise study. Although exercise testing may result in 
exhaustion, rarely do people develop abnormal HR or heart complications during exercise tests. 
The risk of this happening is the same as if the participant would exert themselves during stressful 
situations or during exercise elsewhere. 
We will perform EKG, HR, and blood pressures monitoring during our exercise test. In addition to 
the blood pressure (generally increases) and heart rate (generally increases) changes during 
exercise, we will also monitor arterial saturation. This will be done non-invasively using a pulse 
oximeter. Of note, oxygen levels can decrease with exercise, even in individuals without significant 
cardiopulmonary disease.195, 196 If the arterial saturation falls to below 88% (“severe exercise 
induced hypoxemia”196), we will alert the care provider as this may prompt consideration for 
additional/alternative causes for arterial hypoxemia. 
Venipuncture and IV placement: According to the 2010 WHO guidelines on phlebotomy, major 
risks associated with blood donations include hematoma at the site of venipuncture in 23%, and 
vasovagal reactions and fainting in 1%. The placement of an intravenous catheter would be 
anticipated to increase the risk of hematoma and discomfort slightly. Given that the catheter will 
be in place for a short-period of time, infection is an unlikely complication. Of note, the amount of 
blood to be drawn in the study is less than the amount of blood drawn at routine blood donation 
where the risk of syncope of 1% was defined. 
Arterial tonometry and assessments of oscillometric arterial pressure waveforms are noninvasive 
procedures and do not have any known risks. 
During various procedures (echocardiography, arterial tonometry), we will use adhesive 
electrodes attached to the participant’s skin to record the electrical signal from the heart. These 
may occasionally cause skin itching and irritation. 
Ambulatory pulse wave analysis (Aurora device) may cause mild discomfort in the wrist. 
Pregnancy Risks: We will not be enrolling subjects who are pregnant in this study. Right now, 
there is not enough research on the potassium nitrate to determine the safety of the mother or 
unborn child. A pregnancy test will be given to women of child-bearing potential prior to enrollment 
in the study and administration of the supplement. The pregnancy test will be repeated at the 6-
week visit (prior to initiation of the second treatment period). If a woman is enrolled of child-bearing 
potential, we will ask that they use a medically accepted method of birth control (such as an IUD, 
birth control combination pill, patch, ring, progestin-only pills, Depo Provera Shot, Implanon, 
complete abstinence, or condoms) while they participate in the study. In addition, we will test for 
pregnancy at each study visit and discontinue subjects from the study immediately if she becomes 
pregnant. As subjects with HFpEF are generally older (>55 years old) we do not anticipate this 
concern to occur with our study population. We will not enroll pregnant women or women who are 
lactating. 
11.2.3. Potential Risks of Cardiac MRI sub-study procedures: 

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Subjects who have agreed to participate in the cardiaccardiac MRI sub-study will undergo a 
cardiac MRI with IV gadolinium and IV regadenoson administration. The risks entailed with 
performing a MRI scan are described under lower extremity MRI in the risk section. 
Gadolinium-based contrast agent administration: During the MRI examination, subjects will 
be administered a gadolinium-based contrast agent (GBCA), which allows for the quantification 
of diffuse interstitial fibrosis and the visualization of focal scars. Gadolinium-based contrast agents 
have been linked to increased risk of nephrogenic systemic fibrosis (NSF), a disease that causes 
fibrosis of the skin and connective tissues throughout the body and that can cause death. There 
is no known treatment for NSF. While there seems no increased risk for NSF in subjects with 
normal renal function or only moderate renal insufficiency, risk for NSF seems to increase in 
subjects with severe renal insufficiency (glomerular filtration rate (GFR) <30 mL/min/1.73m2). In 
a retrospective study with Omniscan (one of the five FDA approved GBCAs), 4% of subjects with 
severe renal insufficiency were estimated to be at risk for NSF.197 Allergic reactions, which may 
include rash, sweating, itching, hives, facial swelling, and anaphylactic shock, may occur with 
GBCAs independent of renal function. No increased risk of gadolinium administration is 
anticipated among patients taking the study drug. There is recent data showing that traces of 
gadolinium may remain in the body long-term after contrast administration. However reviews by 
the FDA have not identified adverse health effects from gadolinium retained in the brain or bodily 
tissues after MRI. Furthermore, the gadolinium agent that is used in this study belongs to a group 
of macrocyclic gadolinium based agents that are thought to minimize or eliminate this risk. 
Regadenoson: Regadenoson will be administered intravenously as part of the stress portion of 
the exam in order to induce coronary vasodilatation. Regadenoson is used as part of both cardiac 
MRI and cardiac nuclear stress testing protocols on a daily basis for clinical assessment of 
ischemia. Common side effects of regadenoson include headache, dizziness, nausea, abdominal 
discomfort, chest discomfort, flushing and shortness of breath. 
Venipuncture and IV placement: As described above. 
Potential risk of investigational devices: The Aurora device (pulse wave analysis watch) can 
produce skin irritation at the tonometer site. This usually goes away within few hours of 
discontinuing use. The version of the device that we will use for exercise studies uses adhesive 
electrodes attached to the participant’s skin to record the electrical signal from the heart. These 
may occasionally cause skin itching and irritation. 
The Portomon and Portalite (near infrared spectroscopy, or NIRS) devices have no known risks. 
They are non-invasive devices applied on top ofto the skin to measure the oxygenation of 
underlying tissue. 
As with any clinical research study, there is a potential for breach of confidentiality. Adequate 
measures will be taken to minimize this risk (below). 
11.3. Adequacy of Protection Against Risks 
11.3.1. Recruitment and Informed Consent:  

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Written informed consent will be obtained from the subjects by the investigators prior to entry into 
the research study. This will be performed in accordance with the guidelines and under the 
supervision of the University of Pennsylvania, Corporal Michael J. Crescenz VAMC, and the 
Northwestern University Institutional Review Boards. The study procedures and interventions and 
the associated risks will be explained to the subjects during the informed consent process. Only 
IRB-approved consent forms and related materials will be used. 
11.3.2. Protection against risks associated with the MRI study:  
Subjects will be thoroughly screened for the presence of body metal and excluded from the study 
if they are unable to safely undergo an MRI study. We will exclude patients who are not suitable 
candidates for an MRI study by virtue of having the following absolute or relative contraindications: 
(i) Central nervous system aneurysm clips; (ii) Implanted neural stimulators; (iii) Implanted cardiac 
pacemaker or defibrillator; (iv) Cochlear implant; (v) Ocular foreign body (e.g. metal shavings); 
(vi) Other implanted medical devices: (e.g. drug infusion ports); (vii) Insulin pump; (viii) Metal 
shrapnel or bullet; (ix) Claustrophobia; (x) Extreme obesity rendering the patient unable to fit into 
narrow-bore scanners; (xi) Unwillingness of the patient to undergo an MRI. All patients with 
metallic implants will be individually evaluated prior to MRI. Women with child-bearing potential 
will have a serum pregnancy test to exclude pregnancy before each MRI scan. Pregnant women 
will be excluded from the study. Screening for MRI-related exclusion criteria will done during a 
phone interview (pre-screen) and with an on-site questionnaire  at the time of the scan. 
Once subjects are deemed able to safely undergo an MRI study, these will be performed 
according to standard safety practices as per our institutional standards. Voice contact with 
patients will be maintained throughout the scan. 
All people involved with the study remove all metal from their clothing and all metal objects from 
their pockets. No metal objects are allowed to be brought into the magnet room anytime. In 
addition, once the subject is in the magnet, the door to the room will be closed so that no one 
inadvertently walks into the magnet. 
11.3.3 Protection against risks associated with cardiopulmonary exercise tests:  
These tests will be performed by qualified personnel according to established American Heart 
Association Guidelines192, under ECG monitoring. Personnel with adequate cardiopulmonary 
resuscitation training and resuscitation equipment (crash cart) will be available during these tests. 
Similarly, these tests will be performed in a hospital setting where a full code team can be 
deployed immediately should complications occur. 
11.3.4 Protection against risks of potassium nitrate administration: 
Subjects will be thoroughly advised regarding the potential risks of the study medication and 
precautions needed during its administration. We will exclude individuals who are taking sildenafil 
(Viagra®), tadalafil (Cialis®), or vardenafil (Levitra®) and we will instruct them not to take these 
medications while participating in the study, since it is likely that these medications may 
exaggerate the vasoactive effects of potassium nitrate. Similarly, the use of organic nitrate will be 
contraindicated during participation in this trial. 

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Supine and orthostatic blood pressure measurements: Supine vital signs will be measured at 
the screening visit. The subject will rest in a supine position for a minimum of 3 minutes prior to 
obtaining vital sign measurements. Subjects with a supine systolic blood pressure >110 will be 
enrolled in the study. The subject will then assume a standing position for 3-5 minutes. Vital signs 
(BP and pulse rate) will then be measured while the subject is standing. A reduction in systolic 
blood pressure >20 mmHg will be considered an exclusion criterion for the study. Blood pressure 
measurements will be repeated after approximately 6 weeks of randomized therapy. 
Measurements will also be prompted by reports of any report of orthostatic symptom by study 
subjects at any point during the trial. The presence of symptomatic orthostatic hypotension (20 
mmHg reduction in systolic blood pressure with associated symptom such as dizziness) will 
prompt exclusion of patients from the trial. 
Safety Clinical Laboratory Tests: Laboratory evaluations will be collected prior to study drug 
initiation. Among women who are not surgically sterilized or post-menopausal, a urine pregnancy 
test will be performed at screening (prior to all baseline studies and drug initiation), at the end of 
the first treatment phase (prior to the MRI study and initiation of the second treatment phase) and 
immediately before the MRI study performed at the end of the second treatment phase. 
Methemoglobin levels will be measured before initiation of randomized therapy and approximately 
6 weeks after initiation of randomized therapy in both treatment phases (1 and 2). 
Methemoglobinemia (methemoglobin levels >10%) will prompt discontinuation of patients from 
the trial. Other tests, including a complete blood count, liver enzymes, a basic metabolic panel 
(including serum creatinine for assessments of renal function) will also be performed prior to 
randomization and approximately 6 weeks after initiation of randomized therapy. 
Significant methemoglobinemia was not observed in our pharmacokinetics trial, at the same doses 
used in this trial, in the same patient population. In this study we measured methemoglobin every 
half hour after the first administration as well as during steady-state continued oral administration 
at doses of both 12 mmol/d and 18 mmol/d (same dosing scheme used in the KNO3CK OUT 
HFpEFp trial). 
Side Effect Management Plan: No side effects have been observed in our single-dose study or 
our ongoing pharmacokinetics study (in particular, no hypotension or methemoglobinemia). 
However, a plan for management of side effects will be in place. The use of acetaminophen may 
be used for headaches during the study. Although hypotension is not expected, it should be 
managed as per standard clinical practice. In the unlikely event that methemoglobinemia does 
occur during the trial, we will manage according to best clinical standards.198 As the endogenous 
rate of reconversion from methemoglobin to hemoglobin is approximately 15% per hour, most 
asymptomatic patients can be managed conservatively. Generally, methemoglobin levels >20% 
with symptoms, or > 30% without symptoms, warrant therapy; any patient with levels that are 
elevated to this range will be referred to the emergency room for management.198 
11.3.5 Measures to minimize the risk of breach in confidentiality:  
All records will be treated with strict confidentiality according to HIPAA guidelines (all study 
personnel are trained on HIPAA regulations). Blood samples obtained from subjects will be used 
only for research purposes. Records will be treated with strict confidentiality and stored in a 
secured, limited access area. A randomly assigned number rather than name will identify all 

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collected samples. A secure database of patient information will be maintained. The investigators 
and the sponsor will have access to research information and will follow IRB and institutional 
HIPAA guidelines. Paper files will be saved under lock in a secure IRB-approved area. 
11.3.6 Other measures to minimize risk:  
Phlebotomy, arterial tonometry, NIRS, MRI studies, and Doppler echocardiographic examinations 
will only be performed by appropriately trained personnel as per our institutional standards. 
Subjects will receive instructions in Aurora device use, including written instructions (Appendix 3), 
including instructions to loosen the wrist strap if there is discomfort and discontinue its use if 
discomfort persists. 
11.3.7  Protection against risks associated with the MRI study:  
See above (Section 11.3.2). It is worth noting that the risk of a cardiac MRI at 1.5T is in general, 
lower than at 3T or 7T, given the lower magnetic field. Subjects that qualify for a 3T or 7T scanner 
(for the leg MRI study) also qualify for scanning in a 1.5T scanner (for cardiac MRI during the 
myocardial perfusion ancillary study). However, the opposite is not necessarily true. Some 
situations (such as many intraluminal stents and prosthetic valves) which exclude scanning at 
high field (7T or 3T) are considered safe for 1.5T scanning. If subjects are deemed to be ineligible 
for 7T or 3T scanning in the parent trial, they may still be enrolled in the ancillary myocardial 
perfusion study and undergo a cardiac MRI study (at 1.5T). In all cases, participation in the 
myocardial perfusion / cardiac MRI ancillary study will be approved by Dr. Kuruvilla, who has 
appropriate clinical expertise to assess the safety of this test, according to current clinical 
standards. Dr. Kuruvilla is the Director of Advanced Cardiac Imaging at the CMC VA Medical 
Center and Assistant Professor at the University of Pennsylvania. 
 
Protection against risks associated with IV gadolinium administration: The presence of 
eGFR >30 mL/min/1.73m2 is an exclusion criterion from the parent trial. In addition, GFR will be 
assessed within 3 weeks prior to the cardiac MRI. A GBCA will be only administered for the MRI 
examination if eGFR >40 mL/min/1.73m2. Subjects with a prior history of allergic reactions to 
GBCAs will be excluded from participation. There is recent data showing that traces of gadolinium 
may remain in the body long-term after contrast administration. However reviews by the FDA have 
not identified adverse health effects from gadolinium retained in the brain or bodily tissues after 
MRI. Further the gadolinium agent that is used in this study belongs to a group of macrocyclic 
gadolinium based agents that are thought to minimize or eliminate this risk. 
Protection against risks associated with IV regadenoson administration: Regadenoson 
should be avoided in patients with unstable angina, severe COPD/asthma and patients with 
advanced cardiac heart block. Subjects with 2nd degree AV block, 3rd degree AV block or 
highgrade AV block will be excluded from participation in the ancillary study. Unstable coronary 
syndromes or significant lung disease are exclusion criteria for the parent trial. In all cases, 
screening will be performed as per clinical guidelines prior to all patients receiving regadenoson. 
Cardiac resuscitation equipment and trained staff including a cardiologist will be available before 
administering regadenoson. 

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12. REGULATORY STANDARDS 
12.1. Informed consent 
The site investigator, or a person designated by the site investigator, will fully inform the subject 
of all pertinent aspects of the clinical trial including the review of the informed consent form 
approved by an Institutional Review Board (IRB). Prior to a subject's participation in the clinical 
trial, the Informed Consent Form will be signed and personally dated by the subject or by the 
subject’s legally acceptable representative. Informed consent may be obtained in-person or 
virtually. All subjects will receive a copy of the informed consent form. 
12.2. Institutional Review Board (IRB) 
The site Principal Investigator will submit this protocol to the appropriate IRBs, and will forward to 
the NHLBI a copy of the written and dated approval by the IRB Chairman and committee. The 
study (study number, protocol title and version number), the document reviewed (protocol, 
Informed Consent Form, etc.) and the date of the review will be clearly stated on the written IRB 
approval opinion. During the study, any amendment or modification to the protocol will be sent to 
the IRB. It will also be informed of any event likely to affect the safety of subjects or the continued 
conduct of the study, in particular any change in safety and all updates to the protocol will be sent 
to IRB. 
12.3. Investigational New Drug (IND) application 
This trial will be done under our currently open Food and Drug Administration IND (IND#123785) 
to study this particular drug and preparation in this particular patient population. The IND Sponsor 
will be responsible for interactions with the FDA, with support from the Sponsor Support Unit within 
the Office of Clinical Research at the Perelman School of Medicine, University of Pennsylvania. 
12.4. Source document handling and archiving 
Hard copies of subject’s study records, including signed informed consent forms, HIPPA forms, 
source documents, and other study related materials will be stored in the subject binders, in a 
locked file cabinet in the research coordinator’s study office until archived. Research records will 
be retained for at least 2 years after the completion of this study. All study electronic files will be 
kept for at least 6 years after IRB acknowledgement of study termination. Files are not be 
destroyed or deleted without Sponsor approval. Data will be made available to the study sponsor 
National Institute of Health (NIH) members of the Institutional Review Board, and the Food and 
Drug administration, if requested by any of these entities. 
13. DATA AND SAFETY MONITORING PLAN: 
A data and safety monitoring plan is in place for this study. 
 

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15. APPENDIX 1. SCHEDULE OF EVENTS 
 
SCHEDULE OF EVENTS 
Baseline
 
assessment 
Phase 
1 
1 
wee
k 
6 
week
1-week
 
washout 
Phase 
2 
1 
wee
k 
6 
week
Informed Consent a 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X X 
X 
X 
Eligibility assessment 
X 
Medical History 
X 
Concomitant Medications 
X 
Randomization  
X 
Dietary counseling 
X 
 
Kansas City Cardiomyopathy Questionnaire 
X 
Physical Exam (including orthostatic vital signs and 
anthropometric measurements) 
X 
12-lead ECG 
X 
Laboratory tests (CBC, chem 7, methemoglobin, pregnancy 
test as needed) 
X 
RNA collection with PAXgene tubes 
 
G6PD test as needed 
X 
 
Cognitive assessment 
X 
 
6 minute walk test with O2 saturation 
X 
 
Blood, urine and saliva collection and freezing 
X 
 
2D echocardiogram 
X 
 
Arterial tonometry 
X 
 
Pill count 
 
Drug accountability 
 
Maximal effort exercise test with expired gas 
 
Exercise cardiac output measurements 
 
Plantar flexor exercise / MRI 
 
Assessment of Adverse events 
 
Placement of Aurora device and Actigraph 
X 
 
Retrieval of Aurora device and Actigraph 
 
Note: For subjects participating in the ancillary study, a cardiac MRI will be performed 4-6 weeks 
after drug initiation in each treatment phase, on a different day than the other study procedures. 
 
 

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16. APPENDIX 2. ANTICIPATED STUDY TIMELINE AND KEY DATES 
 
 
KEY DATES 
 
 
 
17. APPENDIX 3 Instructions for wearing the Aurora Device (Pulse Watch) 
 
The Aurora device is designed to record your pulse throughout the day. We ask that you wear 
throughout the day and night, as you may wear a watch. During your first study visit, we will do 
a demonstration to teach you how to place and secure the device to your wrist. 
 
Use of the Aurora Device is very safe. However, there are a few precautions of which you 
should be aware: 
 
- 
The wrist strap will need to be moderately tight for acquisition of good quality pulse 
signals. It should never be so tight, however, as to restrict circulation in the hand. If you 
feel a tingling sensation in the hand or wrist where the Aurora device is being worn, 

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immediately loosen the wrist strap. If the sensation persists, take the device off and 
discontinue use. 
- 
The device will beep several times during the day. When you hear the beep, you should 
touch the two contacts on the front of the device with two fingers from the hand that is 
not wearing the device, as illustrated in the following image: 
 
 
 
- 
The device has been engineered to be robust to normal daily wear conditions, but it is 
not waterproof. Therefore, avoid submerging the device in water or using it outdoors in 
heavy rain. 
- 
The Aurora Device can be cleaned using standard isopropyl alcohol wipes. Do not use 
any other cleaning agents. Do not submerge the device in cleaning agents. 
- 
Do not attempt to open the housing of the device or to dismantle the device. 
  
  
18. APPENDIX 4. HISTORY OF CHANGES 
All changes after version V1 (dated 4/29/16), which was finalized after receiving input from all 
DSMB members and both IRBs, and before any subject enrollment, are documented below. 
Version 2 (Feb 2016): Amended to specifically include 
a) The administration of a low-nitrate diet with study medications during Visit 2 and Visit 3. 
b) The use of the Aurora watch during the symptom-limited maximal effort exercise test 
c) Blood draw at peak exercise 
d) Minor grammatical changes and inconsistencies fixed throughout the protocol 
Version 3 (July 2016): 
a) Changed the language about the randomization procedure (direct communication 
between IDS and Jesse Chittams, without involvement of the study team. 
b) Specified that we will provide subjects with a 14-day supply of study medication at the 
beginning of each phase. 
c) Minor changes in language to meet wording consistent with FDA regulations 

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d) Added unanticipated device-related effects as potential adverse events. 
e) Replaced MEDWATCH format with CIOMS format for SAE reporting 
f) Added a section of potential risks of investigational devices. 
g) Removed DSMB as an appendix, as it is a stand-alone core document. 
h) Removed VaSera measurements to simplify the protocol. 
Version 4 (July 2016) 
a) Updated the chemistry and manufacturing document 
b) Updated the chemistry and manufacturing portion of the protocol 
Version 5 (August 2016) 
a) Altered the inclusion criteria to remove the stipulation that evidence for elevated filling 
pressures must have been demonstrated within the last 12 months. 
Version 6 (October 2016) 
a) Modified the inclusion criteria to include specific criteria for elevated intracardiac filling 
pressures at rest or with exercise. 
b) Modified the exclusion criteria to allow enrollment of individuals with rate-controlled 
(resting HR<100) atrial fibrillation. 
c) Included specific plans to ensure MRI safety for subject who may have metal in his/her 
body. This includes obtaining X-rays or X-ray reports and discussing the case with an 
attending radiologist prior the MRI scan. 
d) Updated the MRI protocol to include phase contrast imaging instead of ASL. 
e) Added the Philadelphia VAMC as an enrollment site, pending VA IRB/ACOS approval and 
site activation. 
f) In response to VA IRB stipulations: 
i. 
Reworded maximal exercise test to “symptom-limited maximal effort exercise test” 
throughout the protocol 
ii. 
Added additional monitoring of serum potassium for participants with a baseline 
serum K between 4.7 and 5.0 mEq/L in the presence of either: (a) 
potassiumsparing diuretic use, or (b) Estimated glomerular filtration rate of 30-39 
mL/min/1.73m2. In this instance, we will check a serum potassium 1 week after 
the implementation of the 18 mmol/d dose as a safety measure. 
iii. 
Added more explicit language about the purpose of KNO3 in the study, in the 
specific aims section: “The purpose of the trial is to test the efficacy of KNO3 on a 
number of clinical and physiologic endpoints in subjects with HFpEF…” 
Version 7 (January 2017) 
a. Clarified that the Aurora and Actigraph devices will be given to the subjects at 
approximately 5 weeks into each interventional phase. 
b. Clarified safety monitoring to be performed during the exercise study, including oxygen 
saturation monitoring. 
c. Added a section (4.3.3. Criteria that will prompt exclusion from the trial at the 6 week visit) 
in response to further VA IRB stipulations 
d. Updated figure 3. Overview of study design and procedures 

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e. Added a sentence describing that a letter may be sent to potential subjects prior to initial 
contact 
f. Added Sujith Kuruvilla, M.D. to the protocol as a sub-investigator 
  
Version 8 (July 20177) 
a. Added cognitive assessments via Cogstate tests. 
b. Added RNA collection (PAXGene tubes) 
c. Added cuff brachial and central BP measurements using BP+ Uscom device. 
d. Removed febuxostat/allopurinol as exclusion criteria. 
e. Removed NT-proBNP testing for visits 2 and 3. 
  
Version 9 (November 2017) 
a. 
Added ancillary study to perform vasodilatorvasodilator cardiac MRI’s in 20 subjects 
enrolled at CMC VA Medical Center and University of Pennsylvania. The cardiac MRI will 
be performed  ~4-6 weeks into each phase. The ancillary study is designed to assess the 
effect of KNO3 on myocardial perfusion (MP) and perfusion reserve (MPR) in 
HFpEFHFpEF subjects. Stress cardiac MRI’s will be performed at 1.5 T scanner. Subjects 
will be administered GBCA (Gadolinium Based Contrast Agent) and Regandenoson. The 
potential risks associated with these procedures and adequacy to protect against these 
risks are also added. 
b. 
Some minor typos were corrected. 
c. 
Ambulatory blood pressure monitoring was removed. 
d. 
The placement of Actigraph (activity monitor) was specified inin the wrist instead of hip.  
e. 
Addition of standardized low nitrate breakfast to baseline visit following the blood draw for 
labs and biomarker samples. 
f. 
SpecificationSpecification of cutpoints for considering a quantitative G6PD activity test 
“positive” for clinically significant G6PD deficiency (<60% of normal). 
g. 
Increase of number of enrolled subjects to 84. 
Version 10 (October 2018) 
a. 
Added laungage for recent FDA guidance regarding GBCA (Gadolinium Based Contrast 
Agent) administration. The potential risks associated with these procedures and 
adequacy to protect against these risks were also updated. 
b. 
Some minor typos and formatting errors were corrected. 
c. 
Clarification of the role of the DSMB and PI reporting requirements 
d. 
Clarification of some exclusion criteria. 
 
Version 11 (March 2020) 
a. Added Srinath Adusumalli, MD, M. SC, to protocol as sub-investigator 
 
Version 12 (07-Oct-2021) 
a. Added clarification of consent procedure