Matching Perfusion and Metabolic Activity in HFpEF
Phase: PHASE2
Conditions: Heart Failure With Preserved Ejection Fraction
Sponsor: University of Pennsylvania

 
 
 
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CLINICAL RESEARCH PROTOCOL 
 
INVESTIGATIONAL PRODUCT(S): 
 
Potassium Nitrate 
Nicotinamide Riboside 
Propionyl-L-Carnitine 
Potassium Chloride 
STUDY 
NUMBER(S): 
IRB Number 
844627 
Other Protocol 
Identifiers  
 
CAMRIS: 735 
CHPS: 
PROTOCOL(S) TITLE: 
Matching Perfusion to Metabolic Activity in HFpEF 
REGULATORY SPONSOR: 
 
University of Pennsylvania 
FUNDING SPONSOR(S): 
National Institutes of Health 
PRINCIPAL INVESTIGATOR 
 
Payman Zamani, MD, MTR 
ORIGINAL PROTOCOL DATE: 
 
VERSION NUMBER: 
v1.0 
VERSION DATE: 
29 January 2021  
 

 
 
 
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1 
Study Summary ........................................................................................................ 7 
1.1 
Synopsis ............................................................................................................... 7 
1.2 
Key Roles and Study Governance ......................... Error! Bookmark not defined. 
1.3 Schema ..................................................................................................................... 10 
2 
Introduction and RationalE .................................................................................... 11 
2.1 
Study Rationale .................................................................................................. 11 
2.2 
Background ........................................................................................................ 11 
2.2.1 
Pharmacokinetics, Pharmacodynamics and Toxicology ......................... 11 
2.2.2 
Assessment for Potential Study Products Drug-Drug, Drug-Device, 
Device-Device Interactions ......................................................................... 14 
2.2.3 
Clinical Adverse Event Profile .......................... Error! Bookmark not defined. 
2.2.4 
Dosing Rationale ................................................ Error! Bookmark not defined. 
2.3 
Risk/Benefit Assessment ........................................ Error! Bookmark not defined. 
2.3.1 
Known Potential Risks ................................................................................ 22 
2.3.2 
Known Potential Benefits ............................................................................ 22 
2.3.3 
Assessment of Potential Risks and Benefits .. Error! Bookmark not defined. 
3 
Study Objectives and Endpoints ........................................................................... 25 
4 
Study Plan ............................................................................................................... 29 
4.1 
Study Design ...................................................................................................... 29 
4.2 
Scientific Rationale for Study Design .............................................................. 29 
4.3 
Justification for Dose ........................................................................................ 29 
4.4 
End of Study Definition ..................................................................................... 29 
5 
Study Population .................................................................................................... 31 
5.1 
Inclusion Criteria ...................................................... Error! Bookmark not defined. 
5.2 
Exclusion Criteria .............................................................................................. 31 
5.3 
Lifestyle Considerations ................................................................................... 32 
5.4 
Screen Failures .................................................................................................. 33 
5.5 
Strategies for Recruitment and Retention ....................................................... 33 
6 
Study Intervention .................................................................................................. 35 
6.1 
Study Intervention(s) Administration ............................................................... 36 
6.1.1 
Study Intervention Description ................................................................... 36 
6.1.2 
Dosing and Administration ......................................................................... 36 
6.2 
Preparation/Handling/Storage/Accountability ................................................ 38 
6.2.1 
Acquisition and accountability ................................................................... 39 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ................................ 40 
6.2.3 
Product Storage and Stability ........................... Error! Bookmark not defined. 
6.2.4 
Preparation ......................................................... Error! Bookmark not defined. 
6.3 
Measures to Minimize Bias: Randomization and Blinding ............................ 40 
6.4 
Study Intervention Compliance ........................................................................ 41 
6.5 
Concomitant Therapy ........................................................................................ 41 
6.5.1 
Rescue Medicine ................................................ Error! Bookmark not defined. 
7 
Study Intervention Discontinuation and Participant 
Discontinuation/Withdrawal .................................................................................. 42 
7.1 
Discontinuation of Study Intervention ............................................................. 42 
7.2 
Participant Discontinuation/Withdrawal from the Study ................................ 42 
 

 
 
 
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7.3 
Lost To Follow-Up .............................................................................................. 42 
8 
Study Assessment and Procedures ..................................................................... 44 
8.1 
Efficacy Assessments ....................................................................................... 44 
8.2 
Safety and Other Assessments ........................................................................ 53 
8.3 
Adverse Events and Serious Adverse Events ................................................ 53 
8.3.1 
Definition of Adverse Events (AE) .................... Error! Bookmark not defined. 
8.3.2 
Definition of Serious Adverse Events (SAE) ... Error! Bookmark not defined. 
8.3.3 
Classification of an Adverse Event .................. Error! Bookmark not defined. 
8.3.4 
Time Period and Frequency for Event Assessment and Follow-Up .. Error! 
Bookmark not defined. 
8.3.5 
Adverse Event Reporting .................................. Error! Bookmark not defined. 
8.3.6 
Serious Adverse Event Reporting .................... Error! Bookmark not defined. 
8.3.7 
Reporting Events to Participants ..................... Error! Bookmark not defined. 
8.3.8 
Events of Special Interest ................................. Error! Bookmark not defined. 
8.3.9 
Reporting of Pregnancy .................................... Error! Bookmark not defined. 
8.4 
Unanticipated Problems .......................................... Error! Bookmark not defined. 
8.4.1 
Definition of Unanticipated Problems (UP) ...... Error! Bookmark not defined. 
8.4.2 
Unanticipated Problem Reporting .................... Error! Bookmark not defined. 
8.4.3 
Reporting Unanticipated Problems To Participants .... Error! Bookmark not 
defined. 
9 
Statistical Considerations ...................................................................................... 63 
9.1 
Statistical Hypotheses ....................................................................................... 63 
9.2 
Sample Size Determination ..................................... Error! Bookmark not defined. 
9.3 
Populations for Analyses .................................................................................. 63 
9.4 
Statistical Analyses ................................................. Error! Bookmark not defined. 
9.4.1 
General Approach .............................................. Error! Bookmark not defined. 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) . Error! Bookmark not defined. 
9.4.3 
Analysis of the Secondary Endpoint(s) ........... Error! Bookmark not defined. 
9.4.4 
Safety Analyses .................................................. Error! Bookmark not defined. 
9.4.5 
Baseline Descriptive Statistics ......................... Error! Bookmark not defined. 
9.4.6 
Planned Interim Analyses ................................. Error! Bookmark not defined. 
9.4.7 
Sub-Group Analyses .......................................... Error! Bookmark not defined. 
9.4.8 
Tabulation of Individual Participant Data ........ Error! Bookmark not defined. 
9.4.9 
Exploratory Analyses ........................................ Error! Bookmark not defined. 
10 Supporting Documentation and Operational Considerations ............................ 66 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ............................ 66 
10.1.1 
Informed Consent Process ......................................................................... 66 
10.1.2 
Study Discontinuation and Closure ........................................................... 66 
10.1.3 
Confidentiality and Privacy ......................................................................... 67 
10.1.4 
Future Use of Stored Specimens and Data ............................................... 67 
10.1.5 
Safety Oversight ........................................................................................... 68 
10.1.6 
Clinical Monitoring ....................................................................................... 68 
10.1.7 
Quality Assurance and Quality Control ..................................................... 68 
10.1.8 
Data Handling and Record Keeping ........................................................... 69 
10.1.9 
Protocol Deviations ..................................................................................... 69 
10.1.10 
Publication and Data Sharing Policy .......................................................... 70 

 
 
 
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10.1.11 
Conflict of Interest Policy ............................................................................ 70 
10.2 
Additional Considerations ...................................... Error! Bookmark not defined. 
10.3 
Protocol Amendment History ........................................................................... 70 
11 References .................................................................... Error! Bookmark not defined. 
12 APPENDIX ............................................................................................................... 71 
12.1 
Schedule of Activities (SoA) ............................................................................. 71 
 
 
 
 
 

 
 
 
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PRINCIPAL INVESTIGATOR SIGNATURE 
 
STUDY SPONSOR: National Institutes of Health  
STUDY TITLE: 
Matching Perfusion to Metabolic Activity in HFpEF – The MPMA HFpEF 
Study 
STUDY ID 
PROTOCOL 
VERSION 
84467 
v1.0 
I have read the referenced protocol. I agree to conduct the study in accordance to this protocol, in 
compliance with the Declaration of Helsinki, Good Clinical Practices (GCP), and all applicable 
regulatory requirements and guidelines. 
Principal 
Investigator Name  
Payman Zamani, MD, MTR 
 
Signature 
 
Affiliation: 
University of Pennsylvania 
 
Date 
 
 
 
 
 
 
 

 
 
 
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Abbreviations 
AE – Adverse events 
ATP – Adenosine triphosphate 
BID – Twice daily 
Cr – Creatine 
CrAT – Carnitine acetyltransferase 
CrCEST – Creatine chemical exchange saturation transfer 
DSMP – Data safety and monitoring plan 
DSMB – Data safety and monitoring board 
GCP – Good clinical practice 
HIPAA – Heath Insurance Portability and Accountability Act 
HFpEF – Heart failure with preserved ejection fraction 
HFrEF – Heart failure with reduced ejection fraction 
HTN - Hypertension 
IDS – Investigational Drug Service 
IRB – Institutional Review Board 
KCCQ – Kansas City Cardiomyopathy Questionnaire 
KCl – Potassium chloride 
KNO3 – Potassium nitrate 
LG – Lateral gastrocnemius 
MRI – Magnetic resonance imaging 
NAD+ – Nicotinamide adenine dinucleotide 
NIRS – Near infrared spectroscopy 
NO – Nitric oxide 
NR – Nicotinamide riboside 
OxPhos – Oxidative phosphorylation capacity 
O2 – Oxygen 
PB – Placebo 
PLC – Propionyl-L-Carnitine 
SOP – Standard operating procedure 
QD – Daily 
SAE – Serious adverse events 
SkM – Skeletal muscle 
SVR – Systemic vascular resistance 
t1/2,Cr – Half-time of creatine recovery 
TCA – Tricarboxylic acid cycle 
TID – Three times daily 
ULN – Upper limit of normal 
UP – Unanticipated problem 
UPenn – University of Pennsylvania 
VT – Ventilatory threshold 
VO2 – Oxygen uptake 
VO2,peak – Peak oxygen uptake 
vPIVOT – Velocity and Perfusion, Intravascular Venous Oxygen saturation, and T2* 
DAVO2 – Arteriovenous O2 content difference 
31P – 31-Phosphate 
 

 
 
 
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1 
STUDY SUMMARY 
1.1 
Synopsis 
Title:  
Matching Perfusion to Metabolic Activity in HFpEF – The MPMA HFpEF 
Study 
Short Title: 
Perfusion to Metabolic Activity in H FpEF 
Study 
Description:  
This study will test whether Potassium Nitrate (KNO3), with and without 
Propionyl-L-Carnitine (PLC) and Nicotinamide Riboside (NR), improves 
submaximal exercise endurance and skeletal muscle oxidative 
phosphorylation capacity (SkM OxPhos) in participants with Heart Failure 
with Preserved Ejection Fraction (HFpEF). This study will also test 
whether the response to supplemental oxygen identifies HFpEF 
individuals who derive additional benefit from combination therapy with 
KNO3 + PLC + NR, as opposed to KNO3 alone. 
Objectives:  
1. Compare the impact on submaximal exercise endurance of 
pharmacologic therapy aimed primarily at increasing intramuscular 
perfusion (KNO3) versus combination therapy to increase 
intramuscular perfusion and mitochondrial reserve (KNO3 + NR + 
PLC).  
2. Assess the relationship between the individual response of 
submaximal exercise endurance to our pharmacologic interventions 
and the individual response of SkM OxPhos to supplemental oxygen 
(O2). 
 
Primary 
Endpoint:  
1. The primary endpoint will be the change in submaximal exercise 
endurance (time to fatigue at 75% of peak workload) between the 
interventional therapies and active control (KCl) 
 
Secondary 
Endpoints: 
1. Change in SkM OxPhos following plantar flexion on MRI  
2. Change in exercise vasodilatory reserve 
3. Change in KCCQ overall summary score 
4. Change in steps per day 
5. Change in the kinetics of O2-consumption 
6. Peak oxygen consumption during submaximal exercise 

 
 
 
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7. Change in intramuscular perfusion following plantar flexion on MRI 
 
Exploratory 
Endpoints: 
Assess the impact of our interventions on: 
(1) Arterio-venous concentration gradients for plasma substrates at 
baseline, 4 minutes of exercise, and at the time of exhaustion 
(2) Respiratory exchange ratio, a non-invasive metric of whole-body 
substrate oxidation obtained from expired gas analysis, assessed 
at 4 minutes of exercise 
(3) Tissue-based mitochondrial respiration  
(4) Tissue-based metabolomics 
(5) Muscle proteome 
Study 
Population: 
This study will be performed in approximately 53 stable outpatient 
participants with symptomatic Heart Failure with Preserved Ejection 
Fraction. 
 
Phase: 
Phase II 
Description of 
Sites/Facilities  
Center for Human Phenomic Sciences 
Presbyterian Medical Center 
University of Pennsylvania 
Enrolling 
Participants: 
This will be a single center study 
Description of 
Study 
Intervention: 
Investigational Medications: 
• 
Potassium Nitrate (KNO3) 6 mmol three times daily 
• 
Propionyl-L-Carnitine (PLC) 1000 mg twice daily 
• 
Nicotinamide Riboside (NR) 300 mg three times daily 
• 
Potassium Chloride (KCl) 6 mmol three times daily 
The order in which subjects will receive the following 3 interventions will 
be randomized and administered in a double-blind fashion: 
1. KNO3 alone 

 
 
 
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2. KNO3 + PLC + NR 
3. KCl 
 
Study Duration: 
60 months 
Participant 
Duration: 
Approximately 7 months 
 
 
 

 
 
 
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1.2 
Schema 
Total anticipated enrollment: 53 participants 
3 period cross-over study during which all participants will receive the following interventions in 
randomized double-blind order, with a 2-week washout period in between phases:  
• 
Potassium Nitrate (KNO3)  
• 
Potassium Nitrate + Nicotinamide Riboside + Propionyl-L-Carnitine (KNO3 + PLC + 
NR)  
• 
Potassium Chloride (KCl), as an active control 
Study Overview: 
 
Visit
Procedures
Primary Objective
Visit 1: 
Baseline Testing
a)
Informed Consent and 
Randomization
b)
Max Effort CPET
c)
Submax verification study
d)
KCCQ
-
Determine Peak VO2
-
Determine peak workload (PW) and 75%PW to be used in 
subsequent submaximal tests
-
Submax workload titration to target ex duration between 3-6 min
6 weeks of Period 1
Visit 2: 
Period 1 Endpoint 
Assessment 1
a)
Muscle Biopsy
b)
Submax (75%PW)  Exercise 
test
c)
KCCQ
-
Determine Phase A submax exercise time
-
Determine Phase A submax VO2 on/off-kinetics
-
Assess muscle metabolome, citrate synthase activity, rates of 
carbohydrate and fatty acid oxidation
Visit 3: (3-7d later)
Period 1 Endpoint 
Assessment 2
Skeletal muscle calf MRI with 
plantar flexion exercise
-
SkM OxPhos – CrCEST
-
Intramuscular perfusion – vPIVOT
2-Week Washout, followed by 6 weeks of Period 2
Visit 4: 
Period 2 Endpoint 
Assessment 1
a)
Muscle Biopsy
b)
Submax (75%PW)  Exercise 
test
c)
KCCQ
-
Determine Phase B submax exercise time
-
Determine Phase B submax VO2 on/off-kinetics
-
Assess muscle metabolome, citrate synthase activity, rates of 
carbohydrate and fatty acid oxidation
Visit 5: (3-7d later)
Period 2 Endpoint 
Assessment 2
Skeletal muscle calf MRI with 
plantar flexion exercise
-
SkM OxPhos – CrCEST
-
Intramuscular perfusion – vPIVOT
2-Week Washout, followed by 6 weeks of Period 3
Visit 6: 
Period 3 Endpoint 
Assessment 1
a)
Muscle Biopsy
b)
Submax (75%PW)  Exercise 
test
c)
KCCQ
-
Determine Phase C submax exercise time
-
Determine Phase C submax VO2 on/off-kinetics
-
Assess muscle metabolome, citrate synthase activity, rates of 
carbohydrate and fatty acid oxidation
Visit 7: (3-7d later)
Period 3 Endpoint 
Assessment 2
Skeletal muscle calf MRI with 
plantar flexion exercise
-
SkM OxPhos – CrCEST
-
Intramuscular perfusion – vPIVOT
One-month washout
Visit 8: (~1 month later)
Skeletal muscle calf MRI with 
plantar flexion exercise:
•
Room Air
•
100% O2
-
Determine the change in SkM OxPhos with 100% oxygen
-
Compare the change in SkM OxPhos with 100% oxygen to the 
difference in submax exercise time between the KNO3 and 
KNO3+NR+PLC arm

 
 
 
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2 
INTRODUCTION AND RATIONALE 
2.1 
Study Rationale 
The incidence of HFpEF is increasing, leading to more patients who are functionally limited and with a 
poor quality of life. While the incidence of heart failure with reduced ejection fraction (HFrEF) has decreased, 
that of heart failure with preserved ejection fraction (HFpEF) continues to rise,1-5 accounting for ~50% of HF 
hospitalizations,4, 6, 7 and disproportionately affecting females.2, 8 Patients with HFpEF suffer mentally and 
physically,9, 10 similar to their HFrEF counterparts.11-16 Yet in contrast to HFrEF, there are no effective 
therapies for HFpEF,17 despite numerous clinical trials.18-20 One reason trials have failed may be the 
significant heterogeneity found across HFpEF patients,21, 22 precluding a “one-size-fits-all” approach to 
treatment. Indeed, this type of approach, where phenotypic differences between subjects are ignored and all 
subjects are treated similarly, may have masked clinically-significant benefit in some.23-25 Yet no study has 
prospectively evaluated a strategy to determine which HFpEF patients are likely to respond to a specific drug 
intervention. In this protocol, we will test novel therapies that specifically target abnormalities identified in 
HFpEF patients and that are associated with impaired exercise capacity. We will also test an MRI-based 
strategy to determine which HFpEF participants are more likely to benefit from our different therapeutic 
approaches. 
2.2 
Background 
The determinants of exercise capacity among HFpEF patients are heterogenous. Reduced aerobic 
capacity is the hallmark of HFpEF, yet 
exactly what limits an individual patient 
may be varied between individuals. In 
an in-depth analysis of oxygen (O2) 
transport during exercise by Houstis et 
al., 79 subjects with HFpEF and 55 
controls underwent cardiopulmonary 
exercise testing with invasive 
hemodynamic measurements.20 The 
authors found significant heterogeneity 
across patients with regard to the 
site(s) of impairment in O2 transport, 
even across participants with similar 
peak oxygen uptake (VO2,peak).20 
Specifically, nearly all of the 
participants studied had ³ 2 significant 
impairments (<80% of control values) 
in the steps of O2 transport and utilization (alveolar ventilation, lung diffusion capacity, cardiac output, 
hemoglobin concentration, skeletal muscle diffusion capacity, and mitochondrial oxidative capacity). These 
data suggest that although exertional intolerance is the common final result, exactly which factors along the O2 
transport pathway are impaired may be different among HFpEF patients, suggesting that there is marked 
heterogeneity in the patient population regarding the factors that limit exercise.  
The skeletal muscle of HFpEF patients may be an important site of exercise limitation.  The study by 
Houstis et al. also identified that the arteriovenous O2 content difference (DAVO2) at peak exercise, a marker 
of skeletal muscle (SkM) oxygen utilization, is consistently impaired in HFpEF patients across several 
studies.20, 26, 27 In our preliminary studies, we evaluated exercise capacity in healthy individuals (n=20), HFpEF 
0
50
100
150
mL O2/min/kg Leg Lean Mass
Healthy
HTN
HFpEF
Peak VO2, Indexed to Leg Lean Mass
5
10
15
20
25
30
mL O2/dL Blood
Healthy
HTN
HFpEF
Peak AVO2 Diff
Overall p<0.001
***
**
***
Overall p<0.001
*
***
Figure 1 – Peak VO2 and the arteriovenous O2 content difference are 
reduced in HFpEF. Healthy (n=20), HTN (n=19), and HFpEF (n=20) individuals 
performed a maximal effort cardiopulmonary exercise test on a supine cycle 
ergometer. Echocardiography was used to determine cardiac output, and the 
DAVO2, a marker of SM oxygen utilization, was solved using the direct Fick 
equation. *** adjusted p£0.001, ** adjusted p£0.01, * adjusted p<0.05 

 
 
 
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subjects (n=20), and age-matched hypertensive subjects (HTN, n=19) during cycle ergometry.28 HFpEF 
subjects had markedly lower peak VO2, compared to either control group. Importantly, HFpEF subjects had 
a diminished DAVO2 (Fig. 1), while peak cardiac output was no different, suggesting to us that the SkM may 
be an important site of exercise limitation in HFpEF.  
The rate of skeletal muscle oxidative phosphorylation 
(OxPhos) is reduced in HFpEF. Focusing our attention on the 
skeletal muscle, we then assessed SkM OxPhos (Healthy=20, 
HTN=17, HFpEF=14) using a novel MRI-based technique 
developed by our collaborators at UPenn: Creatine Chemical 
Exchange Saturation Transfer (CrCEST, Fig. 2).29-33  CrCEST 
measures free Cr concentration, which increases during exercise as 
it releases from phosphocreatine (PCr) to generate ATP, and 
decreases during recovery as the mitochondria generate ATP and 
reform PCr.31, 34 CrCEST imaging has the ability to interrogate 
oxidative capacity in an anatomically-resolved manner, allowing for 
recovery parameters to be determined for individual muscle groups, 
without the confounding influence of non-activated intervening 
tissue (e.g. fat, inactivate muscle) which contributes to 31P 
measurements.29, 31-33  
Using CrCEST, we found that the half-time of Cr recovery (t1/2,Cr) of 
the lateral gastrocnemius muscle, the muscle most activated by our 
plantar flexion protocol, was markedly prolonged in HFpEF 
participants, compared to both healthy and age-matched 
hypertensive controls (Fig. 3).  
Moreover, among all study 
participants, t1/2,Cr correlated 
with Peak VO2, indexed to leg 
lean mass (Spearman’s rho:-
0.28, p=0.047) and the 
ventilatory threshold (VTleg lean, 
Spearman’s rho: -0.34, p=0.01), 
demonstrating an association 
between SkM OxPhos and a 
patient’s aerobic capacity. 
Mitochondrial function is normal in HFpEF. The prolonged SkM 
OxPhos seen in our HFpEF subjects (Fig. 3) may be attributable to 
several different abnormalities: (1) impaired mitochondrial function; (2) 
diminished mitochondrial volume; or (3) diminished O2 availability to 
respiring mitochondria.35 To investigate these possibilities further, we 
invited participants back to undergo a muscle biopsy of the vastus lateralis 
(Healthy=12, HTN=9, HFpEF=11). To examine mitochondrial function, we 
measured O2 flux from freshly isolated mitochondria that were given a 
saturating level of ADP, pyruvate, and malate, finding no difference among 
groups. We also subjected muscle homogenates to tritiated palmitate and 
assessed rates of palmitate flux,36, 37 again finding no difference among groups. We then assessed 
mitochondrial morphology (n=50 mitochondria/subject) on electron microscopy, finding no difference in 
CrCEST Contrast (%) 
Time (s) 
exercise 
25 
20 
15 
10 
5 
0 
-100 
0 
400 
500 
100 
200 
300 
Figure 2 – Example of exercise-induced 
changes in Free Cr, measured by CrCEST. 
One healthy 36-year-old male performed plantar 
flexion exercise for 2 minutes (0.75 Hz). Baseline 
Cr maps are shown in the upper left (white box). 
Serial Cr maps following exercise are displayed 
at 30s intervals, with relative Cr concentrations 
displayed on the bar on the upper right.  
Healthy
HTN
HFpEF
0
100
200
300
400
CrCEST Recovery 
Half Time (s)
Overall p=0.002


Figure 3 – SkM OxPhos is prolonged 
in HFpEF. The linear half-time of Cr 
recovery was calculated following a 
standardized plantar flexion exercise 
protocol. Data are displayed as the 
geometric mean with the 95% CI.  ** 
Bonferroni-adjusted p<0.01 

 
 
 
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individual mitochondrial size, Feret’s diameter, or electron density between groups. While there are limitations 
to these techniques, including the use of isolated mitochondria as opposed to permeabilized fibers,38 these 
findings suggested to us that, at least at rest, isolated mitochondria in HFpEF and controls are morphologically 
and functionally similar.  
However, it could be that HFpEF patients have a lower mitochondrial volume density.35, 39 We therefore 
assessed SkM mitochondrial content, both by measuring citrate synthase activity and by quantifying 
mitochondria volume density on our electron microscopy images. We did not find evidence for decreased 
mitochondrial volume density in our HFpEF samples using either technique. However, a report (Healthy=17,  
HFpEF=20) by Dr. Kitzman’s group at Wake Forest demonstrated reduced citrate synthase activity in HFpEF,40 
suggesting reduced mitochondrial content. We considered whether the discrepancy between the findings 
represents a Type II error, or alternatively, that decreased mitochondrial content, and hence 
mitochondrial reserve capacity, is a HFpEF phenotype not present in all patients. Yet whether this is the 
case, and determining which HFpEF patients have impaired mitochondrial reserve and thus may benefit from a 
mitochondrially-directed intervention, can currently only be done with a muscle biopsy.  
Intramuscular perfusion may be different than bulk blood flow.  Finally, impaired blood flow to the lateral 
gastrocnemius muscle during exercise and recovery could also explain the impaired SkM OxPhos in HFpEF. In 
fact, numerous studies,41-43 including ours,28 demonstrate an impaired vasodilatory reserve during cycle 
ergometry exercise in HFpEF subjects, suggesting that HFpEF patients cannot efficiently redistribute blood 
flow to locomotive exercising muscle. In our initial 
efforts to investigate this possibility, we measured 
popliteal artery bulk blood flow using the same 
plantar flexion exercise protocol (Healthy=7, 
HTN=8, HFpEF=5), repeated immediately after the 
CrCEST studies. Although popliteal artery blood 
flow increased with exercise, we found no 
difference in peak popliteal artery blood flow 
between groups (p=0.26), suggesting that bulk 
arterial blood flow could not explain the 
differences in SkM OxPhos seen among the 
groups.   
Novel MRI technique to assess intramuscular 
perfusion: beyond bulk blood flow. Our 
collaborators have developed a novel technique to 
simultaneously quantify conduit artery flow velocity 
and intramuscular perfusion following plantar 
flexion exercise with high temporal resolution (4s): Velocity and Perfusion, Intravascular Venous Oxygen 
saturation, and T2* (vPIVOT).44-46 In a prior study in healthy individuals, conduit artery blood flow rate peaked 
8±3 seconds after the cessation of plantar flexion exercise, yet intramuscular gastrocnemius perfusion, the 
muscle most activated by plantar flexion, did not peak until 101±53s (Fig. 4). The physiologic concept of 
importance is noting that oxygen can only be consumed after it has reached the mitochondria. To this end, 
measuring intramuscular perfusion, as we propose to do here using vPIVOT, is more physiologically 
relevant than conduit artery bulk blood flow in interpreting SkM OxPhos. 
VO2 kinetics are an important determinant of exercise capacity and are impaired in HFpEF. In our 
preliminary studies, we measured the half-time of VO2 recovery following our maximal effort cycle ergometry 
studies. We found that VO2 recovery was prolonged in HFpEF subjects (Half-time of VO2 recovery: Healthy 
Figure 4 – Conduit artery blood flow characteristics, such as peak timing, 
are different than those of directly measured intramuscular perfusion 
following plantar flexion exercise. From Englund, 2018. 

 
 
 
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53.5±7.3, HTN 63.4±16.5, HFpEF 69.4±17.3 s, p=0.004). Importantly, across all subjects, the half-time of VO2 
recovery correlated with SkM OxPhos on CrCEST (Spearman’s rho: 0.29, p=0.045) and the ventilatory 
threshold (Spearman’s rho: -0.29, p=0.03), reinforcing the notion that recovery kinetics are related to SkM 
OxPhos and portend important relationships to functional capacity.47, 48 Our results are in line with other studies 
that found slowed VO2 kinetics in HFpEF patients.49, 50 In healthy individuals, speeding VO2 kinetics has been 
shown to improve exercise endurance.51-53 However, in HFpEF, the interplay between submaximal exercise 
endurance, SkM OxPhos, intramuscular perfusion, and VO2 kinetics is not well-understood. We seek to fill 
this gap and test the hypothesis that submaximal exercise tolerance in HFpEF patients can be 
increased by improving SkM OxPhos. Improved exercise tolerance will allow HFpEF patients to 
perform his/her activities of daily living more comfortably and enhance the likelihood that a patient 
may engage in prescribed exercise training.54  
We will test this hypothesis by evaluating pharmacologic strategies to improve submaximal exercise in HFpEF 
patients, while also determining the impact of our interventions on SkM OxPhos and VO2 kinetics. The 
therapies to be tested in this study are affordable and expected to be well-tolerated, based on many prior 
studies that utilized these agents. Importantly, even a small improvement in exercise tolerance is expected to 
allow patients to perform more activities of daily living, increasing their quality of life and independence, while 
potentially reducing the burden on the healthcare system. Thus, our study has the potential to have a major 
impact on the lives of millions of patients who suffer from this debilitating condition and who currently are 
without any pharmacologic treatment options. 
2.2.1 
Pharmacokinetics, Pharmacodynamics and Toxicology – Rationale for Dose Selection 
 
a) Rationale and drug dosing for inorganic nitrate, an intervention primarily targeting SkM 
perfusion 
While nitric oxide (NO) has 
many functions, including 
contributing to fuel 
metabolism36 and myocardial 
stiffness,55 NO also 
contributes to the exercise 
vasodilatory response.48, 56-59 
This is particularly important 
for HFpEF patients, in whom 
numerous studies document 
an impaired ability to decrease 
systemic vascular resistance 
with exercise.42 In a pilot 
study, we tested the 
hypothesis that 
supplementation with 
inorganic nitrate, which can be 
reduced to NO in the hypoxic 
and acidic environment of exercising muscle,60-63 improves exercise capacity in HFpEF.64 In a 2x2 cross-over 
trial of 17 HFpEF subjects, we found that a single dose of 12.9 mmol of inorganic nitrate led to significant 
improvements in the vasodilatory reserve during exercise, allowing for greater cardiac output, higher peak VO2, 
and higher ventilatory thresholds (Fig. 5).64 Using near infrared spectroscopy (NIRS) on the calf muscle during 
exercise, we found a tendency for oxyhemoglobin to fall to a lesser degree during exercise following inorganic 
P=0.005
5
10
15
20
Oxygen Consumption (mL/min/kg)
Placebo
Nitrate
Peak VO2
P=0.03
−60
−40
−20
0
% Change Peak Versus Baseline
Placebo
Nitrate
Systemic Vascular Resistance
P=0.006
0
100
200
300
% Change Peak Versus Baseline
Placebo
Nitrate
Cardiac Output
Figure 5 – Inorganic nitrate enhances the vasodilatory reserve in HFpEF, allowing for 
higher cardiac output and peak VO2 in HFpEF subjects (n=17). Note: Four HFpEF subjects 
experienced a decline in peak VO2 with inorganic nitrate compared to PB (non-responders). 
Red line indicates mean difference. 

 
 
 
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nitrate than during exercise following placebo (-11.3% vs. -15.8%, p=0.07), suggesting that intramuscular 
perfusion was improved with inorganic nitrate. Notably, other reports have identified a similar vasodilatory 
effect of inorganic nitrate, including a reduction in blood pressure65-74 and vascular stiffness,71, 75 and an 
increase in vascular conductance during exercise.48, 76, 77 These data suggest to us that the predominant 
effect of inorganic nitrate is to improve intramuscular perfusion during exercise.58, 78  
Relevant to this proposal, we also found a trend towards improved SkM OxPhos using NIRS and a forearm 
exercise paradigm (p=0.083),79, 80 and a trend towards improved VO2 kinetics following submaximal exercise 
(p=0.089). Unfortunately, however, our experimental design precluded an assessment of whether these 
physiologic changes reflected improved flow or improved mitochondrial oxidative capacity, a topic to be 
addressed in the current proposal. One additional study also identified an ergogenic benefit to inorganic nitrate 
specifically in HFpEF. In a cross-over trial, Eggebeen et al. treated 20 subjects with HFpEF with 6 mmol of 
inorganic nitrate daily for 7 days. Submaximal exercise tolerance, defined as the exercise time at 75% peak 
workload, was significantly increased by inorganic nitrate (inorganic nitrate: 449±180 vs. placebo: 363±125 s, 
24% increase, p=0.02).74  Other studies have also shown an improvement in VO2 kinetics with inorganic 
nitrate,51, 52, 81 yet none of these studies provided mechanistic data to explain the benefit. 
KNO3 dosing regimen. We then performed a dose finding study of potassium nitrate (KNO3) capsules 
specifically in HFpEF.82 We demonstrated that 6 mmol of KNO3 given three times daily appeared to be well 
tolerated in HFpEF patients, without clinically significant hypotension or methemoglobinemia. Additionally, we 
found an improvement in exercise time and quality of life.82 Based on our data, we will use a 6 mmol dose of 
KNO3 three times daily in this study. 
 
b. Rationale and drug dosing of nicotinamide riboside (NR) and propionyl-L-carnitine (PLC), as the 
intervention targeting mitochondrial reserve 
As shown in Fig. 5, we found several (n=4, 24%) non-responders in our study of acute inorganic nitrate, 
despite their having increased blood levels of nitric oxide metabolites (e.g. nitrate and nitrite).64 Similarly, in the 
Eggebeen et al. study, ~25% of the HFpEF subjects did not experience an increase in exercise endurance with 
inorganic nitrate.74 To realize the salutary benefits of inorganic nitrate, the skeletal muscle mitochondria 
must have the reserve capacity to utilize the additional oxygen delivered; therefore, we hypothesized 
that individuals who did not respond to inorganic nitrate might have impaired mitochondrial reserve. 
This led us to consider whether we could identify individuals who have a skeletal muscle metabolic signature 
consistent with an impairment in mitochondrial reserve. We executed a prospective skeletal muscle biopsy 
study to answer this question. As above, we did not find a decrease in mitochondrial content. However, we did 
find reductions in intramuscular NAD+, propionyl-CoA, and acetylcarnitine in our HFpEF muscle biopsy 
samples compared to controls (Fig. 6), indicating metabolic perturbations that may lead to energetic deficits 
and impair mitochondrial reserve.   
Rationale for NAD+ supplementation. Nicotinamide adenine dinucleotide (NAD+) is essential for energy 
production given its role in carrying electrons to the electron transport chain, and it is also a necessary co-
factor for the sirtuins, which deacetylate a host of proteins, including those involved in fuel metabolism83-91 and 
mitochondrial biogenesis (e.g. PGC-1a and HIF-1).91-93 NAD+ declines with aging,91, 94-98 and is lower in murine 
models of diabetes and obesity,86 conditions of great importance to HFpEF. Additionally, deficiencies in NAD+ 
limit mitochondrial ATP production from oxidative phosphorylation and render muscle more prone to 
weakness,97 as seen in HFpEF patients.99  

 
 
 
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NAD+ is reduced in HFpEF SkM. We found 
NAD+ concentrations to be significantly 
reduced in resting muscle biopsy tissue of 
HFpEF patients compared to controls 
(Healthy: 0.01±0.003, HTN: 0.01±0.003, 
HFpEF: 0.009±0.003 AU, p=0.039, Fig 6). 
Concentrations of NAD+ correlated with our 
CrCEST metrics of SkM OxPhos (Spearman’s 
rho: -0.47, p=0.01), suggesting that NAD+ 
supplementation has the potential to improve 
SkM OxPhos, perhaps by increasing 
mitochondrial reserve. In line with this, prior 
studies demonstrated several beneficial 
effects of increased NAD+ on the 
mitochondria: improved SkM OxPhos in 
models of aging;91, 92, 97 improved SkM 
oxidative metabolism and glucose tolerance in 
high-fat diet induced obesity;89, 93, 100 improved 
SkM OxPhos and mitochondrial biogenesis in 
mitochondrial disease models;100-105 improved 
exercise endurance in transgenic mouse 
models of increased NAD+,106, 107 increased 
mitochondrial respiration in humans with diabetes108 or mitochondrial myopathy104,  and an increased capacity 
to generate acetylcarnitine in overweight/obese humans.109 Finally, supplementation with NR enhanced 
peripheral blood mononuclear cell mitochondrial respiration, isolated from adults with HFrEF.110 
Table Examining NR Supplementation in Human Subjects (compiled Sept 2020) 
Author 
Population, n 
Dose/Duration 
Primary 
Outcome 
Results 
Adverse Events 
Airhart, 
2017111 
Healthy 
volunteers 
n=8 
250 mg BID 
escalating to 1000 
mg BID  
9 days 
Whole blood [NR] 
and [NAD+]  
1. Whole blood NAD+ 
and NR increased 
None 
Slight decrease in 
[K+] (-0.4 mEq) 
and [Hgb] (-0.4 
g/dL) 
Dellinger, 
2017112 
Healthy adults 
(60-80 years old) 
 
n=120 
 
NR + Pterostilbene 
(NRPT): 
randomized, double 
blind, placebo (PB) 
controlled parallel 
design 
1. 250 mg NR + 50 
mg PT daily 
2. 500 mg NR + 100 
mg PT daily 
3. PB 
8 weeks 
Whole blood 
[NAD+] 
1. Whole blood NAD+ 
increased in dose 
dependent manner 
2. Increased 6 min 
walk distance and 
30s chair stand test 
 
No serious AEs 
AE’s possible 
related to NR+PT 
1. Nausea 
2. Fatigue 
3. Headache 
4. GI upset 
5. Diarrhea 
 
Increased total 
cholesterol and 
.005
.01
.015
.02
.025
AU
Healthy
HTN
HFpEF
NAD+
0
.05
.1
.15
AU
Healthy
HTN
HFpEF
Propionyl−CoA
0
10
20
30
40
Healthy
HTN
HFpEF
Acetylcarnitine (uM)
p=0.039
p=0.033
p=0.028
Figure 6 – Lower NAD+, 
propionyl-CoA, and 
acetylcarnitine in HFpEF 
skeletal muscle. Levels were 
measured using mass 
spectrometry. NAD+ and 
Propionyl-CoA were normalized 
to the nearest internal standard 
(ISTD) and per mg of wet tissue 
weight and expressed as  
arbitrary units (AU). For NAD+, data are presented as means (blue) with 
95% CI (red whiskers); black dots are individual data points. For propionyl-
CoA and acetylcarnitine: box represents 25th-75th percentile; median is the 
red line; black dots indicate outliers.   

 
 
 
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LDL in treatment 
groups 
Martens, 
2018113 
Lean healthy, 
middle-aged and 
older men and 
women 
n= 30 
Crossover: PB vs. 
NR 500 mg BID  
6 weeks 
duration/phase 
 
Peripheral blood 
mononuclear 
cells (PBMC) 
[NAD+] 
1. Oral NR increased 
PBMC [NAD+]  
2. NAAD sensitive 
biomarker of NAD+ 
metabolism 
3. Greater NAD+ 
increase in subjects 
with lower baseline 
PBMC NAD+ 
4. Trend for reduced 
pulse-wave velocity, 
a marker of aortic 
stiffness 
No serious AEs.  
Possible NR AEs:  
• Nausea  
• rash/flushing  
• leg cramp  
• bruising 
Dollerup, 
2018114 
and 
2020115 
Healthy, 
sedentary, obese 
insulin-resistant 
men (40-70 yo) 
n=40 
 
Randomized, double 
blind, parallel design: 
PB vs. NR 1000 mg 
po BID  
12 weeks 
1. Insulin 
sensitivity 
2. Muscle NAD+ 
metabolome 
3. Muscle 
mitochondrial 
respiration 
1. No change in insulin 
sensitivity 
2. No change in muscle 
NAD+ with NR 
3. No change in global 
protein acetylation 
with NR 
4. No change in 
mitochondrial content 
or respiration with 
NR 
No serious AEs 
due to NR 
Possible AEs in 
NR: 
• Pruritus 
• Excessive 
sweating 
• Bloating 
• Transient 
changes in stool 
 
Conze, 
2019116 
Healthy 
overweight adults 
between 40-60 
years old  
n=140 
Randomized double-
blind, parallel design 
1. Placebo  
2. NR 100 mg/d  
3. NR 300 mg/d  
4. NR 1000 mg/d  
8 weeks 
Urinary MeNAM 
levels (NAD 
metabolite) 
1. Urinary MeNAM 
increased in dose 
dependent fashion  
2. PBMC NAD+ levels 
increased in dose 
dependent manner 
3. 1000 mg/day 
concluded to be 
upper limit for NR 
administration 
No dose 
dependent AEs. 
Possible AE: 
nausea, muscle 
pain/soreness 
El Hassan, 
2019117 
Healthy elderly 
(70-80) men  
n=12 
Double-blind, 
placebo controlled, 
cross-over: NR 500 
mg twice daily 
21 days/phase; no 
washout period 
 
1. Increased muscle 
NAAD and MeNAM 
2. Muscle NR and 
NAD were not 
increased by NR 
3. Whole blood NAD+, 
NAAD, and MeNAM 
increased with NR 
4. Genes associated 
with energy 
metabolism (TCA 
cycle/glycolysis/mito 
genes) 
downregulated by 
NR, but no change 
No AEs with NR 

 
 
 
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in glycolytic protein 
expression, 
mitochondrial 
bioenergetics or 
metabolism noted 
5. No change in 
mitochondrial 
respiration or 
content 
6. NR decreased 
inflammatory 
proteins (e.g. IL-6, 
IL-2, TNF-a)  
Dolopikou, 
2019118 
Twelve young 
(~22 yo) and 
twelve old (~72 
yo) healthy men 
 
n=24 
 
Randomized 
placebo-controlled 
single-dose cross-
over trial of NR 500 
mg versus PB, with a 
10d washout 
Single dose 
Peak VO2 
Concentric peak 
torque (leg) 
Isometric peak 
torque (leg) 
Fatigue Index 
1. NR increased RBC 
glutathione and 
reduced urine F2-
isoprostane in older 
patients 
2. NR increased RBC 
NADPH levels in 
both young and old 
3. Older subjects 
exhibited higher 
isometric peak 
torque and less 
fatigue following NR 
Not reported 
Remie, 
2020109 
45-65 years old, 
overweight/obese, 
sedentary men 
and post-
menopausal 
women 
 
n=13 
 
Double-blind 
randomized placebo-
controlled crossover: 
1000 mg NR/d 
6 weeks/phase 
 
Insulin sensitivity 
1. Intramuscular NAD+ 
not different 
between NR and 
PB; however, 
skeletal muscle 
NAAD and MeNAM 
increased 
2. No change in 
skeletal muscle 
mitochondrial 
respiration or 
mitochondrial 
complex proteins 
3. Intramuscular 
acetylcarnitine (C2) 
increased 
4. Slight increase in fat-
free mass with NR 
No AEs or side 
effects related to 
NR 
NAD+ precursor (NR) dosing.  Most cells, except for neurons, cannot import NAD+ directly, instead relying on 
its synthesis from tryptophan or Vitamin B3 analogs. NAD+ can also be recycled intracellularly from its 
byproducts (e.g. nicotinamide, nicotinamide mononucleotide [NMN], or nicotinamide riboside [NR]) in what is 
known as the “salvage pathway” (Fig. 7).87, 119 Both NR and NMN are orally bioavailable,87 and could be used 
to bolster NAD+ levels;119 however, very little data has been generated in humans with NMN 
supplementation.120, 121 On the other hand, substantial evidence supports using NR to increase NAD+ 
metabolism. Our co-investigators demonstrated that the SkM preferentially uses NR for NAD+ regeneration,85 
and that NR supplementation increases mitochondrial NAD+,122 supporting our planned use of NR in this 

 
 
 
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proposal that is focused on the SkM. Importantly, while 
several studies have shown that NR increases whole blood 
NAD+ (see table above), more recent studies have 
additionally demonstrated that NR supplementation leads to 
increased markers of NAD+ metabolism specifically within 
human skeletal muscle.109, 117  
Safety of NR Supplementation: Several studies have 
demonstrated the safety of NR supplementation in 
humans.109, 111, 113-118, 123 In a study of 8 healthy individuals, 
increasing doses of up to 1000 mg po BID for 2 days were 
well tolerated, increasing whole blood NAD+ levels by nearly 
100%.111 In another study, 40 obese sedentary men were 
randomized to 12 weeks of NR 1000 mg po BID versus placebo. NR was well-tolerated, and there was no 
increase in adverse events.114 In 12 elderly men, 1000 mg  of NR daily for 21 days did not lead to any adverse 
events.117 One additional study tested combined NR + Pterostilbene (NRPT) in 120 older adults (age 60-80) for 
8 weeks. No serious adverse events were noted; however, a small increase in cholesterol (LDL and total 
cholesterol) was noted in the combined NR+PT groups.112 In the largest study to date,140 obese middle-aged 
individuals were treated with increasing doses of NR up to 1000 mg/day. This maximal dose was shown to be 
safe, suggesting that 1000 mg/day was consistent with the tolerable upper limit of intake,116 and in line with the 
findings of the European Scientific Committee that suggested an upper limit of 900 mg/day for nicotinamide in 
adults.124  No change in LDL/Total cholesterol was seen in the NR group. Based on these data, we will use a 
dose of NR of 300 mg po TID.  
Rationale and dosing for propionyl-L-carnitine (PLC). Our preliminary data demonstrate a reduction in 
propionyl-CoA and acetylcarnitine in HFpEF (Fig. 6). Acetylcarnitine acts as a supply depot for acetyl-CoA 
when acetyl-CoA generation is limited.125-127 It is produced by the reaction of acetyl-CoA with carnitine, via 
carnitine acetyltransferase (CrAT), and functions to maintain a low intra-mitochondrial acetyl-CoA/CoA ratio 
and adequate availability of CoA for ongoing fat metabolism. Because increasing acetyl-CoA can also reduce 
flux through the PDH complex, maintaining a low acetyl-CoA/CoA ratio by shuttling acetyl-CoA into 
acetylcarnitine may also improve carbohydrate metabolism.128 In fact, L-carnitine has been shown to improve 
insulin sensitivity and carbohydrate metabolism in both murine and human subjects.126, 129, 130 Oral L-carnitine 
supplementation has been shown to increase muscle carnitine levels in athletes, along with increases in PDH 
complex activity, demonstrating the potential utility of oral supplementation.131, 132 In our biopsy studies, muscle 
acetylcarnitine negatively correlated with the half-time of CrCEST recovery (Spearman’s rho: -0.42, p=0.02), 
suggesting that increasing acetylcarnitine could speed SkM OxPhos.  
In one small study of 31 HFpEF subjects, improvements in diastolic function and shortness of breath were 
seen following 3 months of treatment with 1500 mg of L-carnitine daily.133 In a small study of HFrEF patients, 3 
grams/day of L-carnitine for 120 days led to significant increases in the maximal workload achieved during 
cycle ergometry.134 In another study, 2 grams/day of L-carnitine improved exercise capacity at 3 months in a 
randomized trial of 80 HFrEF subjects. Following this blinded period, subjects were continued in their groups 
for 3 years, noting an improvement in survival with L-carnitine. The only adverse effect noted was minor 
gastrointestinal upset in 3 subjects assigned to L-carnitine, though this did not require withdrawal.135  
Propionyl-CoA is generated via the breakdown of odd chain fatty acids and branched chain amino acids 
(BCAA). It is then converted to succinate, providing carbons to be used in the tricarboxylic acid (TCA) cycle.136-
140 During exercise, TCA intermediate concentrations increase,141, 142 perhaps in order to sustain high rates of 
acetyl-CoA oxidation and NADH production.143 This suggests that reductions in propionyl-CoA may slow TCA 
cycling by impairing the expansion of its intermediates. In our preliminary data, muscle propionyl-CoA 
Figure 7 – NAD+ can be synthesized from nicotinamide 
riboside (NR). From Yoshino, 2018. 

 
 
 
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concentration correlated positively and strongly with both submaximal (ventilatory threshold indexed to leg lean 
mass: spearman’s rho: 0.55, p=0.002) and maximal oxygen uptake (peak VO2 indexed to leg lean mass: 
spearman’s rho: 0.59, p=0.0005). As propionic acid is toxic,139, 144, 145 prior investigation has focused on giving 
propionyl-L-carnitine (PLC), which is acted upon by CrAT to yield propionyl-CoA and L-carnitine.127, 138, 144, 146 
As an additional benefit, PLC may have better penetration into the cell due to its more lipophilic properties than 
L-carnitine alone.138, 139, 147, 148 
When given exogenously, PLC is taken up by the heart and skeletal muscle.149 PLC has been studied 
previously in HFrEF, where smaller studies demonstrated improvements in exercise capacity and left 
ventricular performance, using a dose of 1.5 g/day.150, 151 However, in a larger study of over 500 HFrEF 
subjects randomized to 1 gram PLC twice daily for 6 months, PLC did not improve exercise tolerance, though 
supplementation was safe.152 Interestingly, in the subgroup of patients with higher LV ejection fractions (>30% 
vs <30%), an increase in exercise time was seen.  
Table of Human Heart Failure Studies of Oral Propionyl-L-Carnitine Supplementation (Compiled 
September 2020) 
Author 
Population, n 
Dose/Duration 
Primary Outcome 
Results 
Adverse Events 
Mancini, 
1992153 
NYHA II/III 
HFrEF, 
randomized 
double blind 
placebo 
controlled, 
parallel design 
n=60 
500 mg PLC, 
three times 
daily 
180 days 
1. Change in 
exercise time on 
incremental 
cycle ergometry 
study 
2. Change in 
ejection fraction 
• Exercise time 
increased in 
PLC group at 
30 days, 
continuing to 
increase to 
180 days 
• Ejection 
fraction 
improved in 
PLC group 
Not reported 
Caponnetto, 
1994151 
NYHA Class II 
HFrEF 
Randomized, 
double blind, 
parallel design 
n=50 
500 mg PLC, 
three times 
daily 
180 days 
1. Steady-state 
submaximal 
exercise 
endurance 
(workload 
corresponding 
to 70% peak 
heart rate during 
incremental 
test)  
• Exercise 
endurance 
increased, 
starting from 
30d and 
sustained at 
180d 
• PLC decreased 
lactate 
production at 
the end 
exercise 
• LV ejection 
fraction 
No adverse 
events 
occurred 
One PB patient 
developed 
diarrhea which 
resolved 
No clinically 
significant 
biochemical 
abnormalities 
developed 

 
 
 
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improved in 
PLC 
Anand, 
1998150 
NYHA Class 
II/III HFrEF 
Randomized, 
single-blind, 
parallel design 
n=30  
(18 to PLC, 12 
to PB) 
500 mg PLC, 
three times 
daily 
30 days 
1. Impact of IV PLC 
on invasive 
hemodynamics 
acutely and 
following 30d of 
oral 
supplementation. 
Invasive hemo’s 
only in PLC 
group 
2. Peak VO2 on 
incremental 
treadmill study 
1. Chronic PLC 
lowered PA 
pressure 
2. IV PLC 
acutely 
reduced PA 
and PCWP 
pressure 
3. Chronic oral 
PLC reduced 
PA and 
PCWP after 
30d 
4. PLC did not 
impact 
neurohormone 
levels  
5. PLC 
increased 
peak VO2 
(+45%) and 
treadmill time 
(+21%) at 
15d, 
continuing to 
30d 
No 
complications. 
PLC did not 
affect basic 
laboratory tests 
(CBC, liver, 
renal function) 
Investigators 
of the Study 
on PLC in 
Chronic HF, 
1999152 
NYHA Class 
II/III HFrEF  
Phase III, 
double blind 
randomized 
parallel 
multicenter 
design 
 
n=537 (271 to 
PLC, 266 to 
PB) 
1-gram PLC, 
twice daily 
6 months 
1. Maximal 
exercise 
duration on 
incremental 
cycle ergometry 
protocol 
1. No significant 
difference in 
exercise 
duration in 
the overall 
population, 
nor in the 
subgroup of 
individuals 
who 
completed 
the study 
(n=353, 188 
in PLC and 
165 in PB) 
No difference 
in adverse 
events 
between PB 
and PLC. 
Patients with 
any AE: 
PB: 97   
(33.7%) 
 
vs. 
  
PLC: 98 
(34.3%) 

 
 
 
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2. Post-hoc: 
Subjects with 
a baseline 
LVEF 
between 30-
40% had an 
improvement 
in exercise 
duration with 
PLC 
 
p=0.882 
Additionally, several studies have examined the use of PLC for the treatment of claudications in peripheral 
arterial disease (reviewed in154), providing additional safety data. In the largest study, 485 patients with 
peripheral arterial disease and claudication were randomized to PLC 1000 mg po BID or placebo for 12 
months in a parallel design across 38 centers.155 Five subjects died in each of the PLC and PB arms. Adverse 
events requiring treatment discontinuation occurred in 27 in the PLC arm and 30 in the PB group, and adverse 
events not requiring discontinuation occurred in 38 in the PLC group and 98 in the PB group.155 In the second 
largest study, 245 patients were studied in a randomized placebo-controlled parallel design study of up to 3 
grams/day of PLC based on exercise response for 24 weeks across 13 centers.156 Eleven adverse events 
leading to drug discontinuation occurred in the PLC group, as compared to 3 in the placebo group; however, 
the adverse events requiring drug discontinuation in the PLC group were judged to be unrelated to study 
medication. There were 7 adverse events not requiring treatment discontinuation in the PB group and 5 in the 
PLC group – the most common adverse events were nausea and gastric pain. Moreover, minimal benefit was 
demonstrated for increasing the dose of PLC beyond 2 grams/day. Overall, these studies add to the evidence 
that PLC supplementation has an acceptable safety profile, with excellent tolerability. Based on all of the 
above, we will use a dose of PLC 1000 mg po BID. 
 
3 Rationale for potassium chloride (KCl), as an active control  
Potassium is known to impact the vasculature and blood pressure.157 To isolate the benefits of our intervention 
to either inorganic nitrate, or the combination of inorganic nitrate+NR+PLC, we will use potassium chloride as 
an active control. KCl will be given in the same doses as KNO3: 6 mmol three times a day. 
3.1.1 
Known Potential Risks 
a. Potassium Nitrate – Inorganic nitrate has been tested in several human studies in HFpEF to 
date and is very well tolerated.64, 74, 82 In our Dose-Finding study of 12 individuals with HFpEF, 9 
were treated with KNO3 as we propose to do in this study.82 KNO3 did not lead to clinically 
significant hypotension or methemoglobinemia.82 There was a systolic blood pressure lowering 
effect (approximately 12 mmHg),72, 82 though we note that HFpEF patients generally have elevated 
blood pressures.2 Gastrointestinal symptoms and headache were the most common side effects. 
Anecdotally, the gastrointestinal side effects can be mitigated by taking study medications with a 
meal,69 and may be related to the potassium content, as potassium is known to cause GI side 
effects in clinical practice.158  
 
b. Propionyl-L-Carnitine – PLC has been used in humans with heart failure previously.152 In the 
largest study, 537 patients with HFrEF were randomized to PLC versus placebo (271 received 
PLC) at the same dose to be used in this protocol. There was no significant difference in adverse 

 
 
 
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events between subjects receiving PLC versus those receiving PB.152 Nausea and gastric pain 
have been reported in other studies.156 
 
c. Nicotinamide Riboside (NR) - Several studies have reported results on various endpoints using 
NR supplementation in humans.111, 113, 114, 116, 118, 123 In a study of 8 healthy individuals, increasing 
doses of up to 1000 mg po BID for 2 days were well tolerated, increasing NAD+ levels by nearly 
100%.111 More recently, 40 obese sedentary men were randomized to 12 weeks of NR 1000 mg 
po BID versus placebo. NR was well-tolerated, and there was no increase in adverse events with 
supplementation.114 Finally, in the largest study to date,140 obese middle-aged individuals were 
treated with increasing doses of NR up to 1000 mg/day. This maximal dose was shown to be safe, 
suggesting that 1000 mg/day was consistent with the tolerable upper limit of intake,116 and in line 
with the findings of the European Scientific Committee which suggested an upper limit of 900 
mg/day for nicotinamide in adults.124 We therefore selected this dose (NR 300 mg po three times 
daily) to be used in this proposal. 
i. Possible side effects related to NR noted in the above studies include: 
1. GI upset: nausea, vomiting, diarrhea, transient changes in stool, bloating 
2. Muscle soreness/cramping 
3. Rash/flushing 
4. Headache  
 
d. Potassium Chloride: The main side effect of potassium chloride in clinical practice are 
gastrointestinal symptoms.69, 158 We will ask subjects to take study medications with food to 
minimize this. Potassium itself also has an effect on the vasculature, lowering blood pressure 
(average of ~3/2 mmHg).158, 159 We do not expect this to lead to clinically-significant hypotension 
in our HFpEF participants who are generally hypertensive. Subjects with a baseline potassium 
>5.0 mEq/L will be excluded from the study. 
 
We note that our study seeks to use a combination of therapies in one interventional arm: 
• 
Potassium nitrate + Propionyl-L-Carnitine + Nicotinamide riboside 
 
It is possible that there will also be additional/different side effects when the combination of the above 
medications is administered. For example, a small study in trained individuals demonstrated that glycine 
propionyl-L-carnitine increased plasma nitric oxide species, suggesting that there may be an added effect with 
potassium nitrate due to additional nitric oxide release from PLC.160 Another study in HFrEF individuals found 
that PLC reduced systemic vascular resistance,139, 151 which is also a target of KNO3.  
On the other hand, in a mouse model of diet-induced obesity, the combination of NR + L-carnitine 
attenuated weight gain and hepatic steatosis to a greater extent than monotherapy with either agent,161 
suggesting the possibility of additional salutary benefits to the combination group. Overall, these data suggest 
that additional risks/benefits may be present in the combination group that will require close monitoring.   
As enumerated in the Data Safety Monitoring Plan, we will ensure adequate safeguards are in place for 
these side effects to be identified, acted upon as necessary, and documented (please see Data Safety and 
Monitoring Plan for additional details). 
Risks and Benefits Assessment: 
 
The side effect profile of the study drugs, in isolation, demonstrate acceptable tolerability. Our 
preliminary data, and additional data from other patient populations, support the conclusion that the 
supplementation strategies to be tested are rationale. At this time, there are no pharmacologic strategies that 
consistently show a benefit in HFpEF. Even a small improvement in exercise tolerance is expected to allow 

 
 
 
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HFpEF patients to perform more activities of daily living, increasing their quality of life and independence, while 
potentially reducing the burden on the healthcare system. Thus, our study has the potential to have a major 
impact on the lives of millions of patients who suffer from this debilitating condition and who currently are 
without any pharmacologic treatment options. 
  

 
 
 
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4 
STUDY OBJECTIVES AND ENDPOINTS 
Aim 1: 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
Compare the impact on submaximal 
exercise endurance of pharmacologic 
therapy aimed primarily at increasing 
intramuscular perfusion (KNO3) 
versus combination therapy to 
increase intramuscular perfusion and 
mitochondrial reserve 
(KNO3+NR+PLC), as compared to 
active control (KCl) 
 
Submaximal Exercise Endurance: 
Time to exhaustion while exercising at 
75% of peak workload 
Submaximal exercise 
endurance is more 
relevant to a patient’s 
ability to perform 
activities of daily living 
than metrics assessed at 
higher degrees of 
exertion (e.g. ‘peak’) that 
are not routinely 
achieved during daily life.  
Secondary 
 
 
Assess the impact of our interventions 
on skeletal muscle oxidative 
phosphorylation capacity (SkM 
OxPhos) 
MRI-based assessment of SkM 
OxPhos: The kinetics of Creatine 
recovery following exercise, as 
assessed using CrCEST MRI 
spectroscopy 
SkM OxPhos is a major 
topic of interest for this 
proposal. We 
hypothesize that our 
interventions may lead to 
a benefit in this patient 
population, 
predominantly by 
targeting muscle 
perfusion and oxidative 
phosphorylation capacity 
during exercise. 
Assess the impact of our interventions 
on intramuscular perfusion following 
plantar flexion 
MRI-based assessment of SkM 
intramuscular perfusion, as assessed 
using vPIVOT MRI sequences 
Mitochondrial oxidative 
function is intimately 
associated with oxygen 
delivery. Any changes in 
SkM OxPhos induced by 
our interventions are 
best viewed in light of 
changes to intramuscular 
perfusion, allowing a 
better understanding the 
mechanism of 
improvement. 
Assess the impact of our intervention 
on arterial properties during exercise  
Vasodilatory Reserve: Systemic 
vascular resistance (SVR) reserve will 
be calculated as the % change in 
systemic vascular resistance at baseline 
Our interventions, 
particularly KNO3, may 
impact exercise arterial 
properties, which could 
influence exercise 

 
 
 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
compared to the SVR at 4 minutes of 
exercise. 
capacity by altering 
muscle blood flow during 
exercise. 
Assess the impact of our interventions 
on quality of life 
Kansas City Cardiomyopathy 
Questionnaire Overall Summary 
Score 
Patients with HFpEF 
have an impaired quality 
of life that is related to 
their reduced functional 
capacity 
Assess the impact of our interventions 
on the kinetics of oxygen 
consumption (VO2 kinetics) during 
exercise and recovery 
VO2 Kinetics: “On” and “Off” kinetics 
will be modeled during the submaximal 
exercise transient 
VO2 kinetics relate to 
skeletal muscle 
mitochondrial properties 
and also to exercise 
endurance. Through their 
impact on muscle blood 
flow and SkM OxPhos, 
our interventions may 
affect VO2 kinetics.  
Assess the impact of our interventions 
on the maximal/peak rate of oxygen 
consumption (peak VO2) at the time of 
exhaustion during the submaximal 
exercise transient 
Peak VO2 – the maximal rate of oxygen 
consumption determined during the last 
30s of exercise 
Because 75%PW will 
likely be above critical 
power, it is expected that 
subjects will evince an 
additional metabolic cost 
to exercise that will drive 
oxygen consumption to 
maximal/peak rates at 
the time of exhaustion.  
Assess the impact of our interventions 
on ambulatory physical activity 
Steps per day – we will use actigraphy 
to document the average number of 
steps taken per day during the final 
week of each interventional period 
In addition to formal 
measures of quality of 
life and exercise 
tolerance, we will 
measure steps taken per 
day as a more patient-
centric metric of 
ambulatory exercise 
tolerance. 

 
 
 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Tertiary 
 
 
Assess the impact of our interventions 
on substrate metabolism during 
exercise 
(a) We will measure arteriovenous 
concentration gradients for plasma 
substrates (e.g. glucose, lactate, non-
esterified fatty acids, and triglycerides) 
at baseline, 4 minutes of exercise, and 
at the time of exhaustion. (b) We will 
also assess the respiratory exchange 
ratio, a metric of whole-body substrate 
metabolism obtained from expired gas 
analysis, at these same time points. 
Comparisons at 4 minutes of exercise 
will be the focus.  
Inorganic nitrate, PLC, 
and NR all have been 
shown to impact muscle 
metabolism and may 
alter substrate 
preference during 
submaximal exercise. 
Assess the impact of our interventions 
on muscle metabolomics 
Tissue metabolite concentrations: 
Muscle tissue concentrations of NAD+ 
and the NAD+ metabolome, propionyl-
CoA, and the acylcarnitine profile, and 
inorganic nitrate will be measured. 
We will assess the 
efficacy of our 
supplementation strategy 
on altering tissue 
concentrations of 
relevant metabolites.  
Assess the impact of our interventions 
on muscle tissue respirometry 
Ex vivo tissue measures of substrate 
metabolism. We will measure tissue 
rates of substrate (fat and carbohydrate) 
metabolism in isolated mitochondria and 
saponin-skinned fibers. Citrate 
synthase, a marker of mitochondrial 
content, will also be assessed. 
In addition to our 
CrCEST-based 
measures of SkM 
OxPhos, we will also 
perform tissue-based 
measurements to 
complement the in vivo 
findings.  
Assess the impact of our interventions 
on the muscle proteome 
Muscle Proteome: Relative 
abundances of proteins related to NAD+ 
metabolism, mitochondrial biogenesis, 
and mitochondrial OxPhos will be 
measured. 
Our supplementation 
strategy may alter 
concentrations of the 
proteins involved in 
substrate utilization and 
mitochondrial biogenesis 
AIM 2: IMPACT OF SUPPLEMENTAL O2 ON SKELETAL MUSCLE OXIDATIVE PHOSPHORYLATION 
CAPACITY  
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
To assess the response of SkM 
OxPhos to supplemental oxygen 
We will compare SkM OxPhos, 
assessed using the half-time CrCEST 
recovery, under both room air 
(FIO2=0.21) and 100% Oxygen 
(FIO2=1.0) conditions 
If a mitochondrial reserve 
is present, supplemental 
oxygen should speed 
SkM OxPhos. On the 
other hand, if no reserve 
is present, minimal 

 
 
 
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change in SkM OxPhos 
to 100% oxygen should 
be observed. This test  
will identify HFpEF 
individuals who harbor 
additional impairments at 
the mitochondria (O2-
non-responsive) who are 
more likely to benefit 
from mitochondrially-
directed interventions.  
To assess whether the contrast in the 
individual response to our 
pharmacologic interventions (KNO3 
versus KNO3+PLC+NR) on 
submaximal exercise endurance 
correlates to the response of SkM 
OxPhos to supplemental O2 on 
CrCEST 
We will assess the correlation between 
the change in SkM OxPhos to 
supplemental oxygen and the contrast 
in submaximal exercise endurance 
between the KNO3 and the 
KNO3+PLC+NR arms. 
While KNO3 is 
hypothesized to improve 
muscle blood flow, NR 
and PLC are 
hypothesized to 
additionally improve 
mitochondrial reserve. In 
patients with intact 
mitochondrial reserve at 
baseline, the majority of 
the benefit to our 
supplementation strategy 
will be by increasing 
muscle blood flow, thus 
there will be little 
additional benefit to the 
NR+PLC. On the other 
hand, in patients with 
impaired mitochondrial 
reserve, the addition of 
NR+PLC to KNO3 will 
lead to more substantial 
improvements in 
exercise endurance. We 
will use the CrCEST 
response to 100% 
oxygen as a marker of 
mitochondrial reserve, 
which should track with 
the increased exercise 
response to combination 
therapy over KNO3 
alone.  
 

 
 
 
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5 
STUDY PLAN 
5.1 
Study Design 
Overview: 
This will be a single-center randomized, placebo controlled, double-blind, 3-treatment, 3-period cross-over trial 
in 48 HFpEF participants: (A) KCl (active control); (B) KNO3; (C) KNO3+NR+PLC. This design was chosen so 
that each participant receives all interventions in a pre-specified order across 3 periods; thus, within-subject 
comparisons of each pair of interventions are possible. Each interventional period will be 6 weeks in duration, 
separated by a 2-week wash-out period. The Investigational Drug Services (IDS) at UPenn will formulate the 
KCl and KNO3 capsules; the commercially obtained NR and PLC capsules will be over-encapsulated to 
maintain blinding.  
While changes to the mitochondrial proteome occur early after the initiation of an exercise program (1-2 
weeks),162, 163 the timing of mitochondrial biogenesis in humans is less well known, suggesting that the longer 
duration of therapy chosen in this study is warranted.164 Similarly, data are unavailable to specify the washout 
period for a mitochondrial intervention. In a forced bedrest study, mitochondrial content and OxPhos proteins 
all decreased after 7 days, demonstrating rapid mitochondrial plasticity.165, 166 This suggests that our 2-week 
washout period is reasonable, particularly since this washout ensures an 8-week interval for measuring the 
outcomes after completion of one intervention.  
Randomization schemes will be determined by the statistician and transmitted to the pharmacy. We will split 
the set of 6 sequences into two sub-sequences (ABC: BCA: CAB) or its dual (ACB: BAC: CBA) and use the full 
sequence and the sub-sequence to create random blocks of 3 (corresponding to one of the two subsequences) 
or 6 (the complete sequence). To achieve a balanced design, once one sub-sequence is used, it will not be 
used again until its dual is selected. We will enroll 53 subjects to account for drop out. In the case of medical 
necessity, the Principal Investigator can request the randomization codes from IDS. 
 
Following completion of the interventional study enumerated above, subjects will return for a one-day 
MRI visit during which the response of SkM OxPhos to supplemental inspired oxygen (100% O2) will be 
assessed. The results from this MRI visit will be compared to the results obtained during the exercise tests.  
5.2 
Scientific Rationale for Study Design 
HFpEF subjects suffer from multiple comorbidities that may impact exercise capacity.167-169 As such, a cross-
over design, in which each participant receives all 3 interventions in randomized order, can better account for 
these phenotypic differences and more efficiently identify any changes in exercise endurance with our 
interventions, as compared to a parallel design.  
The limitations of a cross over study include the need for an adequate washout period, as discussed above, 
and the more marked impact of dropout on data acquisition.  
5.3 
Justification for Dose 
A discussion of the doses to be used in this protocol are enumerated in Section 2.2.1. The 3 arms to be tested 
during the supplementation portion of the study are: 
(1) Potassium Nitrate alone 

 
 
 
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a. KNO3: 6 mmol three times daily 
b. Placebo A: Two times daily (Placebo for Propionyl-L-Carnitine) 
c. Placebo B: Three times daily (Placebo for Nicotinamide Riboside) 
(2) Potassium Nitrate + Nicotinamide Riboside + Propionyl-L-Carnitine  
a. KNO3: 6 mmol three times daily 
b. Propionyl-L-Carnitine: 1000 mg po two times daily 
c. Nicotinamide Riboside: 300 mg po three times daily 
(3) Potassium Chloride 
a. KCl: 6 mmol three times daily 
b. Placebo A: Two times daily (Placebo for Propionyl-L-Carnitine) 
c. Placebo B: Three times daily (Placebo for Nicotinamide Riboside) 
Because the combination phase includes 3 different compounds, each phase will need to include 3 sets of 
investigational bottles to maintain blinding. Both the KNO3 and the KCl will be made by the UPenn IDS, as we 
have done previously,82 with pharmaceutical grade product obtained from a commercial supplier (e.g. 
Spectrum Laboratory Products (Gardenia, CA)). Both Nicotinamide Riboside and Propionyl-L-Carnitine will be 
obtained commercially and delivered to UPenn IDS. UPenn IDS will over encapsulate the pills in order to 
maintain blinding.  
 

 
 
 
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6 
STUDY POPULATION 
6.1 
Inclusion criteria for patients with HFpEF are:  
1. NYHA Class II-III symptoms 
2. Left ventricular ejection fraction >= 50% 
3. Stable medical therapy for at least 1 month 
4. Prior or current evidence for elevated filling pressures as follows:  
a. Mitral early (E)/septal tissue annular (e’) velocity ratio > 8, in the context of a septal e’ velocity 
<=7 cm/s or a lateral e’ <= 10 cm/s, in addition to one of the following: 
i. Large left atrium (LA volume index > 34 mL/m2)170, 171 
ii. Chronic loop diuretic use for control of symptoms 
iii. Elevated natriuretic peptides within the past year (e.g. NTproBNP > 125 pg/mL in sinus 
rhythm or > 375 pg/mL if in atrial fibrillation)172 
b. Mitral E/e’ ratio > 14171 at rest or during exercise172 
c. Elevated invasively-determined filling pressures previously (resting left ventricular end-
diastolic pressure >= 16 mm Hg or pulmonary capillary wedge pressure >= 15 mmHg; or 
PCWP/LVEDP >= 25 mmHg with exercise)172, 173 
d. Prior episode of acute heart failure requiring IV diuretics 
 
We note that our inclusion criteria are broadly consistent with the current ESC HFpEF diagnosis algorithm.172  
 
6.2 
Exclusion Criteria 
1. Age <18 years old 
2. Pregnancy: Women of childbearing potential will undergo a urine pregnancy test during the screening 
visit. We note that the advanced age of HFpEF subjects (median age of 78 in the Get With the 
Guidelines-HF program2) will make it unlikely that pre-menopausal females will be enrolled. 
3. Treatment with organic nitrates or phosphodiesterase inhibitors that cannot be interrupted 
4. Uncontrolled atrial fibrillation, as defined by a resting heart rate > 100 beats per minute at the time of 
the baseline assessment 
5. Hemoglobin < 10 g/dL 
6. Subject inability/unwillingness to exercise 
7. Moderate or greater left sided valvular disease (mitral regurgitation, aortic stenosis, aortic 
regurgitation), mild or greater mitral stenosis, severe right-sided valvular disease 
8. Known hypertrophic, infiltrative, or inflammatory cardiomyopathy 
9. Clinically significant pericardial disease, as per investigator judgment 
10. Current angina due to clinically significant epicardial coronary disease, as per investigator judgment 
11. Acute coronary syndrome or coronary intervention within the past 2 months 
12. Primary pulmonary artery hypertension (WHO Group 1 Pulmonary Arterial Hypertension) 
13. Clinically significant lung disease as defined by: Chronic Obstructive Pulmonary Disease Stage III or 
greater GOLD criteria (FEV1<50%), treatment with oral steroids within the past 6 months for an 
exacerbation of obstructive lung disease, current use of supplemental oxygen aside from nocturnal 
oxygen for the treatment of obstructive sleep apnea.  
- 
Desaturation to <90% on the baseline maximal effort cardiopulmonary exercise test will 
also be grounds for exclusion 
14. Clinically-significant ischemia, as per investigator’s judgement, on stress testing without either (1) 
subsequent revascularization, (2) an angiogram demonstrating the absence of clinically significant 
epicardial coronary artery disease, as per investigator judgment; (3) a follow-up ‘negative’ stress test, 
particularly when using a more specific technique (i.e., a negative perfusion imaging test following a 
‘positive’ ECG stress test) 

 
 
 
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- 
Exercise-induced regional wall motion abnormalities on the echocardiographic 
assessment during the baseline maximal effort cardiopulmonary exercise test will also 
exclusionary  
15. Left ventricular ejection fraction < 45% on a prior echocardiogram or cardiac MRI 
16. Significant liver disease impacting synthetic function or volume control (ALT/AST > 3x ULN, Albumin 
< 3.0 g/dL) 
17. eGFR < 30 mL/min/1.73m^2 
18. Methemoglobin > 5% 
19. Serum potassium > 5.0 mEq/L on baseline testing 
20. Severe right ventricular dysfunction 
21. Baseline resting seated systolic blood pressure > 180 mmHg or < 100 mmHg 
22. Orthostatic blood pressure response to the transition from supine to standing (>20 mmHg reduction 
in systolic blood pressure 2-3 minutes after standing) 
23. Active participation in another study that utilizes an investigational agent (observational 
studies/registries allowed) 
24. Any condition that, in the opinion of the investigator, will interfere with the completion of the study. 
This may include comorbid or psychiatric conditions that may impede successful completion of the 
protocol, or logistical concerns (e.g. inability to travel to the exercise unit).  
Additionally, specific exclusion criteria exist for the performance of the MRI studies**: 
– ANY intra-luminal implant, filter, stent, or valve replacement 
– ANY type of life assist device, pump, or prosthetic 
– ANY vascular clip or clamp 
– 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 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. 
 
**We note that many contemporary ICD/pacemakers are MRI-compatible. In participants with an 
implantable device (defibrillator or pacemaker), the specific details of the device will be reviewed and 
discussed with our radiologists in order to ensure the safety of the participant with that device during 
scanning at 3T. The participant will only undergo the MRI scan if it is deemed to be safe. 
 
If a subject is interested in participation in the study but has contraindications to the MRI 
portion, the subject can still be enrolled, and the MRI assessments will be omitted. 
6.3 
Lifestyle Considerations 
The following lifestyle changes will be requested during the study: 

 
 
 
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• 
Subjects will be asked to follow a low-nitrate diet, avoiding the excessive intake of certain food items 
such as beets and arugula. 
• 
Subjects will be asked to avoid strenuous exercise for 48 hours prior to each study visit 
• 
A standardized low-nitrate dinner will be provided to each subject prior to each visit 
• 
Subjects will be asked to come to each study visit following an overnight fast 
• 
Subjects will be asked to avoid caffeine for 24 hours prior to each visit due to caffeine’s impact on SkM 
OxPhos and substrate utilization.174, 175  
• 
Subjects will be asked to avoid antibacterial mouthwash during the study. Antibacterial mouthwash 
alters the mouth flora which is a key component in the activation of inorganic nitrate.176-179 
6.4 
Screen Failures 
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently randomly assigned to the study intervention or entered in the study. A minimal set of screen 
failure information is required to ensure transparent reporting of screen failure participants, to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to respond to queries 
from regulatory authorities. Minimal information includes demography, screen failure details, eligibility criteria, 
and any serious adverse event (SAE). 
Individuals who do not meet the criteria for participation in this trial (screen failure) because of a specific 
modifiable or transient factor, such as an acute illness which is expected to improve or a recent change in 
medications, may be rescreened in the future when medically stable. Additionally, if a previously screen-failed 
subject’s clinical status changes or more information becomes subsequently available, a subject can be 
rescreened for inclusion. 
6.5 
Strategies for Recruitment and Retention 
6.5.1 
Recruitment Plans 
1. Participants who have participated in prior research studies with our group: Over the past few years, we have 
successfully completed several studies in HFpEF subjects.28, 64, 82, 180 As part of our consent process, we ask 
for permission to contact participants in the future regarding new studies as they become available. As such, 
we have a list of HFpEF patients who are interested in participating in research.  
2. Active recruitment of new subjects: We will leverage an existing recruitment pipeline for heart failure currently 
used by the Cardiovascular Clinical Research Unit (CCRU) at the University of Pennsylvania. The CCRU 
team includes clinical research coordinators with extensive experience in the screening of patients with heart 
failure with preserved ejection fraction for participation in clinical trials (including REDUCE-LAP and various 
NIH funded Heart Failure trials, such as NEAT-HFpEF, KNO3CKOUT-HFpEF, and INDIE-HFpEF). 
 
Strategies and Tools to Support Study Recruitment. Numerous recruitment avenues are in place and may 
be used in parallel to maximize enrollment. 
 
1. CCRU recruitment databases: The CCRU has extensive experience recruiting interested patients for HFpEF 
trials, including many who have expressed interest in continued engagement with new protocols after having 
positive experiences with our group and/or participation in Heart Failure Clinical Research Network trials and 

 
 
 
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others. 
 
2. Directed advertising: This may include postings on the Cardiovascular Medicine divisional website, email 
blasts, clinics, and cardiovascular clinical laboratories. 
 
3. Focused efforts in high yield environments: We will conduct directed recruitment in settings where HFpEF 
patients are likely to present in the course of routine clinical care, including outpatient cardiology practices, 
the clinical echocardiography laboratory, the clinical stress testing laboratory, and inpatient cardiovascular 
services. 
 
4. PennSeek and other Electronic Medical Record query tools: PennSeek is a tool to search unstructured or 
semi-structured medical documents currently residing in Penn Medicine’s EPIC electronic medical record 
and diagnostic applications (Radiology, Pathology, Cardiology, Ambulatory, etc.) to analyze and mine this 
data for patient care and research. PennSeek is designed to allow both refinement of search criteria and 
immediate review of identified cohort data to achieve the desired degree of specificity. PennSeek is available 
at CCRU workstations and will be used to identify lists of patients that meet eligibility for the proposed trials. 
Other similar search tools are available at UPenn, and new search tools are likely to be developed; these too 
may also be employed to identify potential participants. 
 
5. Real-time subject identification through EPIC: The CCRU has worked with our health system electronic 
health records in the past to identify eligible patients and to enable ‘pop-ups’ when a patient who meets 
selection criteria is in clinic or is admitted to hospital. These efforts may again be employed. In addition, these 
events can generate notifications to the study team, providing real-time identification of potential candidates. 
Once identified, research coordinators may approach potential candidates and/or their physicians. 
 
6.5.2 
Retention Plans 
Our participant retention strategies begin with the introduction to the study and relies on frequent communication 
with our participants about the study protocol and the value they bring by participating in research. We include 
frequent phone calls to the prescreened and enrolled subjects at specified time points, including phone calls 
regarding the status of visits, check-ins for enrolled participants, and reminders for upcoming visits. Moreover, 
the frequent study visits in the describe protocol (8 visits within ~6 months) will allow many contact points 
between the study team and the participant, improving communication and the relationship between the 
participant and the study team. We will provide adequate participant compensation and will use the existing 
facilities in the CHPS unit, which allow flexibility of scheduling according to participants’ needs, greatly facilitating 
study visits, retention, and satisfaction. 
 
Based on recent experience, our retention rate is high (91% to date in the KNO3CK OUT-HFpEF trial). We plan 
to exclude participants who are unlikely to complete study visits due to non-compliance, psychiatric illness or 
major comorbidities that would impede successful completion of the protocol, substance abuse, or other social 
or logistic circumstances that will be judged on a case-by-case basis. 
 
In the event that recruitment is slow and jeopardizing the goals laid out in the milestones, we will consider 
several strategies to boost enrollment. If enrollment falls significantly below accrual benchmarks, we will 
expand recruitment efforts to include Penn Medicine hospitals that are farther from downtown Philadelphia, 
such as Chester County Hospital and Lancaster General Hospital. If enrollment is still below the benchmark, 
we will seek partnership with other local institutions such as Temple University and Lankenau Medical Center.  

 
 
 
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If the muscle biopsy and/or the MRI studies represent barriers to enrollment and subject participation, these 
too may be made optional. 
We also provide subject compensation for patient time and transportation to mitigate the burden of participation 
according to the following schedule: 
(1) Baseline Visit: Cycle exercise: $50 
(2) Visit 2: Muscle Biopsy and cycle exercise: $100 
(3) Visit 3: MRI study: $50 
(4) Visit 4: Muscle Biopsy and cycle exercise: $100 
(5) Visit 5: MRI study: $50 
(6) Visit 6: Muscle Biopsy and cycle exercise: $100 
(7) Visit 7: MRI study: $50 
(8) Visit 8: 2 MRI studies (room air and 100% oxygen): $100 
Total: $600 
 
Participants will be paid via by Greenphire clincard after completing each visit. The study coordinator will keep 
track of patients using an electronic database to build-in schedule reminders. We also maintain close 
communication via phone/email with previous and active research subjects to mitigate patients becoming lost 
to follow-up.  
6.6 
End of Study Definition 
A participant is considered to have completed the study if he or she has completed all phases of the study 
including the last visit or the last scheduled procedure shown in the Schedule of Activities (SoA), Appendix 
Section 12.1. This will include the baseline study visit, the 3 endpoint assessments (submaximal exercise and 
CrCEST MRI studies) following each interventional period, as well as the final MRI visit (CrCEST with and 
without supplemental oxygen), unless the subject chooses to curtail participation earlier for any reason. 
Subjects may also be removed from the study by the PI due to non-compliance, inability/unwillingness to 
comply with study instructions, or other unforeseen circumstances. 
 

 
 
 
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STUDY INTERVENTION 
6.7 
Study Intervention(s) Administration 
6.7.1 
Study Intervention Description 
The 3 arms to be tested during the supplementation portion of the study, and the target doses, are: 
(1) Potassium Nitrate 
a. KNO3: 6 mmol three times daily 
b. Placebo A: Two times daily (Placebo for Propionyl-L-Carnitine) 
c. Placebo B: Three times daily (Placebo for Nicotinamide Riboside) 
(2) Potassium Nitrate + Nicotinamide Riboside + Propionyl-L-Carnitine  
a. KNO3: 6 mmol three times daily 
b. Propionyl-L-Carnitine: 1000 mg po two times daily 
c. Nicotinamide Riboside: 300 mg po three times daily 
(3) Potassium Chloride 
a. KCl: 6 mmol three times daily 
b. Placebo A: Two times daily (Placebo for Propionyl-L-Carnitine) 
c. Placebo B: Three times daily (Placebo for Nicotinamide Riboside) 
 
6.7.2 
Dosing and Administration 
Study drugs will be escalated to their targets after approximately one week, barring limiting side-effects. An 
initial two-week supply of the following regimens will be sent to subjects: 
Initial starting regimens: 
(1) Potassium Nitrate 
a. KNO3: 6 mmol two times daily 
d. Placebo A: Once daily (Placebo for Propionyl-L-Carnitine) 
b. Placebo B: Two times daily (Placebo for Nicotinamide Riboside) 
(2) Potassium Nitrate + Nicotinamide Riboside + Propionyl-L-Carnitine  

 
 
 
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a. KNO3: 6 mmol two times daily 
b. Propionyl-L-Carnitine: 1000 mg po once daily 
c. Nicotinamide Riboside: 300 mg po twice daily 
(3) Potassium Chloride 
a. KCl: 6 mmol two times daily 
e. Placebo A: Once daily (Placebo for Propionyl-L-Carnitine) 
b. Placebo B: Two times daily (Placebo for Nicotinamide Riboside) 
A two-week supply will be given to account for potential delays in the shipment of the 2nd batch of study 
medications. Study medications are to be taken with meals to minimize the risk of GI upset. 
One Week Tolerability Assessment: 
After approximately one week (range 5-9 days to account for weekends/holidays), subjects will be contacted 
using his/her preferred method (for example, telephone, televideo, phone, text or email) to assess tolerability. 
Dietary restrictions will be reinforced during this interaction.  
Subjects will be asked about side effects, including headache, dizziness, GI upset, nausea and 
lightheadedness. The presence of symptoms suggestive of orthostatic hypotension (i.e. sustained 
lightheadedness upon standing that does not dissipate quickly) will prompt an assessment for orthostatic vital 
signs (a drop in the systolic blood pressure of >20 mmHg 2-3 minutes after moving from the supine to standing 
position), and/or hypotension (systolic Bp<90 mmHg). If difficulties/barriers/subject preference preclude coming 
in for an in-person assessment (such as concerns regarding COVID-19), a subject may use a home Bp cuff to 
perform the orthostatic assessment, with guidance from the study staff: 
1. Lie supine for approximately 5 minutes and then check the blood pressure. 
2. Gradually move to a sitting position, rest there for a moment (~10-30s), and then stand upright. 
Subjects may hold on to something for balance.  
3. Check blood pressure 2-3 minutes after standing. 
4. Note the presence of any symptoms at the 2-3-minute mark after standing 
Note – transient lightheadedness that occurs soon after standing, but that resolves soon thereafter does not 
constitute a positive response. 
The presence of an orthostatic response (drop in systolic blood pressure of > 20 mmHg approximately 
2-3 minutes after the transition from supine to standing) or hypotension (systolic blood pressure < 90 mmHg) 
will lead to exclusion from the study if confirmed on a repeat assessment. In cases in which home monitoring 
suggests resting or orthostatic hypotension, the study subject will be encouraged to come to UPenn for a 
formal assessment.  

 
 
 
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On the other hand, if study medications are well-tolerated, or tolerated with mild/minimal side effects, the doses 
will be increased to target: 
Weeks 2-6: 
(1) Potassium Nitrate 
a. KNO3: 6 mmol three times daily 
f. Placebo A: Two times daily (Placebo for Propionyl-L-Carnitine) 
b. Placebo B: Three times daily (Placebo for Nicotinamide Riboside) 
(2) Potassium Nitrate + Nicotinamide Riboside + Propionyl-L-Carnitine  
a. KNO3: 6 mmol three times daily 
b. Propionyl-L-Carnitine: 1000 mg po two times daily 
c. Nicotinamide Riboside: 300 mg po three times daily 
(3) Potassium Chloride 
a. KCl: 6 mmol three times daily 
g. Placebo A: Two times daily (Placebo for Propionyl-L-Carnitine) 
b. Placebo B: Three times daily (Placebo for Nicotinamide Riboside) 
If subjects experience symptoms that do not prompt exclusion from the study (such as mild dizziness or GI 
upset), the dose may be either kept at the Week 1 dose, or down-titrated to the Week 1 dose from the 
target dose if up titration was attempted. The default approach will be to up-titrate all participants to 
target doses unless a compelling reason, which may include the subject’s strong preference, exists. 
 
 
Initial Dose 
Target Dose 
KNO3 
6 mmol BID 
6 mmol TID 
NR 
300 mg BID 
300 mg TID 
PLC 
1000 mg daily 
1000 mg BID 
KCl 
6 mmol BID 
6 mmol TID 
Serum Potassium Monitoring: 
 
Our study interventions all include potassium (KNO3 or KCl). The amount of potassium provided in our 
study combinations is relatively small (<20 mEq/day). Moreover, subjects with a blood potassium greater than 

 
 
 
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5.0 mEq/L, or an eGFR < 30 mL/min/1.73 m2, at baseline will be excluded from the study, minimizing the risk 
that clinically significant hyperkalemia (K > 5.5 mEq/L) will develop as a result of our interventions. As an extra 
safety precaution, we will perform an additional assessment of the serum potassium following drug uptitration 
during the first phase of the study in participants who may be at higher risk for hyperkalemia, defined as: 
 
1. Subjects who have a baseline serum K between 4.8 and 5.0 mEq/L, AND 
2. Either:  
(a) Potassium-sparing diuretic use (e.g. amiloride, spironolactone, triameterene), OR 
(b) Estimated glomerular filtration rate of 30-39 mL/min/1.73m2 
 
In these individuals, we will check a serum potassium ~1 week after the implementation of the 18 mmol/d 
dose. A serum potassium >5.5 mEq/L will prompt exclusion from the study. Noting that the potassium content 
of all 3 interventional regimens is the same (18 mEq/day), this additional safety laboratory assessment will only 
be done following the first study drug intervention. This laboratory assessment may be performed locally if 
more convenient for the participant and does not need to be fasting. 
 
Given the small amount of potassium in our supplementation strategy, we will not plan to routinely alter 
a participant’s pre-existing potassium supplementation, unless a high potassium (>5.0 mEq/L) is identified on 
baseline laboratories, prior to receiving any study medications. Changes to a participant’s background 
medications will be done in conjunction with the participant’s provider, and the potassium level will be 
rechecked following the change but prior to study drug administration. Participants will only be given study 
medications if the potassium upon recheck following withdrawal/reduction of potassium supplementation is < 
5.0 mEq/L. 
6.7.3 
Preparation/Handling/Storage/Accountability 
All study medications will be dispensed by the UPenn IDS in a randomized double-blind manner.  
 
(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. 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-/day, given as one capsule (6 
mmoles) three times a day. A certificate of analysis will be obtained by the manufacturer. 
(B) Potassium Chloride: Potassium chloride, granular, USP (450mg) plus lactose 
monohydrate, spray dried, NF (300mg) will be combined and packed into an identical capsule 
shell. The dose for this trial will be 18 mmoles of KCl per day, given as one capsule (6 mmoles) 
three times a day. A certificate of analysis will be obtained by the manufacturer. 
(C) Nicotinamide Riboside Chloride: NR will be purchased from Chromadex as 300 mg 
capsules. The dose in this trial will be 300 mg three times a day. Capsules will be over-
encapsulated to maintain blinding. A placebo pill of similar weight and color will be constructed 
by UPenn IDS. 
(D) Propionyl-L-Carnitine: PLC will be purchased from Biovy as 1000 mg capsules and will 
be over-encapsulated by UPenn IDS to maintain blinding. A placebo pill of similar weight/color 
will be constructed by UPenn IDS.  

 
 
 
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6.7.4 
Acquisition and accountability 
Both KNO3 and KCl will be prepared by UPenn IDS, as we have done previously.82 The KNO3 and KCl 
powders will be obtained from a commercial supplier via purchase through IDS. The commercial supplier will 
provide a certificate of analysis. IDS will also furnish the placebo capsules for PLC and NR that are of similar 
color and weight to the study medications. Nicotinamide riboside (NR) will be obtained commercially 
(TruNiagen). Propionyl-L-Carnitine will be obtained commercially (Biovy).  
The study team will randomly test and verify the composition of a random set of NR and PLC pills using 
mass-spectrometry and quantitative methods. Sealed bottles will be delivered to UPenn IDS. One NR/PLC pill 
from each lot will be given to the study team by IDS prior to dispensation of study medication from that lot to 
verify the authenticity of the compound using mass spectrometry/quantitative methods. The purity of the 
PLC/NR pills will be compared against PLC and NR obtained from a commercial supplier (e.g. SigmaAldrich) 
who provides a certificate of analysis. These results will be furnished to IDS and the PI prior to dispensation of 
study medications from that lot. We note that Niagen has been given “Generally Recognized As Safe” status. 
During this determination, data on the chemical purity of the compound (>99% purity) was presented 
(https://www.fda.gov/files/food/published/GRAS-Notice-000635--Nicotinamide-riboside-chloride.pdf; accessed 
1/13/2020). 
6.7.5 
Formulation, Appearance, Packaging, and Labeling 
Potassium nitrate and potassium chloride will be obtained commercially (e.g. from Spectrum Pharmaceuticals) 
and encapsulated by the UPenn IDS. Nicotinamide riboside chloride will be obtained commercially from 
Chromadex. Propionyl-L-Carnitine will be obtained commercially from Biovy. The NR and PLC pills will be 
over-encapsulated to maintain blinding. UPenn IDS will construct a placebo pill for NR and PLC that will be 
similar in weight and appearance to the study medications.  
 
6.8 
Measures to Minimize Bias: Randomization and Blinding 
Subjects will be randomized to one of six pre-specified sequences of interventions as shown in the Table 
below.  
Sequences use in the balanced 
3-period 3-treatment crossover 
design 
Sequence 
Order of 
Intervention1 
1 
ABC 
2 
ACB 
3 
BAC 
4 
BCA 
5 
CAB 

 
 
 
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6 
CBA 
1Each letter corresponds to one 
of the three interventions 
The 3x3 crossover design involves 2 interventions plus an active control and 3 periods, leading to a total of 6 
unique sequences (e.g. KCl in period [P] 1; KNO3 in P2; KNO3+NR+PLC in P3). Each sequence thus includes 
all three possible interventions e.g. in Sequence 1, coded ABC, one possible coding is A=KCl alone, followed 
by B=KNO3 alone, followed by C= KNO3+ PLC + NR. UPenn IDS will assign the codes linked to the 
intervention and will provide study medication based on the sequence code to which the subject is randomized. 
For purposes of blinding, none of the study team will have access to the IDS-assigned codes except in an 
emergency or at unblinding. If emergency unblinding is necessary, the investigators will access the treatment 
code by contacting UPenn IDS. This is unlikely given the safety profile of the study medications, and if time 
allows will be done in consultation with the medical monitor. Regardless, in the event of emergency unblinding, 
both the medical monitor and the Data Safety Monitoring Board overseeing this study will be informed.  
RANDOMIZATION PROCEDURE: 
 
The study statistician, Dr. Putt, will prepare a randomization table for inclusion in Redcap. Each subject 
who enrolls and consents will be randomized to the next available sequence based on consecutive rows of the 
table. In the event that a subject consents and enrolls but does not obtain medication, their sequence will be 
assigned to the next available subject. To ensure balance over the study duration, the randomization table will 
be prepared in random blocks of 6 or 12.  
6.9 
Study Intervention Adherence 
Subjects will bring all study medications to their final endpoint assessment for each phase (i.e. Visit 3 for 
Period 1, Visit 5 for Period 2). Study coordinators will manually count the remaining pills in each bottle and 
compare against the number of pills expected to be taken to determine compliance. Non-compliance will be 
defined as taking < 80% of study medications. Remaining study medications will be returned to UPenn IDS for 
disposal. 
6.10 
Concomitant Therapy 
For this protocol, a prescription medication is defined as a medication that can be prescribed only by a properly 
authorized/licensed clinician. Medications to be reported in the Case Report Form (CRF) are concomitant 
prescription medications, over-the-counter medications, and supplements. 
Subjects will continue on all of their previously prescribed medications during this trial. Except in cases of 
clinical necessity, alterations to background medications will be discouraged. Due to concerns regarding 
excessive nitric oxide bioavailability and over-vasodilation, the following medication classes will not be 
permitted during this study: 
• 
Phosphodiesterase-5 inhibitors (e.g. sildenafil or tadalafil) 
• 
Organic nitrates (e.g. isosorbide mononitrate, sublingual nitroglycerin) 
Changes to the medication regimen will be assessed during subject encounters. 

 
 
 
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7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT DISCONTINUATION/WITHDRAWAL 
7.1 
Discontinuation of Study Intervention 
 
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 
• 
Significant non-compliance with study medications (<80%) 
• 
Incorrect enrollment or randomization 
• 
Any other reasons related to participant safety 
• 
Termination of the study by the DSMB or regulatory authorities 
 
The reason for study discontinuation will be recorded on the source documents. All such subjects 
will be asked to complete an early termination visit if possible. During this visit, we will document:  
(1) vital signs 
(2) compliance with the medications, including pill counts  
(3) adverse effects  
(4) specific reason for withdrawal 
 
We note circumstances may make the performance of an in-person early termination visit 
challenging; therefore, this visit may also be performed virtually (e.g. over video conferencing). 
7.2 
Participant Discontinuation/Withdrawal from the Study 
Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may discontinue or withdraw a participant from the study for the following reasons: 
• 
Pregnancy 
• 
Significant non-compliance with study medications (<80%) 
• 
If any clinical adverse event (AE), laboratory abnormality, or other medical condition or situation occurs 
such that continued participation in the study would not be in the best interest of the participant 
• 
If the participant meets an exclusion criterion (either newly developed or not previously recognized) that 
precludes further study participation 
The reason for participant discontinuation or withdrawal from the study will be recorded on a specific Case 
Report Form (CRF). Subjects who sign the informed consent form and who are randomized, but receive no 
study intervention, will be replaced.  
7.3 
Loss To Follow-Up 
A participant will be considered lost to follow-up if he or she fails to return for an endpoint assessment and is 
unable to be contacted by the study site staff. Given the cross-over nature of this study design, the study-team 
will make significant efforts to retain all randomized subjects. 
The following actions will be taken if a participant fails to return for a required study visit: 
• 
The study team will attempt to contact the participant and reschedule the missed visit as soon as 
possible. Study medications may need to be extended, provided no limiting side-effects are present. 

 
 
 
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• 
The study team will counsel the participant on the value and importance of maintaining the assigned 
visit schedule and ascertain if the participant wishes to and/or should continue in the study. 
• 
Participants who are unable to complete a particular study visit will be encouraged to complete 
subsequent visits, and will not be considered lost to follow-up  
• 
Before a participant is deemed lost to follow-up, the study team will make every effort to regain contact 
with the participant (where possible, 3 telephone calls and, if necessary, a certified letter to the 
participant’s last known mailing address or local equivalent methods). These contact attempts should 
be documented in the participant’s study file.  
• 
Should the participant continue to be unreachable, he or she will be considered to have withdrawn from 
the study with a primary reason of lost to follow-up. 

 
 
 
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8 
STUDY ASSESSMENT AND PROCEDURES 
8.1 
Efficacy Assessments 
Pre-Visit Assessment: 
 
Prior to the initial study visit, subjects may be contacted by the study team to discuss the study. 
Subjects may be sent a copy of the informed consent for review. Data may be abstracted from the electronic 
medical record, and/or in conversation with the subject, to determine potential eligibility. Specific data to be 
reviewed and collected may include: 
• 
Name, MRN, date of birth, gender 
• 
Medical history and review of prior office visits, admissions/discharges, procedures, and surgeries 
• 
Results of prior testing (e.g. echocardiograms, heart catheterizations, stress tests) 
• 
Current medications and allergies 
• 
Tobacco, alcohol, and drug use 
Subjects will be asked to come to the Baseline visit in the fasted state, after avoiding caffeine for 24hrs, and 
strenuous activity for 48hrs prior.  
Baseline Visit: 
 
The main objectives of the baseline visit are to obtain written informed consent from the subject, ensure 
subject eligibility, and determine the submaximal workload to be used in future assessments (primary 
endpoint). Study procedures will not commence until after written informed consent has been obtained. The 
following will be performed during the baseline visit: 
• 
Anthropomorphic measurements, including height and weight 
• 
Physical examination, including seated blood pressure measurement and orthostatic blood pressure 
assessment 
• 
Urinary pregnancy test for women of childbearing potential 
• 
Blood analysis and storage: 
o Comprehensive metabolic profile 
o NT-proBNP 
o Complete Blood Count (hemoglobin, white blood cell count, and platelet) 
o Coagulation profile (PT/PTT/INR) 
o Methemoglobin 

 
 
 
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o Additional blood will be obtained for storage (plasma, serum, and whole blood)  
• 
Urine and saliva collection for storage 
• 
Kansas City Cardiomyopathy Questionnaire: Quality of life is significantly impaired in HFpEF patients 
and correlates with functional capacity.12, 19 The Kansas City Cardiomyopathy Questionnaire (KCCQ) will 
be administered, and the KCCQ Overall Summary Score will be calculated and compared.12, 19, 181, 182 
• 
Low-nitrate diet: Educational information and hand-out regarding foods that are high in inorganic 
nitrate, which are to be avoided during this study. 
• 
Comprehensive echocardiography: Echocardiography will 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. 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, approximately 1-cm apical to the mitral valve plane. 
Additional images may 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. 
Additional echocardiographic images may also be taken, as new echocardiographic techniques 
become available, noting that there are no known risks associated with obtaining echocardiographic 
images.  
• 
Arterial tonometry: Arterial tonometry will be performed using a high-fidelity applanation tonometer. 
Assessments may be obtained at the radial, carotid, brachial, and femoral arteries. Waveforms will be 
stored for offline analysis. Waveforms will be calibrated to the brachial blood pressure using a validated 
blood pressure device. Body surface measurements may be made to determine distances between 
anatomic landmarks and measurement sites, such as the suprasternal notch to the carotid, radial, 
femoral, and brachial arteries. 
• 
Dual-Energy X-ray Absorptiometry: A DEXA scan will be performed. This data will be used to normalize 
exercise parameters to lean muscle mass and to assess body composition. 
• 
Maximal effort cardiopulmonary exercise test: Participants will perform a maximal effort supine bicycle 
exercise test with expired gas analysis using a Parvo Medics TrueOne 2400 device. The device will be 
calibrated prior to each study. We will use a supine cycle ergometer designed for stress echocardiography 
(e.g. Stress Echo Ergometer 1505, Medical Positioning, Inc, Kansas City, MO) and a graded-exercise 
protocol, with resistance beginning at 15 W for 3 minutes, increasing to 25 W for 3 minutes, and then 
increasing by 25 W every 3 minutes thereafter until exhaustion.64, 82 Electrocardiographic and vital sign 
monitoring will be performed during this test. Verbal encouragement will be given to encourage maximal 
effort.  
o Echocardiographic images will be obtained during the exercise tests. Lung ultrasound may be 
performed at rest and during exercise to identify B-lines, a marker of congestion.183   
o Peak workload (PW) will be defined as the maximal workload that could be sustained for at least 
30 seconds.  
o Peak oxygen consumption will be defined as the average rate of oxygen consumption over the 
last 30 seconds of exercise. 

 
 
 
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o Note: Subjects will be excluded if the arterial saturation falls to < 90% during this maximal effort 
study or if significant exercise-induced segmental wall motion abnormalities are noted on exercise 
echocardiography that are consistent with inducible ischemia. Given the high prevalence of ‘false-
positive’ ECG stress tests in HFpEF,184 isolated EKG abnormalities, in the presence of preserved 
echocardiographic wall motion, will not be exclusionary.  
• 
Submaximal workload verification study: After a rest interval of at least 1 hour and a standardized 
low-nitrate lunch, subjects will undergo a submaximal exercise test at 75% of the peak workload 
(75%PW) achieved during the preceding maximal effort test. Subjects will begin with 1 minute of 
unloaded (0W) exercise, followed by a step increase to 75%PW, performed until exhaustion.  
o A pedal cadence of 60 RPMs will be maintained, and exhaustion will be determined as the time 
at which the subject indicates that he or she cannot continue, or as an inability to maintain the 
pedal cadence >50 RPM for >10s, despite verbal encouragement.185  
o The cycle ergometer allows for increments of 5W. If the 75%PW does not fall exactly on a possible 
value that can be input into the cycle ergometer (e.g. a number that is not divisible by 5), the 
nearest integer divisible by 5 will be selected (e.g. 92.5 Wà 95W; and 91W à 90W.) 
• 
Workload titration for subsequent submaximal studies: The primary exercise endpoint of this study 
is the time to exhaustion during submaximal exercise. Following the submaximal verification study during 
the baseline visit, we will titrate the workload to be used in subsequent assessments in order to target a 
time to exhaustion of 3-6 minutes, as longer tests may be limited by boredom or discomfort as opposed 
to a physiologic limitation.185 If the subject exercises for >6 minutes during the submaximal workload 
verification study, the workload to be used in subsequent assessments will be increased by 10W or 15%, 
whichever is a greater change (but not greater than the peak workload during the maximal effort test); if 
the exercise time is <3 minutes, the workload will be reduced by 10W or 15%, whichever is a lesser 
change. A similar schema previously used in HFpEF patients led to exercise times within the desired 
range.74 
        Submaximal Workload Titration Table Based on Results of Verification Test: 
Peak Workload 
during Maximal 
Effort Study 
75% PW from 
Maximal Effort Study 
>6 min to Exhaustion on 
SubMax Verification Study 
(+10W or 15%, whichever ∆ is 
greater) 
<3 min to Exhaustion on 
SubMax Verification Study 
(-10W or 15%, whichever ∆ is 
less) 
15 
10 
15* 
5** 
25 
20 
25* 
15 
50 
40 
50 
35 
75 
55 
65 
45 
100 
75 
85 
65 
125 
95 
110 
85 
150 
115 
130 
105 
175 
130 
150 
120 
 
* Targeted workload cannot be greater than peak workload achieved. 
 
** 5W is the minimum loaded workload allowable by the cycle ergometer 
 
o The workload determined after this titration procedure will be used during all subsequent 
submaximal exercise assessments for the subject. 
o We note that some individuals may not be able to maintain a pedal cadence of 60 RPMs. In these 
instances, the self-selected cadence that can be maintained during the submaximal verification 
study will be recorded. During subsequent visits, the subject will be coached to maintain this same 

 
 
 
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cadence such that the exercise conditions (workload and cadence) will be consistent across all 
submaximal exercise endpoint assessments at the end of each period (i.e. Visits 2/4/6).  
 
Baseline Visit – List of Procedures 
o Eligibility assessment 
o Informed consent 
o Medical history and ascertainment of concomitant medications 
o Dietary counseling re: low nitrate diet 
o KCCQ administration 
o Physical exam (including orthostatic vital signs, anthropometric measurements) 
o Laboratory tests, blood, urine and saliva collection 
o Urine pregnancy test, as applicable 
o Echocardiography and arterial tonometry 
o Maximal effort cardiopulmonary exercise test 
o Standardized Low-Nitrate Meal 
o Submaximal workload verification study 
We note that several studies have demonstrated that inorganic nitrate supplementation does not lead to 
clinically significant increases in methemoglobin;82, 186-188 therefore, this will not be serially measured during our 
study. However, subjects with a significantly increased methemoglobin level at baseline (> 5%, where ~1% is 
considered normal),189 will not be enrolled, as these individuals may harbor defects in their endogenous 
capacity to reduce methemoglobin and manage oxidant stress. 
Following the baseline study visit, the data will be verified to ensure inclusion/exclusion criteria are met. 
Subjects will then be randomized, and the study intervention will be delivered to the subject. 
One-week Assessment: 
 
As described in Section 7.1.2, subjects will start on the initial doses of study medications. Subject will 
be contacted approximately one week later to assess tolerability of the study medications and for adverse 
effects. The following may then result:  
(a) The subject describes mild/no side effects at which point study medications will be up-titrated, as 
detailed in Section 7.1.2;  
(b) The subject describes moderate side effects that are tolerable, and the subject and the investigators 
which to continue – in this case, study medications will be maintained at the initial doses;  
(c) The subject describes limiting side effects at which point study medications will be discontinued, an 
early termination visit will be attempted to be arranged, and the subject will be terminated from the 
study. 
Visit 2 – Assessment #1: Muscle Biopsy and Submaximal Exercise Endurance 
After taking study medications for 6 weeks (range 5-7 weeks), subjects will return to the exercise unit. Prior to 
this visit, subjects will receive an actigraphy monitor to be worn for the week prior to the visit. This device will 
collect activity information, including steps taken per day. The average steps per day during the monitoring 
period will be calculated. The subject will return this device at the study visit.  

 
 
 
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Subjects will come to the exercise unit in the morning and in the fasted state, though routinely prescribed 
medications should be taken according to their schedule. Subjects will be asked to refrain from caffeine for 
24hrs prior to this visit, and to avoid strenuous activity for 48 hours prior. A standardized low-nitrate meal will 
be sent to subjects for the evening meal prior to the study visit, and the last doses of study medications will be 
taken in the evening prior to this visit. We note that muscle markers of NAD+ metabolism,117 and blood and 
muscle nitrate82, 190 remain elevated well after the last dose. This approach will allow for trough sampling on the 
morning of the study visit, reflective of our chronic supplementation strategy, and minimize differences in 
muscle metabolite concentration levels consequent to differences in the timing of the last dose of study 
medications and the performance of the biopsy and exercise test.  
The following will be performed: 
• 
Anthropometric measurements 
• 
Physical exam: including seated and orthostatic blood pressure assessments 
• 
Kansas City Cardiomyopathy Questionnaire 
• 
Intravenous catheter placement: 2 venous catheters will be placed: (a) antecubital and (b) a 
retrograde intravenous catheter on the dorsum of the hand 
• 
Blood draw: 
o Comprehensive metabolic panel 
o Complete blood count 
o Storage of blood for later analysis, including whole blood, plasma, and serum 
• 
Saliva and urine collection for storage 
• 
Urine pregnancy test: for female of childbearing potential 
• 
Skeletal muscle biopsy of the vastus lateralis: Prior to exercise, the lateral aspect of the thigh will be 
sterilized and anesthetized using local anesthetic (e.g. lidocaine with sodium bicarbonate). A small 
incision will be made overlying the muscle and extended down through the fascia. A modified 
Bergstrom needle, with suction, will be used to obtain at least 200 mg of muscle tissue.191 Multiple 
passes through the same incision may be required to obtain sufficient muscle.  
o The following studies may be performed on muscle tissue: 
§ 
Mitochondrial respiration – both isolated mitochondrial and permeabilized fiber 
preparations will be made. Respiration may be assessed following the provision of 
carbohydrate and fatty acid substrate. Citrate synthase activity may be assessed as a 
marker of mitochondrial content and used to normalize the respirometry data. 
§ 
Muscle metabolomics – including measurement of the NAD+ metabolome, 
acylcarnitine profile, Acyl-CoA species (including propionyl-CoA), and nitrate/nitrite 
concentrations 

 
 
 
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§ 
Muscle proteomics 
§ 
Tissue processing for H&E and OCT blocks for subsequent 
histologic/immunofluorescent assessments 
§ 
We note that additional studies, such as muscle homogenate 3H-palmitate flux, may be 
performed if additional tissue remains and the budget allows. 
§ 
Remaining tissue will be stored for later analysis. 
• 
Submaximal exercise test until exhaustion – Primary Endpoint: Following the muscle biopsy, and 
after hemostasis has been achieved, subjects will perform a submaximal exercise test at the workload 
determined during the baseline visit (75% PW). Strong verbal encouragement will be given to 
encourage maximal effort until the time of exhaustion. While time to exhaustion is the primary endpoint, 
several other pieces of data will be obtained. We will mainly compare data obtained at 4 minutes, when 
steady state hemodynamic conditions are likely to be present: 
o Arteriovenous Metabolite Concentrations: The hand with the retrograde IV in its dorsum will 
be placed in a heated hand box prior to, and during, cycle ergometry. Heated hand boxes have 
been used to ‘arterialize’ the blood within the vein, allowing us to obtain blood that approximates 
arterial content without the use, and risk, of an arterial catheter. The hand will be placed in the 
hand box prior to exercise (at least 10 min prior to exercise) and remain in the chamber 
throughout exercise for sampling. The hand box air temperature will be set at < 55°C (for 
comparison, typical sauna temperatures are >65 °C). When used appropriately, and for periods 
of up to 4 hours for serial sampling, no skin burns have been reported 
(http://www.uvm.edu/~dmatthew/dem_res/?Page=heated_hand.html, accessed on 9/29/20). 
Arterialized and venous blood will be obtained immediately prior to exercise, at 4 minutes of 
exercise when steady-state conditions likely are present, and at the time of exhaustion. 
Substrates to be measured may include:  
§ 
Glucose 
§ 
Lactate 
§ 
Non-esterified fatty acids 
§ 
Triglycerides  
Other metabolites/hormones/proteins, such as insulin and acylcarnitines, may also be measured. 
o Aortic input impedance: Radial arterial tonometry and echocardiographic cardiac flow from the 
left ventricular outflow tract Doppler signal will be obtained at rest, at 4 minutes, and at the time 
of exhaustion. We will primarily compare differences in arterial hemodynamics at the 4-minute 
time point across submaximal exercise transients (V2/V4/V6). The vasodilatory reserve (the 
percent change in systemic vascular resistance relative to baseline) will be the main endpoint.  
o Respiratory exchange ratio: the ratio of the rate of carbon dioxide production (VCO2) and the 
rate of oxygen consumption (VO2) will be assessed (VCO2/VO2) as a marker of systemic substrate 
metabolism.192 Lower values (RER of 0.7) correspond to greater fatty acid oxidation, as compared 
to carbohydrate (RER of 1.0).192-195 The RER will be compared at 4 minutes of exercise and will 

 
 
 
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be computed as the average value over a 30 second time period.196  
o VO2 kinetics – the rate of increase or decrease in oxygen consumption following the onset or 
cessation of exercise is related to SkM OxPhos.48, 197-200 In HFpEF, VO2 kinetics are slowed,49, 50 
and SkM OxPhos is insufficient to meet energetic demands, leading to greater biochemical 
perturbations (e.g. accelerated lactate production) and ultimately the early cessation of exercise. 
Our interventions may impact VO2 kinetics, as they affect SkM OxPhos. Both “on” and “off” VO2 
kinetics may be modeled from the submaximal studies using state-of-the-art single- and double- 
exponential models, as appropriate.201 
o Myocardial diastolic parameters and surrogate markers of congestion – Given that our 
interventions may impact arterial properties, which in turn, can impact myocardial 
performance,202-208 we will also measure the mitral inflow velocity (E) and tissue doppler signals 
to arrive at the E/e’ ratio, a non-invasive surrogate for left ventricular filling pressures.209, 210 We 
may also interrogate echocardiographic estimates of RV function including the tricuspid 
regurgitant jet velocity211 and the tricuspid annular plane systolic excursion.212 Lung ultrasound 
may also be performed using echocardiography.183, 212  
Note: if the subject is willing, an additional muscle biopsy may be performed at the time of 
exhaustion. This will be done through the same incision as the resting biopsy performed earlier 
in the day, using a fresh sterile needle biopsy set-up, though the needle may be angled in a 
different orientation to sample undisturbed tissue. We note that many studies have employed 
serial muscle biopsies either during one study visit or on a different days following an 
intervention,117, 163, 190, 213-236 demonstrating the acceptability of this practice. We also note that 
the Molecular Transducers of Physical Activity Consortium (MoTrPAC), an NIH sponsored 
collaborative effort to understand the impact of exercise on the human body, employs serial 
muscle biopsies before and at several time points following exercise (pre-exercise, 0.5 hours 
after exercise, 4 hours after exercise, and 24 hours following exercise).236    
Visit 3 – Assessment #2: SkM MRI for SkM OxPhos and Intramuscular Perfusion 
The subject will continue on study medications for an additional 3-10 days prior to returning to UPenn for the 
CrCEST/vPIVOT MRI assessment. Subjects will be given a standardized low-nitrate meal for the evening prior 
to the scan. The last dose of study medications will be taken with this standardized meal in the evening prior to 
the MRI assessment. Subjects will come to the MRI scanner in the morning and in the fasted state, though 
routinely prescribed medications should be taken according to their schedule. Subjects will be asked to refrain 
from caffeine for 24hrs prior to this visit, and to avoid strenuous activity for 48hrs prior. Subjects will be asked 
to arrive at the MRI scanner at least 1 hour prior to their scan time, to avoid the confounding influence caused 
by travel. A standardized low-nitrate meal will be given during this rest period prior to the MRI scan. 
Subjects will be screened for metal objects on their clothes and in their pockets and asked to remove 
such items. Then the subject will be placed on the MRI patient bed and the radiofrequency coil placed around 
the part of the body under investigation. The subject will be given earplugs to dampen the sound made by the 
scanner during the imaging process. The subject will then be placed inside the magnet until the part of the 
body under investigation is in the center. The study will then proceed. The MR operator will be in constant 
two-way voice communication with the subject. The subject will be given periodic updates throughout the 
examination. Subjects will not be asked to remain in the magnet for more than 1.5 hours.  
During the scan, the subject will be asked to perform 2 bouts of plantar flexion exercise against a 
pneumatically controlled foot pedal. Immediately following exercise, SkM OxPhos (CrCEST protocol, following 
the first bout of exercise) and intramuscular perfusion (vPIVOT protocol, following the second bout of 

 
 
 
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exercise) will be assessed during the post-exercise recovery period (Figure 8). When the study is complete, 
the subject will be brought out of the magnet, the coil will be removed, and the subject will be allowed to get 
up slowly. Subjects are encouraged to take their time in getting up from the patient bed in order not to induce 
lightheadedness after the prolonged supine period. Data may be reviewed during the scan session to assess 
for quality. If one portion of the scan is deemed to be of insufficient quality for accurate data interpretation 
(CrCEST or vPIVOT portion), a third exercise bout may be performed at the end of the study to repeat this 
portion, if time allows and the subject is willing. 
 
For each MRI scan, the following represent 
the main measures: 
• 
CrCEST-based metrics of SkM OxPhos: 
o Half-time of CrCEST recovery 
(t1/2,Cr) – primary outcome measure 
for this assessment 
o The linear slope of CrCEST 
recovery over the early phase (first 
2 minutes) of recovery 
o Baseline and increase in CrCEST 
asymmetry with exercise in the 
posterior 
compartment 
muscle 
groups, 
including 
the 
lateral 
gastrocnemius 
• 
vPIVOT measures: 
o Baseline and peak intramuscular 
perfusion of the muscles involved 
in calf plantar flexion exercise, as 
well as the recovery characteristics 
of 
intramuscular 
perfusion, 
particularly 
of 
the 
lateral 
gastrocnemius muscle. 
o Baseline and peak conduit artery 
(e.g. popliteal artery) blood flow 
o Relative 
measure 
of 
capillary 
oxygenation 
(T2*) 
before 
and 
serially after exercise 
 
The primary endpoint of the MRI assessment will be the half-time of CrCEST recovery of the lateral 
gastrocnemius muscle, used as an in vivo metric of SkM OxPhos. Additional endpoints include the slope of 
early (first 2 minutes) Cr recovery, peak lateral gastrocnemius perfusion and its timing and recovery 
characteristics, relative capillary oxygenation (T2*) and its timing and recovery characteristics, and peak 
conduit (popliteal) artery blood flow and its timing and recovery characteristics. 
Scans will be performed on a 3T MRI scanners. The sequences used in this protocol are not FDA approved. 
Appropriate language stating as such has been added to the informed consent, as suggested by CAMRIS. 
Following both the MRI and exercise assessment, the subject will have completed Period 1. All study 
medications will be collected and counted by the study team to determine medication compliance. Study 
medications will then be returned to the UPenn IDS for disposal. 
Figure 8. CrCEST and vPIVOT imaging protocol. Following rest, the scanning 
protocol begins with anatomic and baseline CrCEST imaging. A standardized plantar 
flexion exercise protocol is then started for 2 minutes. Additional CrCEST images are 
obtained every 30s during recovery for 8 minutes. After a rest period, anatomic and 
baseline vPIVOT images are obtained, followed by an identical exercise transient 
and vPIVOT data collection during recovery. In vPIVOT, bulk conduit artery blood 
flow (A – bulk conduit artery flow), intramuscular perfusion (B – Perfusion), and 
relative T2* (C – T2*) are measured serially. Adapted from Englund, 2018.  
2 min @ 7 PSI - Duty Cycle: 0.75 Hz
 
 
 
Rest: 1 hour
2 min Ex
CrCEST
CrCEST Asymmetry (%) 
Time (s) 
exercise 
25 
20 
15 
10 
5 
0 
-100 
0 
400 
500 
100 
200 
300 
l. 
gastroc
m. 
gastroc
soleus
Baseline
Post-Ex
Anatomic
& Baseline
Imaging
2 min Ex
CrCEST
Recovery
vPIVOT
Anatomic
& Baseline
Imaging
vPIVOT
Imaging
A – Bulk Flow
B - Perfusion
C – T2*

 
 
 
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We note that due to scheduling concerns and/or subject availability, the MRI assessment may be performed 
prior to the exercise assessment. In this instance, a delay of at least 2 days will be arranged to minimize the 
small potential for the calf exercise performed during the MRI assessment to impact the cycle ergometry 
exercise study. In all cases, the subject will remain on study medications until both the exercise and MRI 
assessments for the period are completed. 
 
Wash-Out Period and Subsequent Periods: (Visits 4-7) 
Subjects will then enter a 2-week washout period, before beginning Period 2. After the washout, the next study 
intervention in the subject’s sequence will be given to the subject by UPenn IDS. The exact same study 
procedures and protocols enumerated for Period 1 will be repeated, including Assessment #1: Muscle Biopsy 
and Submaximal Exercise Endurance (Visit 4) and Assessment #2: SkM MRI for SkM OxPhos and 
Intramuscular Perfusion (Visit 5). Following Period 2, the subjects will again have a 2-week washout period, 
before entering Period 3, which will again be identical in its performance (Visit 6 – Muscle biopsy and submaximal 
exercise and Visit 7 – SkM MRI), as the subject receives the final treatment in his/her sequence.  
 
Visit 8 – Assessment #3: The Response of SkM OxPhos to Supplemental Oxygen: 
A central goal of this proposal is to understand the relationship between SkM OxPhos and SkM O2 delivery 
with exercise. If oxygen delivery to, and within, the skeletal muscle is impaired and insufficient to meet 
mitochondrial needs, SkM OxPhos will be reduced. Increasing O2 delivery will increase SkM OxPhos only if 
mitochondrial reserve capacity is present and mitochondrial rates of oxidative energy production are less than 
the rate of SkM energy utilization. In healthy individuals for large muscle mass exercise, maximal rates of 
isolated mitochondrial O2 consumption exceed maximal systemic and skeletal muscle VO2 achieved during 
exercise, demonstrating an excess mitochondrial reserve capacity.237-239 Accordingly, in athletes, SkM OxPhos 
and PCr recovery kinetics are speeded with supplemental oxygen, demonstrating functional mitochondrial 
reserve.240 On the other hand, in sedentary individuals, decreased mitochondrial reserve may limit SkM 
OxPhos, such that supplemental oxygen is of lesser benefit.241-243  
Disease state may also impact the SkM response to oxygen. A recent elegant study demonstrated that 
supplemental O2 did not speed SkM OxPhos in sedentary controls, but it did in sedentary diabetics.244 These 
findings were all the more intriguing as diabetic patients were found to have reduced mitochondrial content on 
muscle biopsy,244 demonstrating that even in the context of impaired mitochondrial content, a mitochondrial 
reserve may be present such that SkM OxPhos rates could be increased with supplemental O2.  
At least 30 days from the last dose of study medications, subjects will again come to the MRI scanner for a 
final visit. This will be a single-day, 2x2 cross-over study to assess the impact of supplemental O2 on SkM 
OxPhos in HFpEF. Equal numbers of subjects will be randomized to receive either low (FIO2=0.21, room air) 
followed by high (FIO2=1.0) FIO2 or high followed by low FIO2. Inhaled gas randomization sequence will be 
prespecified and stored in RedCap in order to ensure balance (50% with FIO2=0.21 first and 50% with 
FIO2=1.0 first).  
Subjects may be sent a standardized meal for the evening prior to the MRI scan. Subjects will arrive at least 
one hour prior to their scan time in the fasted state and will then rest, to avoid any confounding influence 
caused by travel. Subjects will be asked to avoid caffeine for 24 hrs prior to the MRI scan,174, 175 and to avoid 
strenuous activity for 48hrs prior. Water intake will not be restricted, and the subjects will take their usually 
prescribed medications according to their schedule. Upon arrival and during rest, a standardized meal will be 
given to the subject. Aside from the inspired gases (FIO2=0.21 or FIO2=1.0), the MRI scanning procedure will 
be identical. The subject and the MRI technician who will interpret the images will both be blinded to the FIO2 

 
 
 
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order. Both scans will be performed on the same day to minimize biologic variability,175, 245-248 with at least an 
hour separating each session to allow for adequate rest and oxygen washout. The same standardized meal 
will be given to the subject an hour before the second scan. We note that previous studies in sedentary 
individuals used between 5-30 minutes of rest between repeat studies.241, 242, 244 Tanks with room air and 100% 
oxygen will be kept in the control room and attached to a non-rebreather facemask with a reservoir to ensure 
constant delivery throughout the scanning protocol. Inhaled gases will be started ~15 minutes prior to each 
scanning sequence for adequate equilibration.249  
(Note: Regulators providing 25L/min of flow are available for 100% oxygen; however, regulators for room air 
are only available that provide a maximum of 15L/min. As the subjects will be breathing the inhaled gases 
through a reservoir, and the contents of the room air gas should be identical to the content of the air in the MRI 
scanner, additional intake of ambient room air should not lead to any significant difference than if regulators 
were able to provide 25L/min of room air. Should a room air regulator that provides 25 L/min of flow become 
available, this will be used). 
Primary Endpoint. The primary endpoint will be the change in SkM OxPhos on CrCEST in response to 
supplemental oxygen (DSkM OxPhos). We hypothesize that two basic HFpEF phenotypes of impaired SkM 
OxPhos will emerge in response to 100% O2:  
1. O2-Responsive (↑DSkM OxPhos): Those with moderate or major SkM OxPhos improvements with O2, 
suggesting available mitochondrial reserve, but a defect in O2 delivery.  
2. O2-non-responsive (↔DSkM OxPhos): Those with no or little improvement in SkM OxPhos with O2, 
suggesting a lack of mitochondrial reserve. 
In addition to defining differences between the O2-responsiveness/non-responsive subjects, we will also 
compare DSkM OxPhos to the contrasts in exercise duration from the interventional portion of this study. The 
difference in submaximal exercise duration between the KNO3 vs KNO3+NR+PLC interventions may be 
attributable to the addition of agents selected to improve mitochondrial reserve (NR+PLC). Therefore, we 
predict that individuals with large improvements in submaximal exercise duration following combination therapy 
versus KNO3 will be O2-non-responsive, harboring a lack of mitochondrial reserve (↔DSkM OxPhos) at 
baseline. In contrast, individuals with small differences in submaximal exercise duration following combination 
therapy versus KNO3 alone will be O2-responsive (↑DSkM OxPhos), demonstrating the presence of 
mitochondrial reserve at baseline.   
It is possible that in the future, the response to supplemental O2 could be used to phenotype HFpEF patients, 
identifying those HFpEF individuals who are more likely to benefit from a mitochondrial intervention and 
enriching clinical trials that otherwise have been hampered by the heterogenous nature of the disease.250 
8.2 
Subject Safety and Risk Assessments  
Potential Risks 
1. Cardiopulmonary exercise test: Exercise testing 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.251 These procedures will be performed by qualified personnel 
according to established American Heart Association guidelines.252, 253 Non-revascularized myocardial 
ischemia, which may increase the risk of cardiovascular complications with exercise,254 is an exclusion 
criterion for the study, as is current angina due to epicardial coronary artery disease. 
Subjects may feel uncomfortable as a result of pushing themselves during an exercise test. 
Subjects will likely feel short of breath and tired 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 

 
 
 
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of exercise. Although exercise testing may result in exhaustion, rarely do people develop abnormal 
heart rhythms or significant cardiovascular complications, such as an acute myocardial infarction,254 
during exercise tests. The risk of this happening is the same as if the participant were to exert 
him/herself during stressful situations or during exercise elsewhere, though subjects are monitored in 
our exercise lab setting.  
We will perform EKG, heart rate, and blood pressure 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 non-invasively using a pulse oximeter. Of note, 
oxygen levels can decrease with exercise, even in individuals without significant cardiopulmonary 
disease.255, 256 If the arterial saturation falls to below 88% (“severe exercise induced hypoxemia”256), we 
will alert the subject and his/her care provider, if possible, as this may prompt consideration for 
additional/alternative causes for arterial hypoxemia and shortness of breath.  
 
2. Echocardiography: Our exercise protocols routinely incorporate echocardiography during exercise for 
determination of cardiac output.64, 82 This is a non-invasive procedure and does not have any known 
significant risks. It is possible that subjects may experience mild, temporary discomfort or skin irritation 
from the echo probe, or from wearing the electrodes needed to monitor heart rate during the 
examination. We may also use the ultrasound machine to obtain images of the lungs for signs of 
congestion.183  
 
3. Skeletal muscle vastus lateralis biopsy: Muscle needle biopsy with suction will be performed,191 
following local anesthesia (for example, with 1% lidocaine with sodium bicarbonate). In a large series of 
13,500 muscle needle biopsies performed at one center, the overall complication rate was 0.16% in 
adult patients: 
 
 
From Tarnopolsky et al. Muscle Nerve 2011; 43: 717. 
 
The most significant risks occurring in adults included arterial bleeding (1/13,626 = 0.007%) and local 
skin infection (8/13,626 = 0.06%). To date, we have performed vastus lateralis biopsies in over 50 
subjects without significant complications. We note that our protocol of pre-exercise muscle biopsies 
has been extensively employed in many prior physiologic studies.217, 218, 220-228, 230, 257-263 Moreover, 
several studies have employed serial muscle biopsies either during one study visit or on a different 
days following an intervention,117, 163, 190, 213-232, 235, 236 demonstrating that this is an acceptable study 
procedure. We note that in many of these studies, muscle biopsies were performed before and after 
exercise, or multiple times during a single exercise transient. In fact, the Molecular Transducers of 
Physical Activity Consortium (MoTrPAC), an NIH sponsored collaborative effort to understand the 
impact of exercise on the human body, employs serial muscle biopsies before and at several time 

 
 
 
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points following exercise (pre-exercise, 0.5 hours after exercise, 4 hours after exercise, and 24 hours 
following exercise).236 
 
Other considerations specific to the muscle biopsy: 
• 
Post-exercise muscle biopsy: In individuals who elect to also allow us to obtain a post-
exercise muscle biopsy, we do not anticipate a significant increase in risk, noting that a 
new sterile needle will be used through the same skin incision as the biopsy performed 
prior to exercise, though we may attempt to angle the needle into a different segment of 
muscle.  
• 
Thrombocytopenia: Biopsies will not be performed on individuals with platelet count < 
100k on baseline laboratories (Visit 1), unless a subsequent check shows that the 
platelet count has recovered or was erroneous.  
• 
Treatment with anticoagulation (e.g. warfarin, dabigatran, apixiban, rivaroxaban, or a 
different anticoagulant): Given the significant prevalence of atrial fibrillation in HFpEF 
(~35% in recent large clinical trials such as PARAGON and TOPCAT264, 265), we expect 
that a substantial proportion of enrolled subjects will be on anticoagulation. In these 
instances, risk stratification regarding the temporary cessation of anticoagulation will be 
performed (see Table):  
 
 
Table from Spyropoulos and Douketis, 2012266 
 
• 
In subjects at high risk for thromboembolic events, anticoagulation will not be 
interrupted, and the muscle biopsy will not be performed.  
• 
In individuals at intermediate (expected to be the majority of subjects) or low 
(expected to be infrequent, given the age and comorbidities in HFpEF subjects) 
risk for thromboembolic events, we will have an informed conversation with the 
subject about his/her risk with withholding anticoagulation for the performance of 
the muscle biopsy. Anticoagulation will only be held if the subject consents to do 
so. We may also discuss this with the subject’s provider. 
• 
For illustrative purposes, an average of 5 days of non-therapeutic anticoagulation 
in a subject with intermediate risk of thromboembolic events would translate to:  
o 4-10%/year risk à 7% average annual risk à 0.02% daily risk à 0.1% 5-
day risk à 1:1000 risk of a thromboembolic event during the peri-biopsy 
period. 

 
 
 
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• 
With the subject’s consent, and generally after a discussion with the subject’s 
provider, anticoagulation will be discontinued prior to the muscle biopsy (~5 days 
prior for warfarin, and 2 days prior for direct oral anticoagulants). This is similar to 
what is done for clinical procedures, such as a routine colonoscopy. 
• 
In the case of warfarin, the INR will be checked prior to the biopsy, and the 
biopsy will be performed if the INR is <1.5266 If the INR is >1.5, we will attempt to 
reschedule the study visit, if possible, or forego the biopsy. 
• 
Warfarin may be restarted on the evening of the biopsy, and oral anticoagulants 
may be restarted 2 days following the biopsy.  
• 
Antiplatelets: Biopsies can be performed while on aspirin.191 Additional antiplatelet 
agents (e.g. clopidogrel, ticagrelor, prasugrel) will be treated similarly to anticoagulation 
and will not be interrupted without a discussion with the subject and his/her provider. 
Biopsies will not be performed in subjects on dual antiplatelet therapy (aspirin, in 
addition to another antiplatelet). 
• 
Allergy to lidocaine or related anesthetic medication. In these cases, the nature of 
the allergy will be discussed with the subject, and the use of an alternative agent may be 
pursued. For example, in subjects with a lidocaine allergy, which is an amide local 
anesthetic, alternatives could include the use of an esther local anesthetic (such as 
tetracaine) or diphenhydramine.267 These decisions will be made in discussion with the 
Investigational Drug Pharmacy and the subject, and in light of the agents that the subject 
may have received previously for local anesthesia. 
 
4. Phlebotomy and blood draws: The CHPS unit in which the studies will be performed employs a 
critical-care trained nurse practitioner and a research nurse who have extensive experience in blood 
draws and maintaining venous catheters. Risks from the peripheral (e.g. antecubital fossa) venous 
catheterization include minor discomfort, minor bruising, bleeding, hematoma and/or fainting associated 
with the drawing of blood. There is also a very small chance (less than 1%) of infection at the blood 
draw site. We anticipate that completion of the entire protocol (all visits) will lead to less blood drawn 
than that which is removed during a standard red-blood cell donation (approximately 1 pint or 473 mL): 
 
 
Baseline Visit 
Period 1: 
Assessment #1 
Period 2: 
Assessment #1 
Period 3: 
Assessment #1 
Resting 
CMP – 5 mL 
CMP – 5 mL 
CMP – 5 mL 
CMP – 5 mL 
 
NTproBNP – 5 mL 
CBC – 3 mL 
CBC – 3 mL 
CBC – 3 mL 
 
Met-hgb – 6 mL 
(Coag – 3 mL)* 
(Coag – 3 mL)* 
(Coag – 3 mL)* 
 
CBC – 3 mL  
 
 
 
Coag – 3 mL  
 
 
 
Storage: 
 
 
 
 
WB: 5 mL  
 
 
 
Plasma: 15 mL  
 
 
 
Serum: 10 mL  
 
 
Submaximal 
Exercise 
  
 
 
 
“Arterialized” & 
Venous Blood 
Rest (0-min): 
 
Rest (0-min): 
Rest (0-min): 
 
 
WB: 5 mL  
WB: 5 mL 
WB: 5 mL 
 
 
Plasma: 20 mL 
Plasma: 20 mL 
Plasma: 20 mL 
 
 
Serum: 15 mL 
Serum: 15 mL 
Serum: 15 mL 

 
 
 
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4-min: 
4-min: 
4-min: 
 
 
WB: 5 mL 
WB: 5 mL 
WB: 5 mL 
 
 
Plasma: 20 mL 
Plasma: 20 mL 
Plasma: 20 mL 
 
 
Serum: 15 mL 
Serum: 15 mL 
Serum: 15 mL 
 
 
Exhaustion: 
Exhaustion: 
Exhaustion: 
 
 
WB: 5 mL 
WB: 5 mL 
WB: 5 mL 
 
 
Plasma: 20 mL 
Plasma: 20 mL 
Plasma: 20 mL 
 
 
Serum: 15 mL 
Serum: 15 mL 
Serum: 15 mL 
 
 
 
 
 
Approx. Total: 
52 mL 
131 mL 
131 mL 
131 mL 
CMP = comprehensive metabolic panel, CBC = complete blood count, Coag = coagulation profile, Met-hgb 
= methemoglobin %, WB = whole blood. * = if subject is on warfarin and muscle biopsy is to be performed 
 
 
5. Dual Energy X-Ray Absorptiometry (DEXA): DEXA scanning will occur during Visit 1 in the dedicated 
and certified lab space of the CHPS unit according to the practice guidelines of the American College of 
Radiology.268 Participants will be instructed to never look directly into laser of the scanning arm that 
passes over the body. In most instances, scans will occur one time. However, in the event that the 
images and data collection from the first scan are not clear, a second scan may occur. However, in any 
event, no more than 2 scans will occur for a participant. The length of a single scan will take 
approximately 15 minutes. The radiation dose received during a DEXA scan is less than that of a chest 
X-ray.269 This data will be used to normalize exercise parameters to lean muscle mass. 
 
6. Kansas City Cardiomyopathy Questionnaire: Participants may become uncomfortable with 
questions or feel sadness as a result of completing questionnaires. 
 
7. Lower Extremity MRI: The non-invasive nature, lack of ionizing radiation, and lack of gadolinium 
contrast make the risks associated with our MRI studies small. MRI scans requires the subject to be in 
a partially enclosed space inside the scanner, which some people may find uncomfortable. The MRI 
scanner produces different noises during a scan. Patients are given special earplugs to reduce the 
noise. The MRI scanner has a strong magnet that attracts certain metals. If anyone has these types of 
metal in their body, the MRI’s strong magnetic field can cause the metal to move, which may cause 
injury. Screening of MRI-related exclusion criteria will be done during the baseline visit and with an on-
site questionnaire at the time of, or prior to, the first scan. Subjects with be thoroughly screened for the 
presence of body metal and excluded from the MRI portion of the study if they are unable to safely 
undergo an MRI. We will not perform MRI scans in subjects who are not suitable candidates for an 
MRI. The presence of the following will be evaluated:  
a. Central nervous system aneurysm clips  
b. Implanted neural stimulators 
c. Implanted cardiac pacemaker or defibrillator  
d. Cochlear implant 
e. Ocular foreign body (e.g. metal shavings)  
f. Other implanted medical devices: (e.g. drug infusion ports)  
g. Insulin pump 
h. Metal shrapnel or bullet 
i. Severe claustrophobia 
j. Extreme obesity rendering the patient unable to fit into narrow-bore scanners  
k. Unwillingness of the patient to undergo an MRI scan. 

 
 
 
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l. Pregnancy: Women with childbearing potential will have a urine pregnancy test as part of each 
period assessment during the interventional medication portion of this study. Attestation of non-
pregnant status will be obtained prior to the MRI scans and during Assessment #3 (MRI with 
and without oxygen). We note that the subject’s assent of non-pregnancy status is an 
acceptable practice put forth by CAMRIS. 
 
All patients with metallic implants will be individually evaluated prior to the first MRI. In cases where 
there is uncertainty, we will consult with our radiologists/MRI technicians. In these cases, the MRI 
will only be performed if deemed to be safe by our radiologists/technicians. Standard safety 
practices, as per our institutional standards, will be followed. Voice contact with patients will be 
maintained throughout the scan. Some individuals may experience leg stiffness/soreness as a 
result of lying on the table in the MRI scanner with his/her leg relatively still in between bouts of calf 
exercise. This is anticipated to be minor and should abate as the participant gets off the table and 
begins to walk around. 
 
**We note that many contemporary ICD/pacemakers are MRI-compatible. In participants with an 
implantable device (defibrillator or pacemaker), the specific details of the device will be reviewed and 
discussed with our radiologists in order to ensure the safety of the participant with that device during 
scanning at 3T. The participant will only undergo the MRI scan if it is deemed to be safe. 
 
8. Confidentiality: There is a potential for a breach of confidentiality. We are committed to protecting 
confidentiality as described in detail below. 
 
9. Active Pharmacologic Therapy: This proposal includes 3 different pharmacologic 
interventions/combinations: (a) potassium nitrate; (b) potassium nitrate + nicotinamide riboside (NR) + 
propionyl-L-carnitine (PLC); (c) potassium chloride. Risks are described in Section 3.1.1. 
 
10. Acute Oxygen Supplementation: There are little data to guide the risk/benefit discussion of acute 
oxygen supplementation in heart failure overall, and no data in HFpEF specifically, in the absence of 
hypoxia.270 As in non-HF patients, hyperoxia can have negative hemodynamic effects in HFrEF 
patients, such as an increase in systemic vascular resistance and reductions in cardiac output.271-273 
Despite this, oxygen supplementation has been shown to increase exercise time in HFrEF subjects.274 
The impact of oxygen in HFpEF is unknown. Over longer periods of time than that in this study (not 
anticipated to exceed 1.5-2 hours), supplemental oxygen can have a detrimental effect on lung 
function. In studies of monkeys, more than 5 days of exposure to 90% oxygen were needed before 
symptoms developed.275 Exposure to FIO2=0.5 in healthy subjects for a mean of 44 hours led to an 
increase in inflammatory mediators found on bronchoalveolar lavage.276 Pre-existing pathology may 
also exacerbate oxygen toxicity. In a study of human subjects who were intubated (therefore 
presumably with some degree of lung injury present), excessive FIO2 above that needed to maintain 
saturations to >92% have been associated with worse oxygenation at 48 hrs.277 Indeed, pre-existing 
lung injury can exacerbate/predispose to oxygen toxicity, worsening lung injury.275 Even in the setting of 
an acute myocardial infarction, where less lung injury is reasonably expected to be present than in 
acute respiratory distress syndromes, supplemental oxygen can have detrimental effects in the 
absence of hypoxia.278, 279 Hyperoxia can be associated with absorption atelectasis due to the higher 
solubility of oxygen in blood than nitrogen.280 In patients with severe COPD and baseline hypoxia, 
supplemental oxygen can lead to a decrease in respiratory drive.281 We note, however, that our study 
will be performed in stable outpatient in whom known significant pulmonary disease (as defined in the 
exclusion criteria) will be exclusionary. Moreover, subjects with clinically significant coronary artery 
disease (as defined above, and per investigator judgement) will be excluded from the study, minimizing 

 
 
 
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the risk of supplemental oxygen leading to myocardial ischemia. Additionally, the increase in arterial 
oxygen content with supplemental oxygen may help mitigate the impact of decreased coronary flow on 
oxygen delivery. During the scan, we will maintain verbal contact with the subjects, checking in on them 
frequently. The study will be stopped if the subject experiences significant untoward or concerning side 
effects such as chest pain.  
 
11. Actigraphy and Step Counts: The actigraphy monitor is a watch-like device. There are no risks 
associated with actigraphy and acquisition of the step count data. It is possible that a subject might get 
mild irritation from the “watch strap” if the monitor is worn on the wrist. In that case, the device may be 
worn on the hip. 
8.3 
Adverse Events and Serious Adverse Events 
Adverse events  
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 protocol following applicable 
regulations. 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 IRB and the Data Safety 
Monitoring Board in a timely fashion (i.e., at continuing review).  
 
 
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. 
 
Serious Adverse Events (SAE): An adverse event or suspected adverse reaction is considered serious if the 
investigator or Medical Monitor 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 in 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 Medical Monitor (i.e., if any one 
of these believes it is serious, it must be considered serious). 
 
Unanticipated Problem:  An Unanticipated Problem (UP) is a medical adverse event OR a non-medical event 
that is:  
(1) unforeseen (unexpected), AND 
(2) suggests that research places subjects at greater risk than was previously known or recognized, AND  

 
 
 
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(3) is related or possibly related to a subject’s participation in research  
Classification of AE 
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. This also includes “unlikely related” 
events (No temporal association or the cause of the event has been 
identified, or the drug or device is unlikely to be implicated, or there is a 
low likelihood that a causal relationship exists) 
 
Related 
There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely. This includes the category of “possibly related 
(reasonable evidence to suggest a causal relationship between the 
drug and adverse event) 
 
 
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 SAE shall be determined according to the 
specified reference document containing safety information (e.g., most recent protocol or 
informed consent). Any AE/ADE that is not identified in nature, severity, or specificity in the 
current study reference document(s) is considered unexpected. Events that are mentioned in 
the reference documents 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. 
 
Classification of Adverse Events Regarding Severity Scale 

 
 
 
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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. Lightheadedness 
7. Worsening renal function 
8. Edema 
9. Shortness of breath at rest or during/after exertion  
10. Fatigue at rest or during/after exertion 
The following are potential expected side effects of Potassium Nitrate (KNO3): 
1. Slight headache 
2. Dizziness 
3. New onset or worsening lightheadedness 
4. Low blood pressure 
5. GI Upset: Stomach discomfort, stomachache, diarrhea, nausea, or vomiting 
6. Worsening shortness of breath 
7. Worsening fatigue 
8. Flushing 
9. Rash 
10. Orthostatic hypotension 
As above, while methemoglobinemia is a theoretical side effect of KNO3, we did not observe it 
in our prior work in HFpEF participants using the same dosing regimen; therefore, we will 
consider any occurrence of clinically significant methemoglobinemia (>5%) to be unexpected.82  
The following are potential expected side effects of Potassium Chloride (KCl): 
1. GI upset 
2. Stomach discomfort 
3. Nausea 
4. Vomiting 
The following are potential expected side effects of Nicotinamide Riboside: 
1. GI Upset: nausea, vomiting, diarrhea, transient changes in stool, bloating 
2. Muscle soreness/cramping 
3. Rash/flushing 
4. Headache  
 
The following are potential expected side effects of Propionyl-L-Carnitine: 
1. Nausea 
2. GI upset/gastric discomfort 

 
 
 
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Recording and Reporting of Adverse Events & Unanticipated Problems (UP) 
The PI 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 PI will be responsible for ensuring that all 
adverse events are noted, followed, and reported to the IRB. UPs will be reported in an expedited manner 
per applicable regulations. 
 
• 
AEs occurring from the time of signed informed consent to one week after the administration 
of the last study drug, or one day after the final MRI visit if participating in MRI studies, will be 
collected.  
• 
AEs will be classified according to the guidelines/definitions specified above.  
• 
We note that given the pharmacology of our study medications, events occurring > 2 days after the 
last dose are unlikely to be related to their administration.82, 123, 282 As an additional safety margin, we 
will consider the possibility of an AE being related to study medications up to 5 days after the last 
dose.  
• 
Any AE rated >=3 in severity and all SAEs must be reported within 1 working day of first becoming 
aware of the event to the medical monitor. The IRB should also be notified per their reporting 
guidelines.  
• 
The medical monitor will make a determination about the necessity to modify the protocol, include 
additional information in the consent form, inform previous participants, temporarily hold enrollment 
of subjects, or terminate the study. In addition, we will report AEs/SAEs to NIH, FDA, and the DSMB 
per their regulations, as applicable. 
• 
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. 
The DSMB will review adverse events at regularly scheduled meetings. Meetings will be convened 
based on landmarks in participant completion, for example:  
o First 5 participants complete 
o 10 participants complete 
o 30 participants complete 
o Enrollment complete (53 participants) 
§ 
Additional meetings may be convened at the request of the DSMB chair 
§ 
The above schedule may be modified by the DSMB 
• 
Events significant enough to necessitate modification of study drug dosing will be captured on an 
appropriate eCRF module.  
Safety Event 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, improvement, or the event is otherwise explained. 
 
The DSMB will review detailed safety data, as detailed above. 
Management of Suspected (Related) 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 to the regulatory authorities (e.g. DSMB, NIH, etc.). The Principal 
Investigator, along with the Medical Monitor, will assess all SAEs and evaluate for “unexpectedness” and 
relationship to study drug. The Principal Investigator is required to complete a report for any event identified 
as serious, study drug related, and unexpected, and submit it to the Medical Monitor within 1 working day of 
the PI becoming aware of it.  

 
 
 
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Pregnancy 
Pregnancy is a contraindication to enrollment in the study. Pregnancy occurring during the study period, 
although not considered an SAE, must be reported within the same timelines as an SAE, and to the IRB per 
local requirements. The pregnancy will be recorded on the appropriate note to file. Study drugs will be 
discontinued immediately, and the 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. 
9 
STATISTICAL CONSIDERATIONS 
9.1 
Statistical Hypotheses 
Statistics for the Trial Evaluating the Impact of Study Medications on Submaximal Exercise Endurance: 
The primary analyses will be intention-to-treat. The 3x3 crossover design involves 2 interventions plus an 
active control and 3 periods, leading to a total of 6 unique sequences (e.g. KCl in period [P] 1; KNO3 alone in 
P2; KNO3+NR+PLC in P3). For each block of 6 participants, the design is ‘balanced’ across periods, with each 
treatment appearing twice in each period. Each subject will have paired measurements of the primary 
outcome, submaximal exercise endurance, on each treatment pair (KNO3 vs KCl), (KNO3+NR+PLC vs KCl) 
and (KNO3 vs KNO3+NR+PLC). We will analyze each treatment pair contrast (e.g. KNO3 vs. KCl) separately 
using least squares regression, to minimize assumptions on the covariance matrix of the repeated 
measurements.283 The regression model will be adjusted for period effects and for baseline submaximal 
exercise endurance.283, 284 The model-based estimates and hypothesis tests will be used to determine which, if 
any, of the individual interventions is/are effective.  
The proposed sample size of study completers is 48 (8 subjects per each of 6 treatment sequences); we will 
enroll 53 subjects to account for dropouts. For the primary outcome, and Bonferroni adjusting for two multiple 
comparisons of each intervention (KNO3 or KNO3+NR+PLC) to control (KCl), the proposed sample size yields 
at least 80% power to detect mean differences in submaximal exercise time on the order of 80 seconds. The 
calculation uses a family-wise two-sided Type I error rate of 0.05 and conservatively assumes a standard 
deviation (SD) of submaximal exercise time of 170 seconds, around 20% larger than the roughly 140 seconds 
reported by Eggebeen et al.74 This effect is hypothesized to be clinically important given the severity of 
impairment in this cohort (mean submaximal exercise duration of 363±125 s seen in Eggebeen et al.).74 The 
comparison of KNO3+NR+PLC versus KNO3  alone is expected to yield somewhat smaller effects; without 
adjustment for multiple comparisons, we have 80% power to detect a mean difference of 70 seconds. In 
addition to hypothesis testing, 95% confidence intervals will be created to evaluate potential effect sizes for 
possible future studies.  
In general, for the secondary endpoints (e.g. CrCEST recovery time, VO2 recovery kinetics, vasodilatory 
reserve, respiratory exchange ratio, steps per day, KCCQ overall summary score), we have 80% power to 
detect reasonable effect sizes (Mean difference/SD) of 0.41. Within each of the physiologic outcome groups 
(e.g. quality of life, muscle metabolomics, arteriovenous substrate gradients, mitochondrial respirometry and 
muscle proteome), we will follow the general strategy described above. In these exploratory analyses, in 
addition to hypothesis testing, we will focus attention on confidence intervals. Conclusions will be based on the 
coherence of the results for the individual endpoints within pre-defined outcome groups.  
The statistical analysis will be blinded to intervention group; exploratory analyses will stratify by sex as a 
biological variable in order to assess whether males and females tend to respond differently to the 
interventions. The study is not powered to detect these differences between groups, but sex effects will be 
more rigorously examined in future studies should differences be apparent. While missing data are expected to 

 
 
 
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be minimal, we will explore the impact of any missing data using multiple imputation.284, 285 Analyses will be 
carried out using the latest version of R or Stata.   
 
Proteomics Analysis: The protein assignment and MS2 intensity values will be generated by Spectronaut 
(Biognosys AG; or similar software) and used to analyze the proteome data. Perseus (or similar software) will 
be used for proteomics data processing and statistical analysis.286 The data will be log2-transformed and 
normalized by subtracting the median for each sample after each intervention. Protein intensities will be 
compared between matched intervention pairs using paired Student’s t-tests. Proteomic comparisons between 
KNO3, KNO3+NR+PLC, and KCl will identify differentially abundant proteins, and volcano plots will be 
generated using EnhancedVolcano package in R (or similar software).  
 
Comparisons will be made between individual proteins that are key regulators of fatty acid oxidation (e.g. 
CD36, FABP3, SLC27A6, FABP5, CPT1B, CPT2, CRAT, ACADVL, ACADM, ACADS, ACADSB, HADHA, 
HADHB, ECHS1, HADH, ACAA2, ACAD9, ECI1, ECI2, DECR1, ECH1, ACC), mitochondrial biogenesis (e.g. 
PGC-1a, HIF-1, NRF1/2, ERRa, TFAM, MXI1, C-MYC, AMPK, ANT, and mTOR), NAD+ metabolism 
(SIRT1&SIRT3,83 FOXO,91 CD38,287 PARPs,108 NAMPT, NAMNT, NRK, and CX43288), and energy fuel 
metabolism, with adjustment for multiple comparisons. Additionally, lists of differentially abundant proteins will 
be used for downstream bioinformatic analysis using the STRING database (string-db.org) and MetaCore 
(Clarivate Analytics) to identify pathway enrichment, and its associated false discovery rate,289-291 for each 
comparison (other similar software may be used). 
Metabolomics Analysis: Period-adjusted levels of metabolites will be compared between matched intervention 
pairs using paired Student’s t-tests. Candidate metabolites will be selected based on an FDR no greater than 
0.05.  
Visit 7 – Assessment #3: The response of SkM OxPhos to Supplemental Oxygen: 
The design and analysis plan is similar to that of the main trial, except that the design is an AB:BA (two-
treatment, two-period) crossover trial with each letter corresponding to two different oxygen levels (FIO2=0.21 
or FIO2=1.0). Briefly, Dr. Putt will provide a coded randomization sequence in RedCap, which the study 
coordinator will access on the day of each subject’s study.  
Statistics for the Impact of Supplemental Oxygen on SkM OxPhos.  
The primary endpoint will be the change in SkM OxPhos on CrCEST in response to supplemental oxygen 
(DSkM OxPhos) using paired statistics. We will then graph the distribution of SkM OxPhos for the individual 
FIO2 levels as well as for the change in FIO2 levels, as a function of FIO2 order. We hypothesize that the 
HFpEF population represents a mixture of two ‘classes’ of response to O2 i.e., ‘responders’ and ‘non-
responders’. Under this hypothesis, the variability in SkM OxPhos under 100% FIO2 should far exceed that 
seen under room air, with some individuals showing a pronounced increase in SkM OxPhos with 100% FIO2 
compared to room air, and others showing little change. To formally test this hypothesis, we will fit the change 
in SkM OxPhos to a latent class model. We will fit two models, one with a single class, and one with two 
classes and compute the Akaike Information Criteria (AIC). We will then repeatedly permute the assignment of 
the randomization sequence (Room air followed by 100% O2 or vice versa) in order to determine the 
distribution of the AIC under the null hypothesis of a single class of response to 100% O2. The observed AIC 
will be compared to the permutation distribution in order to determine a p-value.  Additionally, we will use the 
latent class model to estimate the proportion of O2-responders by assigning individuals to a class based on the 
larger of their individual probabilities of class membership. A 95% confidence interval for this proportion will be 

 
 
 
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determined using the bootstrap method. Based on individual participants’ class assignment, we will estimate 
the threshold in the change in SkM OxPhos corresponding to a ‘responder’ versus a ‘non-responder’.  
Comparison with data from Submaximal Exercise Studies: 
We will then regress the outcome variable, the change in skeletal muscle oxidative phosphorylation capacity 
on CrCEST (DSkM OxPhos) with 100% oxygen supplementation, on the difference in submaximal exercise 
duration for combination therapy versus KNO3, adjusting for baseline exercise duration. Submaximal exercise 
duration post-intervention will be pre-adjusted for period effects. We hypothesize a negative slope; individuals 
with large improvements in submaximal exercise duration between combination therapy and KNO3 should be 
O2 non-responsive (↔DSkM OxPhos); whereas, individuals with limited improvement in submaximal exercise 
duration between combination therapy and KNO3 are likely O2-responsive (↑DSkM OxPhos). With 48 subjects 
and a Type I error rate of 0.05, we have 80% power to detect moderate Pearson correlations on the order of 
0.39 or larger (%& of at least 0.15).  
Further analyses will allow us to probe our findings. First, we will regress submaximal exercise endurance on 
combination therapy as a function of submaximal exercise endurance on KNO3. We anticipate a large positive 
intercept term and a moderate slope as combination therapy is expected to be most effective in rescuing the 
mitochondrial deficit in individuals with low response to KNO3 alone. Alternatively, combination therapy may 
uncover mitochondrial reserve across the spectrum of KNO3 response; in this case we anticipate a positive 
intercept term and a null slope. We will regress DSkM OxPhos on period-adjusted exercise duration separately 
for each of the three interventions. We hypothesize a null slope for KCl, a strong positive slope for KNO3, and a 
positive slope to KNO3 for the combination therapy (which also contains KNO3). The strong positive slope for 
KNO3 is anticipated based on previous findings; we cannot yet predict the results for the combination as this 
will depend on which subjects are most impacted by improvements in mitochondrial reserve. Linearity will be 
assessed by including spline terms in the model and comparing the spline-based models and the linear model 
using an information criteria (AIC or BIC). Analyses will be repeated, stratified by male versus females, and 
assessed for evidence of sex-specific differences in the response.  
The secondary analysis will consider individual level DSkM OxPhos and include only those individuals in the 
lower and upper quartiles of DSkM OxPhos. The outcome variable will be submaximal exercise endurance 
under each of the treatment regimens. Using a two-group t-test, we will assess whether there are differences in 
the mean exercise endurance for each treatment group between the upper and lower quartiles of DSkM 
OxPhos. We predict that mean submaximal exercise endurance under the KNO3 intervention will be higher in 
the upper versus lower quartiles of DSkM OxPhos response; mean submaximal exercise endurance under the 
combination intervention will be higher in the lower versus upper quartiles of DSkM OxPhos response. 
Using only the 24 subjects with the highest and lowest quartiles of DSkM OxPhos, we have 80% power to 
detect differences in the mean submaximal exercise endurance of 203 seconds. The calculation assumes a 
familywise Type I error rate of 0.05 adjusted for the two different models and standard deviation of submaximal 
exercise endurance of 170 seconds. 

 
 
 
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10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
10.1 
Regulatory, Ethical, and Study Oversight Considerations 
10.1.1 
Informed Consent Process 
10.1.1.1 
Consent Procedures and Documentation 
 
Written informed consent will be obtained from participants 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 Institutional Review Board. The study procedures, interventions, and the associated risks will be 
explained to participants during the informed consent process. Only IRB-approved consent forms will be used. 
We expect that all privacy practices put in place by those entities should translate into sufficient privacy for all 
participants. All informed consent procedures will be carried out by personnel approved to work on the study. 
The consent process will be carried out in a private area. Each potential participant will be given the consent 
form to read for him/herself and given adequate time to read the entire consent form. Study personnel will go 
through the consent form with the potential participant, answering any questions. We will explain the HIPAA form 
to all potential participants. Potential participants will also be informed of the voluntary nature of their participation 
and the lack of direct benefits. If the participant agrees to participate, an approved study team member will 
witness the participant sign and date the consent form, and then verify the process by signing the form as well. 
At no point will any undue influence be applied to the potential participant by any means including the 
embellishment of monetary compensation or understatement of risks associated with this study. The language 
to be used in the consent form will be written such that all potential participants can understand what is being 
asked of them, should they voluntarily choose to participate in the study. Consent forms will be kept in a locked 
file cabinet within the Principal Investigator’s research space. 
 
Although not directly targeted, economically or educationally disadvantaged persons, and/or employees or 
students of the University of Pennsylvania will not be denied enrollment and any special protections and/or 
additional safeguards will be undertaken in order to protect the rights and welfare of these subjects from 
coercion or undue influence, as appropriate. 
 
10.1.2 
Study Discontinuation and Closure 
This study may be temporarily suspended or prematurely terminated by the Data Safety and Monitoring Board, 
the Medical Monitor, or the PI if there is sufficient reasonable cause. Written notification, documenting the 
reason for study suspension or termination, will be provided by the suspending or terminating party to study 
participants, investigators, and the funding agency. If the study is prematurely terminated or suspended, the 
Principal Investigator (PI) will promptly inform study participants, the Institutional Review Board (IRB), and the 
sponsor (NIH) and will provide the reason(s) for the termination or suspension. Study participants will be 
contacted, as applicable, and be informed of changes to study visit schedule. 
Circumstances that may warrant termination or suspension may include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping 
• 
Insufficient compliance to protocol requirements 

 
 
 
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• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
The study may resume once concerns about safety, protocol compliance, and data quality are addressed, and 
satisfy the sponsor, DSMB, and the IRB. 
In terminating the study, the Principal Investigator will assure that adequate consideration is given to the 
protection of the subjects’ interests. 
10.1.3 
Confidentiality and Privacy 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, the 
sponsor, and the UPenn IRB. This confidentiality is extended to cover testing of biological samples and genetic 
tests in addition to the clinical information relating to participants. Therefore, the study protocol, documentation, 
data, and all other information generated will be held in strict confidence.  
All research activities will be conducted in as private a setting as possible. 
The study monitor, other authorized representatives of the sponsor, representatives of the Institutional Review 
Board (IRB), or regulatory agencies may inspect all documents and records required to be maintained by the 
investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy records for 
the participants in this study. The clinical study site will permit access to such records. 
The study participant’s contact information will be securely stored for internal use during the study. At the end 
of the study, all records will continue to be kept in a secure location for as long a period as dictated by the 
reviewing IRB, institutional policies, or sponsor requirements. 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will be 
maintained indefinitely in an electronic database. This will not include the participant’s contact or identifying 
information. Rather, individual participants and their research data will be identified by a unique study 
identification number. The study data entry and study management systems used by clinical sites and by the 
research staff will be secured and password protected. At the end of the study, all study databases will be de-
identified and archived.  
If any data or samples are shared with collaborators at UPenn or elsewhere, only de-identified samples/data 
will be given, without any linking information.  
10.1.4 
Future Use of Stored Specimens and Data  
Data collected for this study will be analyzed and stored at UPenn. After the study is completed, the de-
identified, archived data will be available at UPenn for use by other researchers including those outside of the 
study. Permission to transmit data and/or share de-identified samples (e.g. muscle, blood, images, exercise 
data, etc.) to collaborators will be included in the informed consent. 
With the participant’s approval and as approved by local Institutional Review Boards (IRBs), de-identified 
biological samples will be stored at UPenn. These samples could be used to research the causes of HFpEF, its 
complications, and other conditions for which individuals with HFpEF may have and that could improve with 

 
 
 
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treatment. We cannot anticipate all of the possible future uses of the data and samples. Collaborators and 
study investigators may be provided with information linking the biological specimens with the phenotypic data 
from each participant, while maintaining the blinding/confidentiality of the identity of the participant. 
During the conduct of the study, an individual participant can choose to withdraw consent to have biological 
specimens stored for future research. In this case, no future specimens will be collected. However, biosamples 
and specimens already collected may be retained and used.  
When the study is completed, access to study data and/or samples will be provided through the Principal 
Investigator.  
Commercial Products: Samples/data may be shared with other entities, including for-profit companies, for 
research purposes. Any company/entity (at UPenn or elsewhere) receiving information/samples related to this 
study may ultimately discover products, drugs, tests, etc. as a result, which may lead to commercial products. 
Study participants will not be entitled to compensation for these discoveries. This information has been 
included in the informed consent.  
10.1.5 
Safety Oversight 
Safety oversight will be under the direction of a Medical Monitor and a Data and Safety Monitoring Board 
(DSMB) composed of individuals with the appropriate expertise, including a cardiologist and a statistician. 
Members of the DSMB should be independent from the study conduct and free of conflict of interest, or 
measures should be in place to minimize perceived conflict of interest. The DSMB will meet regularly to assess 
safety data on each arm of the study. The DSMB will operate under the rules of an approved charter that will 
be written and reviewed at the organizational meeting of the DSMB. At this time, each data element that the 
DSMB needs to assess will be clearly defined. The DSMB will provide its input to the Principal Investigator and 
the Medical Monitor.  
10.1.6 
Clinical Monitoring 
A Data and Safety Monitoring Plan has been assembled for this study and is enumerated within a separate 
document.  
 
10.1.7 
Quality Assurance and Quality Control 
All monitoring and audits are to be performed according to ICH GCP E6(R2). 
We will perform internal quality management of study conduct, data and biological specimen collection, 
documentation and completion. As in the Data Safety and Monitoring Plan, an individualized quality 
management plan will be developed to describe a site’s quality management. 
Quality control (QC) procedures will be implemented beginning with the data entry system, and data QC 
checks will be run on the database. Any missing data or data anomalies will be communicated to the study 
team for their resolution. 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is 
conducted, data are generated, specimens are collected, documented (recorded), and reported in compliance 
with the protocol, International Conference on Harmonization Good Clinical Practice (ICH GCP), and 

 
 
 
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applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices 
(GMP)). 
The investigational site will provide direct access to all trial related sites, source data/documents, and reports 
for the purpose of monitoring and auditing by the sponsor, Medical Monitor, and inspection by local and 
regulatory authorities. 
10.1.8 
Data Handling and Record Keeping  
10.1.8.1 
Data Collection and Management Responsibilities  
Data collection is the responsibility of the clinical trial staff under the supervision of the Principal Investigator. 
The investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness of the data 
reported. 
All source documents should be completed in a neat, legible manner to ensure accurate interpretation of data. 
Hardcopies of the study visit worksheets will be provided for use as source document worksheets for recording 
data for each participant enrolled in the study. Data recorded in the electronic case report form (eCRF) derived 
from source documents should be consistent with the data recorded on the source documents. 
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse reactions data) 
and clinical laboratory data will be entered into REDCAP. The data system includes password protection and 
internal quality checks, such as automatic range checks, to identify data that appear inconsistent, incomplete, 
or inaccurate. Clinical data will be entered directly from the source documents.  
10.1.8.2 
Study Records Retention  
Study documents should be retained for a minimum of 2 years after the completion of the study. These 
documents should be retained for a longer period, however, if required by local regulations. 
10.1.9 
Protocol Deviations 
The PI and the study team should document all scenarios where the protocol is not followed and provide 
details which may include: 
• 
Who deviated from the protocol? 
• 
What was the deviation? 
• 
When did the deviation occur? 
• 
How did the deviation happen? 
• 
What is the impact of the deviation? 
• 
A root cause analysis of why the deviation occurred 
If the assessment results in a determination that any of the following are potentially affected, the deviation 
would be considered of significant impact: 
• 
having the potential to adversely affect subject safety; OR 
• 
increases risks to participants; OR 

 
 
 
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• 
adversely affects the integrity of the data; OR 
• 
violates the rights and welfare of participants, OR 
• 
affects the subject’s willingness to participate in research, OR 
• 
there is a potential for an overall impact on the research that should be shared with the IRB for 
consideration and development of next best steps to address it 
10.1.10 
Publication and Data Sharing Policy 
1) Trial Registration: The clinical trial proposed in this application will be registered in www.ClinicalTrials.gov 
prior to initiation. Registration will be updated to reflect significant protocol amendments during the course of 
the study. Trial results will be uploaded at the time of publication. Informed consent documents will include a 
specific statement relating to posting of clinical trial information at ClinicalTrials.gov. The University of 
Pennsylvania has an internal policy in place to ensure that clinical trials registration and results reporting occur 
in compliance with policy requirements.  
 
2) Publication: Study results will be published in a peer-reviewed publication. The final, peer-reviewed journal 
manuscript will be submitted to PubMed Central upon acceptance for publication.  
 
Data sharing may be considered upon reasonable request to the Principal Investigator, with appropriate 
agreements in place. 
10.1.11 
Conflict of Interest Policy 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the design, conduct, 
analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who have 
a perceived conflict of interest will be required to have such conflicts managed in a way that is appropriate to 
their participation in the design and conduct of this trial. 
10.2 
Protocol Amendment History 
 
Version 
Date 
Description of Change  
Brief Rationale 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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Version 
Date 
Description of Change  
Brief Rationale 
 
 
 
 
 
 
 
 
11 APPENDIX  
11.1 
 Schedule of Activities (SoA) 

 
 
 
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Procedures 
Screening 
Day -7 to -1 
Baseline Visit  
Day 1 
Study Visit 2  
Day 42  +/-7 d 
Study Visit 3 
Day 3  +/- 1 day 
Study Visit 4 
Day 21 +/-1 day 
Study Visit 5 
Day 28 +/-1 day 
Study Visit 6 
Day  35 +/-1 day 
Study Visit 7 
Day 42 +/-1 day 
Study Visit 8 
Day 49 +/-1 day 
Informed consent 
 
X 
 
 
 
 
 
 
 
Demographics 
X 
X 
 
 
 
 
 
 
 
Medical History 
X 
X 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
 
Dietary Counseling re: 
Low Nitrate Diet 
X 
X 
X 
 
X 
 
X 
 
 
Concomitant 
Medications 
 
X 
X 
 
X 
 
X 
 
 
Physical Exam 
(height, weight) 
 
X 
X 
 
X 
 
X 
 
 
Orthostatic Vital Signs 
 
X 
X 
 
X 
 
X 
 
 
Kansas City 
Cardiomyopathy 
Questionnaire 
 
X 
X 
 
X 
 
X 
 
 
Complete Blood Count 
 
X 
X 
 
X 
 
X 
 
 
Comprehensive 
Metabolic Profile  
 
X 
X 
 
X 
 
X 
 
 
Coagulation Profile 
 
X 
Only 
select 
cases 
 
Only 
select 
cases 
 
Only 
select 
cases 
 
 
Methemoglobin % 
 
X 
 
 
 
 
 
 
 
NTproBNP 
 
X 
 
 
 
 
 
 
 
Urine 
Collection/Storage 
 
X 
X 
 
X 
 
X 
 
 
Saliva 
Collection/Storage 
 
X 
X 
 
X 
 
X 
 
 
Blood 
Collection/Storage 
 
X 
X 
 
X 
 
X 
 
 
Urine Pregnancy Test 
(if applicable)  
 
X 
X 
 
X 
 
X 
 
 

 
 
 
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Procedures 
Screening 
Day -7 to -1 
Baseline Visit  
Day 1 
Study Visit 2  
Day 42  +/-7 d 
Study Visit 3 
Day 3  +/- 1 day 
Study Visit 4 
Day 21 +/-1 day 
Study Visit 5 
Day 28 +/-1 day 
Study Visit 6 
Day  35 +/-1 day 
Study Visit 7 
Day 42 +/-1 day 
Study Visit 8 
Day 49 +/-1 day 
Echocardiogram and 
Arterial Tonometry 
 
X 
X 
 
X 
 
X 
 
 
Maximal Effort 
Exercise Test 
 
X 
 
 
 
 
 
 
 
Submaximal Exercise 
Test 
 
X 
X 
 
X 
 
X 
 
 
Muscle Biopsy 
 
 
X 
 
X 
 
X 
 
 
CrCEST/vPIVOT MRI 
Assessment 
 
 
 
X 
 
X 
 
X 
X 
Administration of 
Supplemental Oxygen 
during MRI 
 
 
 
 
 
 
 
 
X 
Adverse Event 
Assessment 
 
X 
X 
X 
X 
X 
X 
X 
X 
Complete Case 
Report Forms (CRFs) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 

 
 
 
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END OF DOCUMENT

 
 
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