SGLT2i and KNO3 in HFpEF - The SAK HFpEF Trial
Phase: PHASE2
Conditions: Heart Failure With Preserved Ejection Fraction
Sponsor: University of Pennsylvania

 
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CLINICAL RESEARCH PROTOCOL 
 
INVESTIGATIONAL PRODUCT(S): 
 
Empagliflozin 
Potassium Nitrate 
Potassium Chloride 
STUDY 
NUMBER(S): 
IRB Number 
849401 
Other Protocol 
Identifiers  
 
CHPS: pending 
PROTOCOL(S) TITLE: 
SGLT2i And KNO3 in HFpEF – SAK HFpEF Trial 
REGULATORY SPONSOR: 
 
University of Pennsylvania 
FUNDING SPONSOR(S): 
National Institutes of Health 
PRINCIPAL INVESTIGATOR 
 
Payman Zamani, MD, MTR 
ORIGINAL PROTOCOL DATE: 
 
VERSION NUMBER: 
Version 3 
VERSION DATE: 
11 October 2021  
 

 
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1 
Study Summary ...................................................................................................... 8 
1.1 
Synopsis ............................................................................................................ 8 
1.2 
Key Roles and Study Governance ......................... Error! Bookmark not defined. 
1.3       Schema ............................................................................................................ 11 
2 
Introduction and RationalE .................................................................................. 12 
2.1 
Study Rationale................................................................................................ 12 
2.2 
Background ...................................................................................................... 12 
2.2.1 
Pharmacokinetics, Pharmacodynamics and Toxicology ........................ 12 
2.2.2 
Assessment for Potential Study Products Drug-Drug, Drug-Device, 
Device-Device Interactions............................... Error! Bookmark not defined. 
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 .............................................................................. 18 
2.3.2 
Known Potential Benefits .......................................................................... 18 
2.3.3 
Assessment of Potential Risks and Benefits .. Error! Bookmark not defined. 
3 
Study Objectives and Endpoints ......................................................................... 21 
4 
Study Plan ............................................................................................................. 24 
4.1 
Study Design .................................................................................................... 24 
4.2 
Scientific Rationale for Study Design ............................................................ 24 
4.3 
Justification for Dose ...................................................................................... 24 
4.4 
End of Study Definition ................................................................................... 24 
5 
Study Population .................................................................................................. 26 
5.1 
Inclusion Criteria .................................................... Error! Bookmark not defined. 
5.2 
Exclusion Criteria ............................................................................................ 26 
5.3 
Lifestyle Considerations ................................................................................. 27 
5.4 
Screen Failures ................................................................................................ 28 
5.5 
Strategies for Recruitment and Retention ..................................................... 28 
6 
Study Intervention ................................................................................................ 30 
6.1 
Study Intervention(s) Administration ............................................................. 31 
6.1.1 
Study Intervention Description ................................................................. 31 
6.1.2 
Dosing and Administration ....................................................................... 31 
6.2 
Preparation/Handling/Storage/Accountability ............................................... 32 
6.2.1 
Acquisition and accountability ................................................................. 34 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ............................... 34 
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 ............................ 34 
6.4 
Study Intervention Compliance ...................................................................... 35 
6.5 
Concomitant Therapy ...................................................................................... 35 
6.5.1 
Rescue Medicine ............................................... Error! Bookmark not defined. 
7 
Study Intervention Discontinuation and Participant 
Discontinuation/Withdrawal ................................................................................ 37 
7.1 
Discontinuation of Study Intervention ........................................................... 37 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................... 37 
 

 
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7.3 
Lost To Follow-Up ........................................................................................... 38 
8 
Study Assessment and Procedures .................................................................... 39 
8.1 
Efficacy Assessments ..................................................................................... 39 
8.2 
Safety and Other Assessments ...................................................................... 47 
8.3 
Adverse Events and Serious Adverse Events ............................................... 47 
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 ................................................................................... 56 
9.1 
Statistical Hypotheses ..................................................................................... 56 
9.2 
Sample Size Determination .................................... Error! Bookmark not defined. 
9.3 
Populations for Analyses ................................................................................ 56 
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 ........................... 57 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ........................... 57 
10.1.1 
Informed Consent Process........................................................................ 57 
10.1.2 
Study Discontinuation and Closure .......................................................... 57 
10.1.3 
Confidentiality and Privacy ....................................................................... 58 
10.1.4 
Future Use of Stored Specimens and Data .............................................. 59 
10.1.5 
Safety Oversight ........................................................................................ 60 
10.1.6 
Clinical Monitoring ..................................................................................... 60 
10.1.7 
Quality Assurance and Quality Control .................................................... 60 
10.1.8 
Data Handling and Record Keeping ......................................................... 60 
10.1.9 
Protocol Deviations ................................................................................... 61 
10.1.10 
Publication and Data Sharing Policy ........................................................ 61 

 
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10.1.11 
Conflict of Interest Policy .......................................................................... 61 
10.2 
Additional Considerations ..................................... Error! Bookmark not defined. 
10.3 
Protocol Amendment History ......................................................................... 62 
11 References ................................................................... Error! Bookmark not defined. 
12 APPENDIX ............................................................................................................. 63 
12.1 
Schedule of Activities (SoA) ........................................................................... 63 
 
 
 
 
 

 
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PRINCIPAL INVESTIGATOR SIGNATURE 
 
STUDY SPONSOR: National Institutes of Health  
STUDY TITLE: 
SGLT2i and KNO3 in HFpEF – SAK HFpEF Trial 
STUDY ID 
PROTOCOL 
VERSION 
 
V3.0 
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 
DKA – Diabetic ketoacidosis 
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 
EMPA - Empagliflozin 
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 
NOm – Nitric oxide metabolites 
OxPhos – Oxidative phosphorylation capacity 
O2 – Oxygen 
PB – Placebo 
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* 
AVO2 – Arteriovenous O2 content difference 

 
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31P – 31-Phosphate 
6MW – 6-minute walk distance 
 

 
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1 
STUDY SUMMARY 
1.1 
Synopsis 
Title:  
SGLT2i and KNO3 in HFpEF – The SAK HFpEF Trial 
Short Title: 
The SAK HFpEF Trial 
Study 
Description:  
This study will test whether Empagliflozin (Empa), with and without 
Potassium Nitrate (KNO3), improves submaximal exercise endurance 
and skeletal muscle oxidative phosphorylation capacity (SkM OxPhos) in 
participants with Heart Failure with Preserved Ejection Fraction (HFpEF).  
Objectives:  
1. Compare the impact on submaximal exercise endurance of Empa, 
with and without KNO3, as compared to active control (Potassium 
Chloride, KCl). 
2. Assess the impact of Empa, with and without KNO3, on SkM OxPhos 
using novel in vivo MRI-based imaging techniques that quantify SkM 
OxPhos and intramuscular perfusion with exercise 
3. Assess the impact of Empa, with and without KNO3, on SkM 
respiration, the metabolome, and the proteome. 
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: 
Skeletal Muscle MRI: 
1. Change in SkM OxPhos following plantar flexion on MRI 
2. Change in intramuscular perfusion following plantar flexion on MRI 
Metabolic Efficiency: 
3. Change in the kinetics of O2-consumption with submaximal exercise 
4. Change in VO2 efficiency with submaximal exercise 
5. Change in the respiratory exchange ratio and venous concentration 
of substrates at standardized times during submaximal exercise 
6. Change in tissue measures of substrate metabolism, muscle 
proteome, and the muscle metabolome. 

 
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Hemodynamic: 
7. Change in the exercise vasodilatory reserve with submaximal 
exercise 
Quality of Life: 
8. Change in KCCQ overall summary score 
9. Change in steps per day 
 
Exploratory 
Endpoints: 
Assess the impact of our interventions on: 
(1) Markers of intramyocardial filling pressure during exercise 
(2) Citrate synthase, a surrogate for mitochondrial content 
 
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: 
 
Empagliflozin (Empa) 10 mg daily 
 
Potassium Nitrate (KNO3) 6 mmol 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: 

 
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1. Empa + KCl  
2. Empa + KNO3  
3. KCl + Placebo for Empa 
 
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:  
 
Empagliflozin + Potassium Chloride (KCl) 
 
Empagliflozin + Potassium Nitrate (KNO3)  
 
Potassium Chloride (KCl) + Placebo for Empa 
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

 
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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,(18-21) suggesting that new 
treatment approaches are needed.   
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 differ 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.(22) 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).(22) 
Specifically, nearly all of the 
participants studied had  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. Moreover, because multiple 
abnormalities are present, improving any one abnormality has limited potential in improving exercise 
capacity.(22) On the other hand, therapies that target multiple pathways simultaneously stand a better 
chance to meaningful improve exertional intolerance in HFpEF patients.  
The skeletal muscle of HFpEF patients may be an important site of exercise limitation. The study by 
Houstis et al. also identified that the systemic arteriovenous O2 content difference (AVO2) at peak exercise, a 
marker of skeletal muscle (SkM) oxygen utilization, is consistently impaired in HFpEF patients across several 
studies.(22-24) In our preliminary studies, we evaluated exercise capacity in healthy individuals (n=20), HFpEF 
subjects (n=20), and age-matched hypertensive subjects (HTN, n=19) during cycle ergometry.(25, 26) HFpEF 
subjects had markedly lower peak VO2, compared to either control group. Importantly, HFpEF subjects had 
a diminished AVO2 (Fig. 1), while peak cardiac output was no different,(25, 26) suggesting to us that the 
SkM may be an important site of exercise limitation in HFpEF.  
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 
AVO2, 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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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).(26-31)  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.(29, 32) 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.(27, 29-31)  
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).(26)  
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.  
HFpEF skeletal muscle demonstrates reductions in the relative levels 
of mitochondrial complexes and enzymes in the OxPhos pathways. 
We then initiated a SkM biopsy program to investigate changes within the 
SkM of our HFpEF patients. We performed comprehensive LC-MS/MS 
proteomic analyses on muscle tissue from 12 HFpEF subjects, 9 HTN, 
and 13 healthy controls.(26) Proteins were extracted and digested into 
peptides, and then analyzed using LC-MS/MS, as reported previously by 
our team.(26, 33) In our comparison of HFpEF versus similarly-aged HTN 
SkM, a total of 2832 proteins were quantified, of which the relative 
abundance of 327 proteins was different between groups (p<0.05, Fig. 4). Using the Gene Ontology (GO) 
database,(34, 35) we found that the top biologic processes represented by these differentially abundant 
proteins were predominantly related to energy fuel metabolism and oxidative phosphorylation (false discovery 
rate [FDR]<1e-48).(26) The proteomic data corroborate the functional findings, giving additional support 
for the existence of mitochondrial and SkM OxPhos abnormalities in HFpEF. 
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.  
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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Little research has focused specifically on the mitochondria in HFpEF. A recent phase 2b study 
evaluated the impact of neladenoson bialanate, a partial adenosine A1 receptor agonist with potential 
mitochondrial effects, in 
HFpEF.(36) Unfortunately, 
the drug did not 
demonstrate any 
improvements in the six-
minute walk distance, a 
marker of submaximal 
exercise capacity. 
Supporting data for this 
compound were largely 
drawn from animal studies 
in ischemic HFrEF and 
adults with angina,(37-39) 
two models with unclear 
relevance to HFpEF. As 
pointed out by the authors, 
the neutral findings suggest 
that either neladenoson 
bialanate: (a) did not 
effectively improve the 
mitochondrial abnormalities 
present in HFpEF 
participants, or (b) that any 
mitochondrial improvements 
caused by the drug did not 
translate into changes in six-
minute walk distances.(36) 
Unfortunately, the design of 
the study precluded an 
ability to discern which 
conclusion should be drawn. 
The current proposal will 
test pharmacologic agents 
that specifically target 
abnormalities 
demonstrated in our 
HFpEF patients. Within 
the context of a rigorous clinical trial, we will have the phenotypic depth to explore and understand the 
mechanistic basis of our interventions.  
In summary, HFpEF is a multifaceted disease that includes impediments to SkM OxPhos, impacting 
exercise tolerance. We reason that combinations of pharmacologic agents, which specifically target 
deficits identified in our HFpEF patients, will be needed to have a meaningful impact on exercise 
capacity. 
SGLT2i have changed the paradigm of how “antihyperglycemic” agents are viewed. The medical 
community witnessed a paradigm-shifting discovery with sodium-glucose cotransporter-2 inhibitors (SGLT2i). 
Following concerns for an increased risk of myocardial infarction with rosiglitazone,(40) the FDA mandated 
-Log10 P
0
1
2
3
4
-6
-3
0
3
Log2 Fold Change
Biologic 
Process (GO)
False 
Discovery 
Rate
Generation of precursor 
metabolites and energy
7.76e-53
Oxidation-reduction process
4.32e-49
Small molecule metabolic 
process
7.62e-47
Cellular respiration
3.34e-44
Mitochondrial organization
1.85e-41
Complex IV
Complex I
Complex V
TCA Cycle 
Enzymes
Fatty acid & 
Ketone Metabolism
Proteostasis
Contractile 
Proteins
PDH, BCKDH
AKGDH
A
B
C
Figure 4 – Volcano plot, network map, and biologic enrichment of proteins significantly 
different between HFpEF and HTN participants. (A)Volcano plots were constructed with blue 
dots representing proteins with significantly different relative levels between HFpEF and HTN 
participants (p<0.05), and red dots and the gene names listed for proteins that had significantly 
different levels with an absolute log2 fold-change (FC)>1. (B) All proteins with significantly 
different relative levels between HFpEF and HTN participants (p<0.05), and their log2 fold-
change (FC), were entered into the String Database (string-db.org). Interrelated proteins are 
displayed along with the connection between proteins and groups. Related proteins are shaded 
a similar color. The halo around each protein represents the log2 FC, with blue indicating a 
relative decrease in protein level in HFpEF participants as compared to HTN participants, and 
red representing an increase. Though not inclusive, specific clusters of proteins related to 
energy fuel metabolism are enumerated. (C) Enrichment of the top 5 biologic (GO) processes, 
along with the false-discovery rate are listed. AKGDH= alpha-ketoglutarate dehydrogenase 
complex; BCKDH=branched-chain alpha-keto dehydrogenase complex; PDH=pyruvate 
dehydrogenase; TCA=tricarboxylic acid cycle.  

 
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cardiovascular outcomes trials during the development of antihyperglycemics.(41) Consequently, the EMPA-
REG OUTCOME trial was conducted in ~7000 Type II diabetics,(42) finding a marked reduction in outcomes, 
driven primarily by reduced heart failure hospitalizations. Several subsequent studies of other SGLT2i 
(canagliflozin and dapagliflozin) recapitulated these remarkable findings.(43-49) Importantly, the 
antihyperglycemic effects of these agents are modest (generally 0.5-1% reduction in HbA1C) and unlikely to 
explain the observed benefit.(47, 50)  
Even more provocatively, the DAPA-HF trial randomized subjects with HFrEF, with and without diabetes, to 
dapagliflozin (DAPA) and found that DAPA reduced HF exacerbations and cardiovascular death by ~25% over 
a median of 18 months,(51, 52) alongside improvements in quality of life.(53, 54) The benefit of DAPA was 
demonstrated regardless of diabetes status(55),age,(56) gender,(57) or the use of other guideline-directed 
medical therapy.(58) Similar findings in HFrEF have subsequently been demonstrated for empagliflozin,(59-61) 
another SGLT2i that is more selective for the SGLT2i receptor.(62) 
The SOLOIST-WHF Investigators examined the effect of sotagliflozin, a combined SGLT2i and gut SGLT1- 
inhibitor, in Type 2 DM participants who were hospitalized for worsening heart failure. While the trial was ended 
early due to loss of funding, an analysis of 1222 randomized patients who were followed for 9.0 months 
showed that the rate of primary endpoint (total cardiovascular deaths and hospitalizations/urgent visits for heart 
failure) was reduced by sotagliflozin. Two-hundred and fifty-six of these participants (~20% of the study 
population) had an LVEF >=50%, and there was no heterogeneity of the treatment effect by ejection fraction, 
suggesting a potential role in HFpEF.  
Given these studies, SGLT2i are approved for the treatment of HFrEF patients; however, the utility of SGLT2i 
in HFpEF remains unknown. Two ongoing studies are examining the impact of SGLT2i on cardiovascular 
outcomes in HFpEF (DELIVER; NCT03619213 and EMPEROR-Preserved; NCT03057951). While both studies 
will also look at quality of life measures, neither is testing the impact of SGLT2i on exercise capacity despite its 
important relationship to quality of life and independence.(10, 63) 
SGLT2i have the potential to improve exercise by improving mitochondrial function and energy fuel 
metabolism through several mechanism, many of which target abnormalities specifically described in 
human HFpEF:  
1. Increased mitochondrial biogenesis: In a human HFpEF SkM biopsy study, citrate synthase activity and 
porin expression, two markers of mitochondrial volume density,(64) were both reduced in HFpEF subjects 
as compared to age-matched controls, suggesting impaired mitochondrial biogenesis in HFpEF.(65) EMPA 
has been shown to increase the AMP/ATP ratio, leading to activation of AMP-kinase (AMPK),(66-68) a 
master regulator of mitochondrial energy homeostasis.(69, 70) Amongst its many targets, AMPK activation 
leads to an increase in PGC-1𝛼 activity,(69, 71, 72)  potentially leading to an SGLT2i-induced increase in 
SkM mitochondrial biogenesis.(73-75) In both murine and porcine models of ischemic HFrEF, EMPA 
increased PGC-1𝛼 and AMPK activation,(67, 68, 76) leading to increased mitochondrial biogenesis and 
volume.(76, 77) Through this mechanism, EMPA has the potential to target the impaired SkM OxPhos 
seen in our HFpEF participants. 
2. Increased fatty acid oxidation: AMPK also phosphorylates a host of enzymes including acetyl-CoA 
carboxylase (ACC),(67, 69, 70, 78) leading to a reduction in malonyl-CoA and CPT-1 inhibition.(79, 80) 
Enzymes involved in fatty acid import are also increased,(70) collectively leading to increased fatty acid 
oxidation (FAO).(69-71) SGLT2i may also attenuate a HIF-1𝛼 mediated decrease in FAO in diabetes.(81, 
82) In murine models of heart failure due to coronary artery ligation, EMPA attenuated the post-infarction 
decrease in exercise capacity,(83) increased the levels of transporters involved in fatty acid import,(76) and 
increased mitochondrial FAO.(83) In a porcine model of ischemic HFrEF, EMPA increased myocardial fatty 
acid uptake.(68) In several human studies of diabetic patients, SGLT2i have been shown to increase 
resting FAO.(84-86) Pertinent to this proposal, one of the main adaptations to aerobic conditioning is an 

 
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increase in fatty acid oxidation,(87-92) and animal studies in heart failure,(93, 94) including HFpEF,(95) 
demonstrate impaired FAO in this condition. This suggests that perhaps by increasing rates of FAO, 
SGLT2i could improve the markedly reduced exercise tolerance seen in HFpEF participants.  
3. A shift away from carbohydrate. As glucose levels fall, insulin declines, leading to an increase in the 
glucagon:insulin ratio,(84-86, 96-98) and an increase in lipolysis.(85, 96) In turn, acetyl-CoA production at 
the liver increases, leading to ketone body production and export,(96) which can then be oxidized at the 
SkM.(99-101) Several studies have shown an increase in plasma ketones with SGLT2i in both animal(67, 
68, 76, 83, 102) and human studies.(84, 96, 98, 103-105) EMPA has also been shown to increase 
utilization of ketone bodies,(68, 76) providing an alternative carbon source for energy production.(106) 
Glycogen stores are intrinsically linked to exercise endurance.(107-110) An SGLT2i-induced shift away 
from carbohydrate utilization could have a glycogen-sparing effect,(99, 111, 112) allowing its depletion to 
be delayed, and prolonging exercise capacity.(99, 109, 113-115) In addition to proteomics, we also 
assessed the phosphoproteome using LC+MS/MS, as done by our Co-Investigators previously,(33) and 
compared differences in protein phosphorylation between HFpEF and HTN subjects. While the main 
differences in phosphorylation were on contractile proteins, we also found reductions in phosphorylation on 
serine-293 (log2 fold-change: -2.18 for HFpEF versus HTN, p<0.05) and serine-232 (log2 FC: -1.55, 
p<0.05) on pyruvate dehydrogenase (PDHA1). Phosphorylation at these sites inhibits PDH activity;(116, 
117) therefore, the reduced phosphorylation suggests greater carbohydrate oxidation in our resting HFpEF 
SkM samples, which were obtained in the fasted state, as compared to HTN. Interestingly, in a porcine 
model of HFrEF, EMPA decreased PDH activity,(68) suggesting that EMPA might target the increased 
PDH activity implied by our phosphorylation studies. Additionally, EMPA has been shown to decrease 
myocardial glucose uptake in humans with Type II DM.(105) Collective, these data suggest that SGLT2i 
have the potential to diminish reliance on glucose for energy production.  
4. Increased hemoglobin concentration. Several studies, including our own, have documented a lower 
hemoglobin concentration ([Hgb]) in HFpEF subjects.(25, 118, 119) SGLT2i increase [Hgb],(105, 120-125) 
potentially through increased erythropoietin levels.(104, 121, 123, 125, 126) Increased [Hgb] would 
increase oxygen delivery to the SkM for any given blood flow, supporting increased rates of SkM OxPhos. 
5. Replenish metabolite deficiencies identified in HFpEF – In our muscle biopsy program, we identified 
decreased levels of propionyl-CoA and acetylcarnitine (C2-carnitine) in the SkM of HFpEF participants (Fig 
5). Propionyl-CoA is formed via the breakdown of 
branched chain amino acids and odd chain fatty 
acids and can provide additional carbon backbones 
to the tricarboxylic acid cycle (anapleurosis).(127, 
128) Acetylcarnitine interacts with carnitine 
acetyltransferase (CrAT) to determine the acetyl-
CoA/CoA ratio, which regulates glycolytic flux at 
pyruvate dehydrogenase, provides a readily 
available source of CoA in times of need, and assists 
in the efflux of fatty acid moieties from the 
mitochondria.(129-132) In an in-depth phenotyping 
study of diabetics with cardiovascular disease, 25 
participants were given Empa 10 mg daily, with 
serum drawn at baseline and after 1 month.(98) 
Empa increased circulating levels of Propionyl-CoA 
and acetylcarnitine, along with significant increases 
in many of the TCA cycle intermediates, suggesting 
that Empa might replenish some of the deficits we 
identified in HFpEF SkM. 
Healthy
HTN
HFpEF
0.00
0.05
0.10
0.15
Propionyl-CoA
AU
Median, IQR
p=0.033
Healthy
HTN
HFpEF
0
10000
20000
30000
40000
50000
Acetyl-Carnitine
Nanomolar
p=0.028
Median, IQR
Figure 5 – Intramuscular metabolites in SkM biopsy 
samples. Biopsy samples were taken from HFpEF (n=11), HTN 
(n=9), and Healthy (n=11) SkM and subjected to MS-based 
analysis. Propionyl-CoA and acetyl-carnitine were lower in 
HFpEF SkM. 

 
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Despite these potential benefits, EMPA did not improve 6-minute walk distance in EMPERIAL-
PRESERVED. As illustrated above, SGLT2i have several metabolic effects that could translate into an 
ergogenic benefit, specifically in HFpEF patients. Despite this, results from the EMPERIAL-PRESERVED study 
showed that 12 weeks of EMPA did not improve six-minute walk (6MW) distance in 315 symptomatic HFpEF 
patients.(133) It is certainly possible that the choice of endpoint may have played a role in masking an 
ergogenic benefit, especially considering that SGLT2i have been shown to improve peak VO2 in HFrEF,(134) 
yet also did not increase 6MW in this patient population.(133) While 
the 6MW test is convenient, its administration must be rigorously 
standardized,(135) as different degrees of encouragement influence 
the test.(136) Additionally, the 6MW is self-paced and impacted by 
patient motivation, yet provides no objective metric of effort.(135) 
Moreover, and key to this proposal, there are other physiologic 
reasons, centered on nitric oxide bioavailability, that potentially could 
explain why EMPA did not yield an ergogenic benefit.  
Nitric oxide bioavailability may be needed to unlock the benefits 
of SGLT2i in HFpEF. We,(137, 138) as well as others,(139, 140) 
have demonstrated impaired nitric oxide (NO) bioavailability in 
HFpEF. In two studies, we demonstrated a reduction in serum nitric 
oxide metabolites (NOm) in HFpEF patients, compared to those with 
HFrEF and individuals without heart failure (Fig 6).(137, 138) 
Additionally, in a human myocardial biopsy study, HFpEF hearts had 
lower tissue NOm levels.(139, 140) We demonstrated that supplementation with inorganic nitrate, in the form of 
potassium nitrate (KNO3), significantly increased serum concentrations of NOm;(141, 142) other work showed 
that inorganic nitrate supplementation also increases muscle NOm.(143) While NO has many effects, this 
proposal will focus on 2 beneficial effects of NO that could specifically bolster the effects of SGLT2i: (1) the 
impact of NO on FAO; and (2) the impact of NO on SkM blood flow. 
Nitric oxide is required for efficient fatty acid oxidation. A central mechanism for the postulated ergogenic 
benefit of SGLT2i is an increase in FAO. However, efficient FAO requires nitric oxide. Previously, our Co-
Investigators demonstrated that S-nitrosylation, a NO-mediated post-translational modification, of proteins 
participating in FAO, markedly increased the rate of palmitate oxidation.(144) Moreover, mice deficient in 
endothelial nitric oxide synthase (eNOS-/-) lacked S-nitrosylation of FAO proteins, which coincided with reduced 
liver, heart, and SkM FAO.(145, 146) Treatment of eNOS-/- mice with NO-donors restored both FAO protein S-
nitrosylation and FAO efficiency.(146) With restoration of FAO, the physical activity of the eNOS-/- mice 
improved to approximate that of wild-type mice, suggesting an important link between FAO and physical 
activity.(146) In rats, nitrate supplementation has been shown to increase soleus SkM FAO by increasing the 
activity of key enzymes involved in fatty acid metabolism, such as 3-hydroxylacyl-CoA dehydrogenase, PGC-
1𝛼, and PPAR𝛼, and by decreasing malonyl-CoA content.(80, 147) These data demonstrate a central role 
for NO in efficient FAO, suggesting that without sufficient NO-signaling, SGLT2i cannot improve FAO 
to its full potential, hence limiting its ergogenic benefit. 
Figure 6 – Serum nitric oxide metabolites are 
lower in HFpEF. Comparison of NOm levels 
between subject without heart failure (HF) and 
subjects with HFpEF and HFrEF. From 
Chirinos…Zamani, JAHA 2016. 

 
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Increased nitric oxide bioavailability increases SkM blood flow during exercise, which is necessary to 
support greater FAO. Increased reliance on FAO would, of necessity, require greater oxygen delivery to the 
SkM, as more O2 is required to generate the same amount of ATP from fatty acids as compared to 
carbohydrates (e.g. lower P:O ratio for fatty acids).(148) This is all the more relevant as several studies have 
identified constrained blood flow to the SkM during both large(25, 149, 150) and small(151) muscle mass 
exercise in HFpEF patients. 
Because NO contributes to the 
exercise vasodilatory 
response,(152-156) we tested 
whether increasing NO 
bioavailability could improve 
SkM exercise blood flow. In a 
2x2 cross-over trial of 17 
HFpEF subjects, we found that 
a single dose of 12.9 mmol of 
inorganic nitrate, which can be 
reduced to NO in the hypoxic 
and acidic environment of 
exercising muscle,(157-160) 
led to significant improvements 
in the vasodilatory reserve 
during exercise, allowing for 
greater cardiac output, higher peak VO2, and higher ventilatory thresholds (Fig. 7).(141) Using near infrared 
spectroscopy on the calf muscle during exercise, we found a tendency for oxyhemoglobin to fall to a lesser 
degree following inorganic nitrate than following placebo (-11.3% vs. -15.8%, p=0.07), suggesting an 
improvement in exercise intramuscular perfusion with inorganic nitrate.  
These data suggest that combining inorganic nitrate with SGLT2i may uncover a metabolic and 
ergogenic benefit in HFpEF patients by increasing SkM blood flow and supporting increased rates of 
fatty acid oxidation during exercise. 
HFpEF patients remain extremely limited.(161) Even small improvements in exercise tolerance could allow 
patients to perform more activities of daily living, increasing quality of life and independence, and enhancing 
the likelihood that a patient engages in prescribed exercise training.(162) All of these factors can reduce the 
burden on the healthcare system. Thus, our study has the potential to impact the lives of millions of patients 
who currently suffer from this debilitating condition, without any effective pharmacologic treatment options. 
2.2.1 
Known Potential Risks with SGLT2i 
a. Empagliflozin –  
i. Overall considerations: In the EMPERIAL program, participants with HFpEF and HFrEF 
were randomized to Empa 10 mg po qd for 12 weeks versus PB.(133) The rates of 
premature medication discontinuation were no different between Empa and PB: 8.3% in 
PB, 9.7% with Empa in HFrEF, and 7% with Empa in HFpEF. There was no difference in 
the frequency of AEs, and serious adverse events (SAE) were less frequent with Empa. 
Similarly, “any adverse event” and “serious adverse events” were no different in the 
EMPEROR-Reduced study of HFrEF participants randomized to Empa versus PB.(59) 
Adverse events were also not more frequent in the dapagliflozin arm of the DAPA-HF trial 
in HFrEF participants.(51)  
ii. Genital infections: Empagliflozin has been studied in many clinical trials of diabetics and 
patients with heart failure (with and without diabetes). In the EMPA-REG OUTCOME trial, 
Figure 7 – Inorganic nitrate enhances the vasodilatory reserve in HFpEF subjects (n=17) 
allowing for higher cardiac output and peak VO2. Red line indicates mean difference. 

 
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7020 diabetic participants were randomized to 10 mg or 25 mg of Empa versus placebo. 
As described above, Empa reduced rates of cardiovascular outcomes including HF 
hospitalization, without significance dose heterogeneity.(42) Genital infections were more 
frequent in the Empa groups (6.4% vs. 1.8%). In EMPEROR-Reduced, a study in heart 
failure patients, the incidence of urinary tract infections was no different between Empa 
and PB (Empa 4.9% versus PB 4.5%); the incidence of genital infections was also no 
different (Empa 1.7% versus PB 0.6%).  
iii. Risk of Hypoglycemia and Diabetic Ketoacidosis (DKA):  
1. The EMPA-REG OUTCOME trial enrolled 7020 Type II DM to Empa versus PB. 
Hypoglycemic episodes requiring assistance (Empa 1.3% versus PB 1.5%) and 
diabetic ketoacidosis (DKA, Empa 0.1% versus <0.1% in PB) were no different 
between Empa and PB in the EMPA-REG OUTCOME Trial.(42)  
2. The DAPA-HF Trial enrolled 4744 participants with HFrEF to dapagliflozin 10 mg 
daily versus placebo; approximately 40% of participants had diabetes mellitus, 
and about half of study participants were on biguanide medications (e.g. 
metformin).(51) Hypoglycemia requiring the assistance of another person was 
extremely rare (0.2% in either group) and did not differ significantly between 
groups. Similarly, DKA was extremely infrequent, occurring in 0.1% of those 
randomized to Dapa and 0 of those randomized to PB.(51)  
3. In EMPEROR-Reduced, 3730 HFrEF participants were randomized to Empa 10 
mg daily versus placebo. Approximately 50% of participants had diabetes. In the 
Empa group, HbA1c was reduced by 0.28±0.03%, as compared to -0.12±0.03 in 
PB (Absolute difference of -0.16% in favor of greater reduction with Empa). 
Hypoglycemic events were defined as plasma glucose <=70 mg/dL or that 
required assistance. The overall incidence of hypoglycemic events was 1.4% in 
the Empa group as compared to 1.5% in the PB group. Looking at diabetic 
participants only, the incidence of hypoglycemic events was 2.2% in Empa 
versus 2.4% in PB; lower rates were seen in non-diabetics (0.7% in Empa versus 
0.6% in PB). Overall, the incidence of hypoglycemic events was no different 
between groups. There were 0 episodes of DKA throughout the trial.(59) 
4. Large meta-analyses of SGLT2i use in diabetics shows that while rare, there is an 
increased risk of DKA with SGLT2i use (~1 per 1000 person years for 
SGLT2i).(163) 
iv. Amputations: Earlier studies with canagliflozin, another SGLT2i, suggested an increased 
risk of amputations in diabetics.(44) Subsequent analyses showed that this was specific 
to Cana and not present for Empa or Dapa.(164, 165) A more recent study using Cana in 
diabetics with nephropathy, which instituted regular foot exams, did not identify an 
increased risk of amputation, though Cana did significantly reduce the risk of worsening 
renal function.(166)  
v. Dose considerations: Because the 10 mg dose of Empa has been shown to lower 
adverse events in both diabetics and HFrEF participants,(42) and lead to metabolic 
changes,(98) this dose will be used throughout this project. 
b. Potassium Nitrate – Inorganic nitrate has been tested in several human studies in HFpEF to 
date and is very well tolerated.(141, 142, 167) In our Dose-Finding study of 12 individuals with 
HFpEF, 9 were treated with KNO3 as we propose to do in this study.(142) KNO3 did not lead to 
clinically significant hypotension or methemoglobinemia.(142)  
i. Bp effects: There was a systolic blood pressure lowering effect (approximately 12 
mmHg),(142, 168) though we note that HFpEF patients generally have elevated blood 
pressures.(2)  

 
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ii. Other symptoms: 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,(169) and may be related to the potassium content, as potassium 
is known to cause GI side effects in clinical practice.(170)  
c. Potassium Chloride:  
i. GI side effects: The main side effect of potassium chloride in clinical practice are 
gastrointestinal symptoms.(169, 170) We will ask subjects to take study medications with 
food to minimize this.  
ii. Bp effects: Potassium itself also has an effect on the vasculature, lowering blood 
pressure (average of ~3/2 mmHg).(170, 171) 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 the combination of Empa + KNO3 has not been studies before and so data regarding additional 
adverse effects with the combination are not available. 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 with inorganic nitrate, and the data in the field regarding SGLT2i, support the conclusion that 
the supplementation strategies to be tested in HFpEF are rationale and target HFpEF-specific deficits. 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 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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3 
STUDY OBJECTIVES AND ENDPOINTS 
Aim 1: Compare the impact of Empa, without and without KNO3, on submaximal exercise endurance 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
Compare the impact on submaximal 
exercise endurance for Empa, with 
and without pharmacologic therapy 
aimed at increasing nitric oxide 
signaling (KNO3), 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 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 
SkM mitochondrial 
properties, O2 delivery, 
and exercise endurance. 
Through their impact on 
these mechanisms, our 
interventions may affect 
VO2 kinetics.  
Assess the impact of our intervention 
on VO2 efficiency 
VO2 Efficiency: Ratio of total work 
performed to total oxygen consumed 
above basal metabolic rate during the 
exercise study.(26, 172) 
VO2 efficiency reflects 
the muscle’s capacity to 
convert O2 into 
mechanical work.  
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 
compared to the SVR at 4 minutes of 
exercise. Aortic input impedance will 
also be determined to further 
characterize changes in pulsatile load. 
Both KNO3 and Empa 
may have vascular 
effects that could 
influence exercise, 
cardiac performance, 
and SkM blood flow and 
O2 delivery.  
 
Assess the impact of our interventions 
on venous substrate concentrations 
during exercise 
We will measure blood concentrations 
of substrates (e.g. glucose, lactate, non-
esterified fatty acids, and triglycerides) 
at baseline, 4 minutes of exercise, and 
at the time of exhaustion. Comparisons 
at 4 minutes of exercise will be the 
focus.  
Empa and KNO3 may 
alter SkM substrate 
metabolism during 
exercise. 

 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Assess the impact of our interventions 
on the respiratory exchange ratio 
We will also assess the respiratory 
exchange ratio, a metric of whole-body 
substrate metabolism, obtained from 
expired gas analysis. Comparisons at 4 
minutes of exercise will be the focus. 
Empa and KNO3 may 
alter SkM substrate 
metabolism 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 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. 
Tertiary 
 
 
Assess the impact of our interventions 
on intramyocardial filling pressure 
during submaximal exercise 
E/septal e’: Mitral early inflow velocity 
(E) to septal tissue Doppler velocity (e’) 
is non-invasive marker of 
intramyocardial filling pressures that 
correlates with invasively-derived 
metrics.(173, 174)  
Through multiple 
mechanisms, SLGT2i 
may reduce 
intramyocardial filling 
pressures at rest and 
with exercise, which 
could serve as another 
mechanism for improved 
exercise tolerance. 
AIM 2: Compare the impact of Empa, with and without KNO3, on SkM OxPhos and Intramuscular 
Perfusion using MRI 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
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 (half-time of 
CrCEST recovery), 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 altering 

 
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SkM energy fuel 
metabolism 
Secondary 
 
 
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 
capacity 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. 
AIM 3: Assess the impact of Empa, with and without KNO3, on SkM respiration, the metabolome, and 
the proteome 
Assess the impact of our interventions 
on muscle tissue respirometry, 
specifically FAO 
Ex vivo tissue measures of substrate 
metabolism. We will measure tissue 
rates of substrate (fat and carbohydrate) 
metabolism. Citrate synthase, a marker 
of mitochondrial content, will also be 
assessed. 
In addition to our 
CrCEST-based 
measures of SkM 
OxPhos, we will 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 fatty 
acid and ketone oxidation will be 
measured, as well as proteins related to 
mitochondrial biogenesis. 
We postulate that 
Empa+KNO3 will lead to 
greater improvements in 
FAO than either Empa 
alone or active control, 
as reflected by changes 
in the muscle proteome. 
As Empa may alter 
mitochondrial 
biogenesis, we will also 
measure proteins key to 
the process of 
mitochondrial expansion.  
Assess the impact of our interventions 
on the muscle metabolome 
Muscle Metabolome: Targeted 
quantitative metabolomics will be 
performed, including the acylcarnitine 
profile (specifically C4-OH carnitine, a 
marker of ketone metabolism, and 
acetylcarnitine [C2]), malonyl-CoA, and 
glycolytic intermediates 
Changes in substrate 
metabolism may be 
reflected in changes in 
substrate concentrations 
within the muscle.  

 
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4 
 STUDY PLAN 
4.1 
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) Empa; (C) Empa + KNO3. 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; Empa will be purchased by IDS and over-encapsulated to maintain blinding.  
While changes to the mitochondrial proteome occur early after the initiation of an exercise program (1-2 
weeks),(175, 176) the timing of mitochondrial biogenesis in humans is less well known, suggesting that the 
longer duration of therapy chosen in this study is warranted.(177) 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.(178, 179) Similar findings of 
decreased mitochondrial oxidative capacity were noted during a 12-day unilateral leg suspension 
protocol.(180) 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. 
4.2 
Scientific Rationale for Study Design 
HFpEF subjects suffer from multiple comorbidities that may impact exercise capacity.(181-183) 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.  
4.3 
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) Empagliflozin alone (4 pills daily) 
a. Empa 10 mg daily 
b. Potassium Chloride 6 mmol three times daily 
(2) Empagliflozin + Potassium Nitrate (4 pills daily)  

 
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a. Empa 10 mg daily 
b. KNO3: 6 mmol three times daily 
(3) Potassium Chloride alone (4 pills daily) 
a. KCl: 6 mmol three times daily 
b. Placebo: Placebo for Empa to be given once daily 
Both the KNO3 and the KCl will be made by the UPenn IDS, as we have done previously,(142) with 
pharmaceutical grade product obtained from a commercial supplier (e.g. Spectrum Laboratory Products 
(Gardenia, CA)). Empagliflozin will be purchased commercially by UPenn IDS and over encapsulated to 
maintain blinding. A similarly appearing placebo will also be made for Empagliflozin by UPenn IDS.  
 

 
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5 
STUDY POPULATION 
5.1 
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, defined as: no addition/removal/changes in 
antihypertensive medications or beta-blockers in the preceding 30 days and continuation of a stable 
diuretic regimen, if applicable 
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)(184, 185) 
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)(186) 
b. Mitral E/e’ ratio > 14(185) at rest or during exercise(186) 
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)(186, 187) 
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.(186)  
 
5.2 
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 program(2)) 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, 

 
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particularly when using a more specific technique (i.e., a negative perfusion imaging test following a 
‘positive’ ECG stress test) 
- 
Exercise-induced regional wall motion abnormalities on the echocardiographic 
assessment during the baseline maximal effort cardiopulmonary exercise test will also 
be 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 < 45 mL/min/1.73m^2. We note that while the FDA packing insert suggests a lower limit of 45 
mL/min/1.73 m2 for Empa, the EMPERIOR Reduced trial enrolled HFrEF participants with an eGFR 
>= 20 mL/min/1.73m2.(59)  
18. Methemoglobin > 5% 
19. Serum potassium > 5.0 mEq/L on baseline testing 
20. Type I Diabetes 
21. History of ketoacidosis 
22. Current use of, or prior intolerance to, an SGLT2i 
23. Ongoing maintenance of a ‘Ketogenic Diet’ (low carbohydrate, high fat) 
24. Allergy to beets 
25. Severe right ventricular dysfunction 
26. Baseline resting seated systolic blood pressure > 180 mmHg or < 100 mmHg 
27. Orthostatic blood pressure response to the transition from supine to standing (>20 mmHg reduction 
in systolic blood pressure 2-3 minutes after standing, or a fall in SBP to < 90 mmHg) 
28. Active participation in another study that utilizes an investigational agent (observational 
studies/registries allowed) 
29. 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 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. 

 
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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. 
5.3 
Lifestyle Considerations 
The following lifestyle changes will be requested during the study: 
 
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.(188, 189)  
 
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.(190-193) 
 
Subjects will be asked to avoid a ketogenic diet (high fat, low carb) or excessive alcohol.(194, 195) 
 
Subjects will be asked to withhold study medications during periods of fasting or illness that inhibit 
adequate oral intake, as these situations may increase the risk for DKA.(194, 195) 
5.4 
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 that 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. 
5.5 
Strategies for Recruitment and Retention 
5.5.1 
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.(25, 26, 141, 142, 196) 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 

 
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used by the Division of Cardiology and 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 
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 may conduct directed recruitment in settings where HFpEF 
patients are likely to present during 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. 
 
5.5.2 
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 (7 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. We plan to exclude participants who are unlikely to 
complete study visits due to non-compliance, psychiatric illness or major comorbidities that would impede 

 
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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.  
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 
Total: $500 
 
Participants will be paid via by Greenphire clincard after completing each visit. The study coordinator will keep 
track of patients using an electronic database, and we may build-in schedule reminders. We also maintain 
close communication via phone/email with previous and active research subjects to mitigate participants 
becoming lost to follow-up.  
5.6 
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, and the 3 endpoint assessments (submaximal exercise 
and CrCEST MRI studies) following each interventional period, 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 
5.7 
Study Intervention(s) Administration 
5.7.1 
Study Intervention Description 
The 3 arms to be tested during the supplementation portion of the study, and the target doses, are: 
(1) Empagliflozin alone (4 pills daily) 
a. Empagliflozin 10 mg daily  
b. Potassium Chloride: 6 mmol three times daily 
(2) Empagliflozin + Potassium Nitrate (4 pills daily) 
a. Empagliflozin 10 mg daily  
b. KNO3: 6 mmol three times daily 
(3) Potassium Chloride (4 pills daily) 
a. KCl: 6 mmol three times daily 
b. Placebo: A placebo for Empagliflozin will be taken once daily 
 
5.7.2 
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) Empagliflozin alone 
a. Empagliflozin 10 mg daily  
b. Potassium Chloride: 6 mmol twice times daily 
(2) Empagliflozin + Potassium Nitrate  
a. Empagliflozin 10 mg daily  
b. KNO3: 6 mmol twice times daily 
(3) Potassium Chloride 
a. KCl: 6 mmol two times daily 
b. Placebo: A placebo for Empagliflozin will be taken once daily 

 
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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 in the supine 
position. 
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 and without another identifiable 
cause (for instance, if a concurrent GI illness is ongoing, study medications can be paused until the participant 
recovers, before restarting with close contact/instruction from the study team). In cases in which home 
monitoring suggests resting or orthostatic hypotension, the study subject may be encouraged to come to 
UPenn for a formal assessment.  
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) Empagliflozin alone 
a. Empagliflozin 10 mg daily  
b. Potassium Chloride (KCl): 6 mmol three times daily 
(2) Empagliflozin + Potassium Nitrate  

 
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a. Empagliflozin 10 mg daily  
b. Potassium Nitrate (KNO3): 6 mmol three times daily 
(3) Potassium Chloride 
a. Potassium Chloride: 6 mmol three times daily 
b. Placebo: A placebo for Empagliflozin will be taken once daily 
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 
Empagliflozin 
10 mg daily 
10 mg daily 
KNO3 
6 mmol twice daily 6 mmol three times daily 
KCl 
6 mmol twice daily 6 mmol three times daily 
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 
5.0 mEq/L, or an eGFR < 45 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 because 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. Chronic use of a potassium-sparing diuretic (e.g. amiloride, spironolactone, triameterene) 
 
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/medication alteration is < 5.0 mEq/L. 

 
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5.7.3 
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) Empagliflozin: Empagliflozin will be purchased by UPenn IDS from a commercial 
pharmaceutical supplier (e.g. Boehringer Ingelheim). Pills will be over-encapsulated to maintain 
blinding. A placebo of similar appearance and weight will be constructed by IDS. 
5.7.4 
Acquisition and accountability 
Both KNO3 and KCl will be prepared by UPenn IDS, as we have done previously.(142) 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. UPenn IDS will also purchase IDS from a commercial supplier. IDS will furnish 
the placebo capsule for Empagliflozin, which will be similar in color and weight the over-encapsulated 
Empagliflozin pill.  
5.7.5 
Formulation, Appearance, Packaging, and Labeling 
Potassium nitrate and potassium chloride will be obtained commercially (e.g. from Spectrum Laboratory 
Products) and encapsulated by the UPenn IDS. UPenn IDS will purchase Empagliflozin from a commercial 
supplier (e.g. Boehringer Ingelheim) and over-encapsulated the pills to maintain blinding. UPenn IDS will 
construct a placebo pill for Empagliflozin that will be similar in weight and appearance to the over-encapsulated 
Empagliflozin pill.  
5.8 
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 

 
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1 
ABC 
2 
ACB 
3 
BAC 
4 
BCA 
5 
CAB 
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; Empa alone in P2; Empa + KNO3 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=Empa alone, followed by C= Empa + KNO3. The study statistician will provide the sequence 
orders. UPenn IDS will assign the codes linked to the intervention and will provide study medication based on 
the sequence order to which the subject is randomized. For purposes of blinding, none of the study team will 
have access to the IDS-assigned codes. Furthermore, the study statistician will not release the sequence 
orders to the Study Team. If emergency unblinding is necessary, the investigators will access the treatment 
identity by contacting UPenn IDS and receive information regarding the specific participant’s active treatment 
at that time (and without other information as to the sequence). This is expected to be infrequent given the 
safety profile of the study medications. If time allows, any unblinding 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, that 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.  
5.9 
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, or UPenn IDS, 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. 
5.10 
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. 

 
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Subjects will continue all 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. 
 
Background Diabetic Medications and Diuretics 
Given the overall low incidence of hypoglycemic events (Reviewed in Section 2.2.1), the study protocol does 
not include plans to modify background diabetes therapy. Furthermore, given that SGLT2i did not increase the 
risk of volume depletion in large HFrEF studies (DAPA HF: Dapa 1.2% versus 1.7% in PB;(51, 197); 
EMPEROR-Reduced: Empa 10.6% versus PB 9.9%), the study protocol does not include plans to modify 
background diuretic therapy.  

 
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6 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT DISCONTINUATION/WITHDRAWAL 
6.1 
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 participant 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 or 
the telephone). 
6.2 
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.  
 
If a participant decides to withdraw from the study AFTER he/she has been randomized, this participant 
will be included in the intention to treat cohort (regardless of whether the participant took any study 
medication or not). 
 
If a randomized participant decides to withdraw from the study before taking any medication, as 
reported to a study coordinator and/or through return of their medication, the subject will be ‘replaced’ 
on the study by increasing the total number of subjects accrued to the study. The randomization 
sequence of the subject who withdraws will not be re-used, i.e., the next individual randomized will be 
assigned the sequence subsequent to that of the participant who withdrew. 

 
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6.3 
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. 
 
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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7 
STUDY ASSESSMENT AND PROCEDURES 
7.1 
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 
o Additional blood will be obtained for storage (plasma, serum, and whole blood)  

 
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 
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, 198) The Kansas City Cardiomyopathy Questionnaire 
(KCCQ) will be administered, and the KCCQ Overall Summary Score will be calculated and 
compared.(12, 198-200) 
 
Low-nitrate diet: We will provide educational information and a 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 may 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, may be assessed in the 4-chamber view. 
Tissue Doppler imaging may 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) may 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 may 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 may 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 metabolic cart (for example, 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.(25, 26, 141, 142) Electrocardiographic and vital sign monitoring will be performed during the 
test. Verbal encouragement will be given to encourage maximal effort.  
o Echocardiographic images may be obtained during the exercise tests. Lung ultrasound may be 
performed at rest and during exercise to identify B-lines, a marker of congestion.(201)   
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.  
o A pedal cadence of 60 RPMs will be targeted, 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 

 
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cadence >50 RPM for >10s, despite verbal encouragement.(202)  
 
We note that some individuals may not be able to maintain a cadence of 60 RPMs. In these 
instances, we will coach the subject to maintain the highest possible sustainable cadence over 
the course of the exercise test.  
 
o Note: Subjects will be excluded if the arterial saturation falls to < 90% on a reliable assessment 
during this maximal effort study or if significant exercise-induced segmental wall motion 
abnormalities develop on exercise echocardiography that are consistent with inducible ischemia. 
Given the high prevalence of ‘false-positive’ ECG stress tests in HFpEF,(203) 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 (0 Watts) 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.(202)  
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 W 95W; and 91W  90W) 
o Note: In participants who could not maintain a pedal cadence > 60 RPM during the maximal effort 
incremental study, the highest sustained cadence during the maximal effort study will be used 
during the submaximal exercise assessment. 
 
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.(202) 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 was previously used in HFpEF patients.(167) 
        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 

 
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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 
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;(142, 204-206) 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),(207) 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;  

 
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(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 approximately 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.  
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 blood and muscle nitrate(142, 208) 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: 1-2 venous catheters will be placed. 
 
Blood draw: 
o Comprehensive metabolic panel 
o Complete blood count 
o Storage of blood for later analysis, including 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.(209) Multiple 
passes through the same incision may be required to obtain sufficient muscle.  
o The following studies may be performed on muscle tissue: 

 
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 
Mitochondrial respiration – both isolated mitochondrial and permeabilized fiber 
preparations may 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 acylcarnitine profile, Acyl-CoA 
species (including malonyl-CoA), glycolytic intermediates, ketones, and nitrate/nitrite 
concentrations 
 
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 Blood Metabolite Concentrations: 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 (but not limited to):  
 
Glucose 
 
Lactate 
 
Non-esterified fatty acids 
 
Triglycerides 
 
Acylcarnitines  
 
Ketones  
Other metabolites/hormones/proteins, such as insulin and glucagon, may also be measured. 
o Aortic input impedance: Radial arterial tonometry and echocardiographic cardiac flow from the 
left ventricular outflow tract Doppler signal may 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 

 
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rate of oxygen consumption (VO2) will be assessed (VCO2/VO2) as a marker of systemic substrate 
metabolism.(148) Lower values (RER of 0.7) correspond to greater fatty acid oxidation, as 
compared to carbohydrate (RER of 1.0).(148, 210-212) The RER will be compared at 4 minutes 
of exercise and will be computed as the average value over a 30-second time period.(172)  
o VO2 kinetics – The rate of increase or decrease in oxygen consumption following the onset or 
cessation of exercise is related to SkM OxPhos.(156, 213-216) In HFpEF, VO2 kinetics are 
slowed,(26, 217, 218) 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.(219) 
o VO2 Efficiency: Because 75%PW will likely be above where lactate levels can be maintained at 
a steady state (above ‘critical power’), it is expected that subjects may evince an additional 
metabolic cost (‘VO2 slow component’) that will drive VO2 to maximal/peak rates of oxygen 
consumption during the submaximal execise studies.(220-222) By improving SkM blood flow, it 
is possible that the study interventions could decrease VO2 at the same work rate by reducing the 
slow-component. On the other hand, increased fatty acid oxidation could lead to greater oxygen 
consumption for the same work-rate due to the lower ATP-generated-to-O2-consumed ratio for 
fats as compared to carbohydrate. Net VO2 efficiency, defined as the ratio of total work performed 
to total oxygen consumed (above baseline metabolic rate) during the exercise study will be 
computed,(141, 142, 172) reflecting the muscle’s capacity to convert oxygen into mechanical 
work.(172)  
o Myocardial diastolic parameters and surrogate markers of congestion – Given that our 
interventions may impact arterial properties, which in turn, can impact myocardial 
performance,(223-229) 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.(230, 231) We may also interrogate echocardiographic estimates of RV function 
including the tricuspid regurgitant jet velocity(232) and the tricuspid annular plane systolic 
excursion.(233) Lung ultrasound may also be performed using echocardiography.(201, 233) 
These markers will primarily be compared at 4 minutes of exercise between interventions. 
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 different days following an 
intervention,(78, 87, 89, 91, 108, 143, 176, 208, 234-253) 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).(252)    
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 

 
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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 may 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 will be placed 
around the part of the body under investigation. The subject may 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 
two-way voice communication with the subject. The subject may 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 may 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 
exercise) may 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 to avoid 
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. Additional ancillary sequences may also be performed to 
gather additional information, if time allows.  
 
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(26) 
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 
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*
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.  

 
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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 or UPenn IDS to determine medication 
compliance. Study medications will then be returned to the UPenn IDS for disposal. 
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 an approximate 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 an ~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.  
 
7.2 
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.(254) These procedures will be performed by qualified 
personnel according to established American Heart Association guidelines.(255, 256) Non-
revascularized significant epicardial coronary disease, which may increase the risk of cardiovascular 
complications with exercise,(257) 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 because of the exercise test. Various other complaints, 
such as nausea, lightheadedness, and other aches and pains are also possible because 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,(257) during exercise 
tests. The risk of this happening is the same as if the participant were to exert him/herself during 
stressful situations or 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 

 
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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.(258, 259) If the arterial saturation falls to below 88% on a reliable assessment (“severe 
exercise induced hypoxemia”(259)), 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,(25, 26, 141, 142) and other parameters. 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.(201)  
 
3. Skeletal muscle vastus lateralis biopsy: Muscle needle biopsy with suction will be performed,(209) 
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 serious complications. We note that our protocol of pre-exercise muscle biopsies has 
been extensively employed in many prior physiologic studies.(108, 238, 240-249, 253, 260-266) 
Moreover, several studies have employed serial muscle biopsies either during one study visit or on  
different days following an intervention,(87, 89, 91, 108, 143, 176, 208, 234-250, 252, 253) 
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 
visit. 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 points following exercise (pre-exercise, 0.5 hours after exercise, 4 
hours after exercise, and 24 hours following exercise).(252) 
 
Other considerations specific to the muscle biopsy: 
 
Post-exercise muscle biopsy: In individuals who 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 

 
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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 TOPCAT(267, 268)), 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, 2012(269) 
 
 
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  7% average annual risk  0.02% daily risk  0.1% 5-
day risk  1:1000 risk of a thromboembolic event during the peri-biopsy 
period. 
 
With the subject’s consent, and possibly 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 <1.5.(269) If the INR is >1.5, we will attempt 
to reschedule the study visit, if possible, or forego the biopsy. 

 
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 
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.(209) 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 possibly 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 ester local anesthetic (such as 
tetracaine) or diphenhydramine.(270) These decisions will be made in discussion with 
Investigational Drug Services and the subject, and in light of the agents that the subject 
may have received previously for local anesthesia (e.g. during dental procedures or skin 
biopsies). 
 
4. Phlebotomy and blood draws: The CHPS unit in which the studies will be performed employs a 
research staff with 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): (values are approximate) 
 
 
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: 
 
 
 
 
Plasma: 15 mL  
 
 
 
Serum: 10 mL  
 
 
Submaximal 
Exercise 
  
 
 
 
Venous Blood Rest (0-min): 
 
Rest (0-min): 
Rest (0-min): 
 
 
Plasma: 20 mL 
Plasma: 20 mL 
Plasma: 20 mL 
 
 
Serum: 15 mL 
Serum: 15 mL 
Serum: 15 mL 
 
 
4-min: 
4-min: 
4-min: 
 
 
Plasma: 20 mL 
Plasma: 20 mL 
Plasma: 20 mL 
 
 
Serum: 15 mL 
Serum: 15 mL 
Serum: 15 mL 
 
 
Exhaustion: 
Exhaustion: 
Exhaustion: 
 
 
Plasma: 20 mL 
Plasma: 20 mL 
Plasma: 20 mL 
 
 
Serum: 15 mL 
Serum: 15 mL 
Serum: 15 mL 
 
 
 
 
 
Approx. Total: 
47 mL 
113 mL 
113 mL 
113 mL 
 
 
 
Grand Total: 
~386 mL 

 
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CMP = comprehensive metabolic panel, CBC = complete blood count, Coag = coagulation profile, Met-hgb 
= methemoglobin %. * = if subject is on warfarin and muscle biopsy is to be performed 
 
 
5. Dual Energy X-Ray Absorptiometry (DEXA): DEXA scanning may 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.(271) Participants will be instructed to never look directly into the 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 during the visit 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.(272) 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 because of completing the 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 require 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. 
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. 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 because 
of lying on the table in the MRI scanner with his/her leg relatively still in between bouts of calf 

 
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exercise. This is anticipated to be minor and should abate as the participant gets off the table and 
begins to walk around. Some individuals may experience lightheadedness upon sitting up from the 
table. We may ask participants to get up slowly and sit for a bit so that this dissipates. 
 
**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 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) Empagliflozin with potassium chloride; (b) Empagliflozin + potassium 
nitrate; (c) potassium chloride. Risks are described in Section 2.2.1. 
 
10. 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. 
 
7.3 
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 the event is 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. 

 
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• 
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 the research places subjects at greater risk than was previously known or recognized, AND  
(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 
affecting clinical status requiring hospitalization or prolongation of 
hospitalization. 
4 
Life-threatening or disabling 
5 
Fatal AE 
 
Classification of Adverse Events Regarding Severity Scale 

 
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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, data 
safety and monitoring plan, and/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. 
 
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.(142)  
The following are potential expected side effects of Potassium Chloride (KCl): 
1. GI upset 
2. Stomach discomfort 
3. Nausea 
4. Vomiting 

 
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The following are potential expected side effects of Empagliflozin: 
1. Urinary tract infections 
2. Genital mycotic infections 
3. Diabetic ketoacidosis 
4. Hypoglycemia 
5. Slight worsening of renal function (though we note that long-term studies in diabetics and in HFrEF 
participants show that SLGT2i are beneficial for renal function over the long term (59, 166, 273, 
274))  
6. Volume depletion 
7. Orthostatic hypotension 
8. Increased urination 
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 will be collected.  
 
AEs will be classified according to the guidelines/definitions specified above.  
 
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 decide 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 15 participants complete 
o 30 participants complete 
o 40 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. 

 
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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.  
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. 
8 
STATISTICAL CONSIDERATIONS 
8.1 
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; Empa + KCl in 
P2; Empa + KNO3 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 (Empa + KCl vs. Empa + KNO3), (Empa + 
KCl + KCl vs. KCl) separately using least squares regression, to minimize assumptions on the covariance 
matrix of the repeated measurements.(275) The regression model will be adjusted for period effects and for 
baseline submaximal exercise endurance.(275, 276) 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 (Empa + KCl or Empa + KNO3) 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.(167) 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.).(167) 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, venous substrate concentrations, mitochondrial respirometry and 
muscle proteome), we will follow the general strategy described above. In these exploratory analyses, in 

 
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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 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 be minimal, we 
will explore the impact of any missing data using multiple imputation.(276, 277) 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.(26, 278) 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 
Empa vs. Empa + KNO3, 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-1, HIF-1, NRF1/2, ERR, TFAM, MXI1, C-MYC, AMPK, ANT, and mTOR), 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,(26, 35, 279, 280) 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.  
9 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
9.1 
Regulatory, Ethical, and Study Oversight Considerations 
9.1.1 
Informed Consent Process 
9.1.1.1 
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. The informed consent may 
be sent to potential participants ahead of time to allow participants more time to review the material. 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 

 
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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. 
 
9.1.2 
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 
 
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. 
9.1.3 
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.  

 
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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.  
9.1.4 
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 which individuals with HFpEF may have and that could improve with 
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.  

 
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9.1.5 
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.  
9.1.6 
Clinical Monitoring 
A Data and Safety Monitoring Plan has been assembled for this study and is enumerated within a separate 
document.  
 
9.1.7 
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 
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. 
9.1.8 
Data Handling and Record Keeping  
9.1.8.1 
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. 

 
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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.  
9.1.8.2 
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. 
9.1.9 
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 
 
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 
9.1.10 
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.  
 

 
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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. 
9.1.11 
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. 
Of note, Dr. Chirinos, a Co-Investigator on the study team, is named as Inventor on a patent application that 
has been assigned to Penn regarding the use of potassium nitrate in the treatment of HFpEF. As a content 
expert in HFpEF and in arterial hemodynamics, areas of direct relevance to this proposal, Dr. Chirinos adds 
significant scientific value to the study team and will provide scientific input and advice on study matters 
(“Protocol Advisor”). Dr. Chirinos and his lab will assist in developing/refining software that he developed to 
model biologic signals (e.g. tonometry, echocardiography, and gas exchange data) and serve as a “Core Lab 
Director.” Hemodynamic/biologic signal analyses will be performed in a blinded manner and prior to data 
unblinding, negating the potential to introduce bias. His group will not provide ‘clinical’ reads on study data. Dr. 
Chirinos will not participate directly in enrollment or consenting subjects for this study. His involvement in the 
study and the potential for its financial implications will be enumerated in the informed consent document and 
will also be reviewed by the Conflict of Interest Standing Committee at the University of Pennsylvania.  
9.2 
Protocol Amendment History 
 
Version 
Date 
Description of Change  
Brief Rationale 
2 
8/20/21 
DSMB meeting plan updated to 
include additional visit 
Request of DSMB 
3 
9/28/21 
1)Exclusion criteria updated to 
exclude subjects on SGLT2i 
2)Clarified replacement 
procedure 
1) Request of DSMB 
2) Clarify ambiguity 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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10 APPENDIX  
10.1 
 Schedule of Activities (SoA) 
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 
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 
 

 
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Urine Pregnancy Test 
(if applicable)  
 
X 
X 
 
X 
 
X 
 
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 
Adverse Event 
Assessment 
 
X 
X 
X 
X 
X 
X 
X 
Complete Case 
Report Forms (CRFs) 
X 
X 
X 
X 
X 
X 
X 
X 

 
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