Nitrite Benefits to Mediate Fatigability in Older HFpEF Patients
Phase: PHASE2
Conditions: Heart Failure
Sponsor: Gladwin, Mark, MD

 
 
 
 
Nitrite benefits to mediate fatigability in older HFpEF patients 
 
 
 
 
 
 
 
 
 
Daniel E. Forman, MD 
Principal Investigator 
 
 
 
 
 
 
Professor of Medicine 
University of Pittsburgh Medical Center 
University of Pittsburgh School of Medicine 
Chair, Geriatric Cardiology Section 
 
 
 
 
NCT Number: 02918552 
 
Version 10.0 
 April 9, 2018 
 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 2 
TABLE OF CONTENTS  
PROTOCOL SYNOPSIS………………………………………………………………………….....3-9 
 
1. 
STUDY OBJECTIVE, SPECIFIC AIMS, BACKGROUND AND SIGNIFICANCE…..9-13 
 
1.1 OBJECTIVE…………………………………………………………………………….…..9 
 
1.2 SPECIFIC AIMS…………………………………………………………………………....9 
 
1.3 BACKGROUND………………………………………………………………………...9-10 
 
1.4 SIGNIFICANCE…………………………………………………………………….....10-13 
 
2. 
RESEARCH DESIGN AND METHODS……………………………………………...…13-35 
 
 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGN…………………………..…13 
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN………………………………....13-23 
 
 
2.2.1 TABLE OF STUDY VISITS AND ASSESSMENTS………………...……25 
 
2.3 STUDY DRUGS……………………………………………………………...............26-32 
 
2.4 STUDY ENDPOINTS………………………………………………………………... ….32 
 
2.5 STATISTICAL ANALYSIS………………………………………………………...….32-34 
 
3. 
HUMAN SUBJECTS…………………………………………………………………...…34-36 
 
 
3.1 SUBJECT POPULATION…………………………………………………………..……34 
 
3.2 INCLUSION CRITERIA…………………………………………………………..…. 35-36 
 
3.3 EXCLUSION CRITERIA………………………………………………………   ...……36 
 
4. 
IRB APPROVAL AND FDA AMENDMENTS……………………………………….....36-37 
 
5. 
RECRUITMENT AND INFORMED CONSENT PROCEDURES………………….....37-38 
5.1 RECRUITMENT METHODS………………………………………………………...37-38 
 
5.2 INFORMED CONSENT PROCEDURES………………………………………...….…39 
 
6. 
POTENTIAL RISKS AND BENEFITS…………………………………………………..39-51 
 
 
6.1 POTENTIAL RISKS………………………………………………………………......39-45 
 
6.2 ALTERNATIVE TREATMENTS………………………………………………….……...45 
 
6.3 POTENTIAL BENEFITS………………………………………………………………....46 
 
6.4 DATA SAFETY MONITORING… …………………………………………………...46-51 
 
6.5 RISK MANAGEMENT PROCEDURES………………………………………….....49-51 
 
7. 
COSTS AND PAYMENTS……………………………………………………………..........53 
 
 
7.1 COSTS……………………………………………………………………………………..53 
 
7.2 PAYMENTS………………………………………………………………………………..53 
 
8. 
QUALIFICATIONS AND SOURCE OF SUPPORT………………………………..….53-57 
 
 
8.1 QUALIFICATIONS OF THE INVESTIGATORS………………………………..….53-57 
 
8.2 SOURCE OF SUPPORT……………………………………………………………..….57 
 
9. 
REFERENCES………………………………………………………………………….…58-67 
 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 3 
 
PROTOCOL SYNOPSIS 
 
Protocol Title:
Nitrite benefits to mediate fatigability in older HFpEF patients
Protocol Number: 
 
IND 115926 
NCT Number:
02918552 
Version # and Date:
Version 10.0. / April 9, 2018
Clinical Phase:
Phase II clinical investigation
Investigational Drugs: 
Nitrogen 14 Sodium Nitrite (14N Sodium Nitrite) 
Trial Site:
University of Pittsburgh Single-Center Randomized Trial 
IND Sponsor: 
Mark T. Gladwin, MD 
Professor & Chairman of the Department of Medicine 
Director, Pittsburgh Heart, Lung, Blood and  
Vascular Medicine Institute 
Department of Medicine 
1218 Scaife Hall 
3550 Terrace St, Pittsburgh, PA  15261 
412-648-9636
Investigator: 
Daniel E. Forman, MD 
Professor of Medicine 
Chair, Geriatric Cardiology Section  
University of Pittsburgh 
3471 Fifth Avenue, Suite 500 
Pittsburgh, PA  15213 
Sub-Investigators: 
Jessica Bon Field, MD 
Mark T. Gladwin, MD  
Nancy Glynn, PhD 
Bret Goodpaster, PhD 
John Gorcsan, MD 
Hoby Hetherington, PhD, MS 
Gavin Hickey, MD 
Kara Hughan, MD, MHSc 
Theodore Huppert, PhD 
Manisha Jhamb, MD, MPH 
Andrea Levine, MD  
Anne Newman, MD, MPH 
Subashan Perera, PhD 
Michael Risbano, MD 
Adam Santanasto, MPH, PhD 
Sruti Shiva, PhD  
Marc Simon, MD, MS 
Frederico Toledo, MD 
 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 4 
Study Monitor: 
Mark T. Gladwin, MD 
Chairman, Department of Medicine 
University of Pittsburgh School of Medicine 
3550 Terrace St. - 1218 Scaife Hall 
Pittsburgh, PA  15261 
Research Facilities: 
UPMC Montefiore Hospital (UPMC Montefiore) Clinical 
Translational Research Center (CTRC) 
3459 Fifth Ave. 6 North 
Pittsburgh, PA  15213 
 
UPMC Montefiore Endocrinology and Metabolism Research 
Center (EMRC) 
3459 Fifth Ave. NW 823 
Pittsburgh, PA  15213 
 
UPMC Presbyterian Hospital (UPMC Presbyterian) 
 
Cardiac Catheterization Lab  
 
Magnetic Resonance Research Center 
200 Lothrop St 
Pittsburgh, PA  15213 
 
University of Pittsburgh Pepper Center–SMART Center 
(Senior Mobility Aging Research Training) 
3471 Fifth Ave. Suite 1200 
Pittsburgh PA  15213 
 
University of Pittsburgh Vascular Medicine Institute 
Biomedical Science Tower E1240 
200 Lothrop St. 
Pittsburgh, PA  15261 
Clinical Laboratories: 
UPMC Presbyterian Hospital 
3477 Euler Way 
Pittsburgh, PA  15213 
Study Rationale: 
Heart failure (HF) is epidemic with aging and prevalence of HF 
is steadily increasing1 as the population of older adults expands. 
While age stands out as a leading risk factor for HF incidence 
as well as for poor HF prognosis2, few HF trials focus specifically 
on aging physiology as a key determinant of the disease and as 
a pertinent therapeutic target. Consistently, HF trials have 
tended to focus primarily on central mechanisms of cardiac 
pumping dysfunction3 despite the fact that HF-outcomes are 
strongly related to functional decrements that are largely 
mediated 
by 
age4 
and 
disease-related5 
peripheral 
manifestations. HF-related skeletal muscle myopathy is a 
component of HF that diminishes physical function, and which 
is likely exacerbated by sarcopenia, vascular stiffening, and 
other aspects of aging such that exercise intolerance is 
disproportionate among older HF populations as well as its 
insidious clinical implications6.  Fatigability is rooted in HF with 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 5 
preserved ejection fraction (HFpEF) pathophysiology, and while 
it often determines a patient’s experience of his/her disease, it 
has not been addressed as a key part of management. We 
propose a prospective, randomized, controlled, double-blinded 
trial to study the utility of nitrite (NO2) therapy in older (age ≥70 
years) HFpEF patients. Subjects (N=18) will be assessed at 
baseline and after 4 weeks of NO2 therapy. Thus we will study 
the utility of NO2 therapy as a highly innovative means to modify 
fatigability. We hypothesize that NO2 therapy will reduce 
perceived and performance fatigability indices and improve 
peak oxygen uptake (VO2) in HFpEF patients and that 
decreased fatigability indices will correspond to increased daily 
activity as measured by actigraphy. We will also clarify the 
mechanisms by which NO2 benefits are mediated. We also 
hypothesize that the primary mechanism by which NO2 
decreases fatigability is improved mitochondrial bioenergetics 
as measured by in vivo 31P Magnetic Resonance Spectroscopy 
(MRS) and in vitro mitochondrial respiration and Adenosine 
Triphosphate (ATP) generation. Thus, among HFpEF patients 
with pulmonary hypertension, NO2 additionally mitigates 
fatigability by reducing pulmonary pressures and improving right 
ventricular-pulmonary artery coupling. Secondary aims will not 
only elucidate important NO2 properties, but also catalyze 
broader focus on the principles of patient-centered care by 
aligning a mechanistic perspective regarding NO2 benefits and 
targets for therapy with the patient-centered experience of 
fatigability. 
Study Objectives: 
Studies have demonstrated NO2 therapy increases ATP 
synthesis in skeletal muscle mitochondria concomitant with 
reduced whole-body oxygen cost during steady state (SS) 
exercise7. Our own work has demonstrated safety and efficacy 
of a FDA-IND approved sodium nitrite (10 mg and 20 mg) 
capsule, and its utility to upregulate the NAD-dependent 
deacetylase sirtuin-3 adenosine monophosphate (SIRT3-AMP) 
pathway of skeletal muscle of younger HF patients8. It now 
seems exceptionally logical and opportune to: 
 
apply these insights to older HF patients 
 
delineate mechanisms of disease and aging that 
respond to NO2 therapy  
 
delineate indices of perceived and performance fatigue.
 
Study Hypothesis: 
1. We 
hypothesize 
that 
NO2 
therapy 
will 
reduce 
performance and perceived fatigability indices and 
improve peak VO2 in HFpEF patients as measured by 
cardiopulmonary exercise testing (CPET).  
2. We hypothesize that decreased fatigability indices will 
correspond to increased daily activity as measured by 
actigraphy. 
3. We hypothesize that the primary mechanism by which 
NO2 decreases fatigability is improved bioenergetics as 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 6 
measured by in vivo 31P MRS and in vitro mitochondrial 
respiration and ATP generation. 
4. We hypothesize that among HFpEF patients with 
pulmonary hypertension, NO2 additionally mitigates 
fatigability by reducing pulmonary pressures and 
improving right ventricular-pulmonary artery coupling.  
 
Study Design: 
This is a prospective, randomized, double blind controlled trial 
of oral sodium nitrite vs. placebo in adults (n=18), ≥70 years of 
age with HFpEF. 
 
Subjects who meet the Inclusion Criteria and none of the 
Exclusion Criteria will receive oral sodium nitrite or placebo for 
4 weeks. We will stratify dosing as below: 
Drug:   
a. oral formulation of sodium nitrite 40 mg/placebo three 
times daily (tid), once in the morning and again in the early 
afternoon and again in the evening, approximately 6 hours 
apart in subjects age 70 to <80 years with low  risk for 
hypotension 
 
b. oral formulation of sodium nitrite 20 mg/placebo three 
times daily (tid), once in the morning and again in the early 
afternoon and again in the evening, approximately 6 hours 
apart in subjects ≥80 years of age, or subjects age 70 to 
<80 years with risk factors for hypotension 
 
 
 
Subjects will be evaluated as outpatients with the first dose of 
study drug/placebo. Blood pressure (BP), heart rate (HR), 
respiratory 
rate 
(RR), 
oxygen 
saturation 
(SpO2), 
methemoglobin % (MetHb) and plasma NO2 will be measured at 
baseline and during the first two hours after dosing. Frequent 
safety monitoring will occur during the time subjects are on drug 
as well as after completion of the post drug assessments, by 
phone and in clinic to assess adverse events (AE), interval 
histories, and medication compliance and to monitor NO2 and 
methemoglobin levels. 
Study Aims 
Aim 1:  To complete a randomized, placebo-controlled trial in 
HFpEF patients aged ≥70 years to assess benefits of 1 month 
of NO2 vs. placebo therapy to reduce perceived and 
performance fatigability and to improve aerobic functional 
capacity. 
Aim 2:  To delineate factors which underlie fatigability and the 
utility of NO2 to modify them. 
 
Planned Sample Size:  
18 subjects:  9 sodium nitrite and 9 placebo 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 7 
Duration of Treatment: 
4 weeks plus range 2-3 weeks for post drug outcome 
assessments 
Major Inclusion Criteria:  
Age ≥70 years 
 
Diagnosis of HFpEF [adapted from the 2016 European 
Society of Cardiology (ESC) Guidelines 
(http://eurheartj.oxfordjournals.org/content/37/27/2129#sec-
12)] to include: 
1. Prior diagnosis of HF via one of these: 
 medical record diagnosis by attending cardiologist 
 verbal confirmation of HFpEF with attending cardiologist 
 PI review of medical record to confirm HFpEF 
AND  
2. EF% ≥40 
 
Clinically stable (euvolemic; baseline HR <100 bpm) and 
without hospitalization or invasive cardiac procedure for 6 
weeks 
 
Patients using 81 milligram (mg) aspirin (ASA) will be 
eligible, but will be asked to hold the medication for 3 days 
prior to biopsy. This technique has previously been used 
with consistent safety. Patients will also be asked to avoid 
non-steroidal anti-inflammatory medications (NSAIDs) for 2 
days prior to the biopsy. 
 
Patients using anti-thrombin and anti-platelet therapy will 
plan to modify prior to muscle biopsies individually in 
coordination with the participant's primary cardiologist. 
Major Exclusion 
Criteria: 
 
Allergy to lidocaine 
 
BP >180/95 or <100/60 mm Hg (either systolic or diastolic) 
 
Anemia: Hgb <11.0 (♂),10.0 (♀) gm/dl 
 
Dementia or inability to give informed consent 
 
End-stage malignancy 
 
Severe orthopedic exercise limitation 
 
Use of chronic oral corticosteroids or other medications that 
affect muscle function. 
 
Chronic alcohol or drug dependency.   
 
Any bleeding disorder that would contraindicate biopsy such 
as history of clinically significant bleeding diathesis (e.g., 
Hemophilia A or B, Von Willebrand's Disease or congenital 
Factor VII deficiency). 
 
Psychiatric hospitalization within the last 3 months 
 
Major cardiovascular event or procedure within the prior 6 
weeks 
 
HF secondary to significant uncorrected primary valvular 
disease (except mitral regurgitation secondary to left 
ventricular dysfunction). If valve replacement has been 
performed, patient may not be enrolled for 12 months after 
this procedure.

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 8 
 
Severe uncorrected primary valvular heart disease (if valve 
replacement has been performed, patients will not be eligible 
for at least 12 months) 
 
Mechanical valve replacement requiring warfarin 
 
Severe peripheral or pulmonary artery disease 
 
Currently taking clopidogrel for a recent stent placement 
and/or a complex atherosclerotic lesion such that holding 
clopidogrel creates disproportionate risk.  
 
Current use of organic nitrates or phosphodiesterase type 5 
inhibitors (PDE5s)  
 
Unable to hold warfarin or use bridging therapy, or to hold 
aspirin for 3 days (81 mg) or (325 mg), prior to muscle biopsy 
or thienopyridine medications for 5 days prior to muscle 
biopsy. 
 
Subjects with diabetes whose HgbA1c >10.0% 
 
Other chronic unstable disease such as active neoplasm, 
end stage chronic kidney, liver or other organ disease  
 
Relative Exclusions 
 
Subjects with a non- 3T MRI compatible pacemaker or 
implantable cardio defibrillator or identified to have other 
metal in their body will be excluded from the Magnetic 
Resonance Spectroscopy study. 
 
Subject who use PDE5s for erectile dysfunction and are 
willing to withhold use 24 hours prior and during entire 
dosing period may be enrolled. 
 
Subjects may choose not to do the pre- and post- right heart 
catheterization exercise test. 
Study Endpoints: 
Overall, study endpoints pre- and post- nitrites will include:  
1. Measures of physical function as measured by non-
invasive CPET (nCPET) aerobic capacity, SS fatigability 
assessment, 6 minute walk test (6MWT), near infrared 
spectroscopy (NIRS) assessments of blood flow, Short 
Physical Performance Battery (SPPB), handgrips, 
accelerometry and invasive CPET (iCPET): 
a. gait speed 
b. strength (endurance, power) 
c. balance 
d. pulmonary pressure and ventricular-pulmonary 
artery coupling 
 
2. Skeletal muscle bioenergetics:  spectrophotometric, 
mitochondrial respiration, polymerase chain reaction 
(PCR) including ribonucleic acid (RNA) isolation, protein 
isolation, electron microscopy     
 
3. Serology:  inflammatory peptides (interleukin (IL) 1, IL-6, 
IL-15, TNFα; adipokines (leptin and adiponectin); vitamin 
D, glomerular filtration rate (GFR), brain natriuretic 
protein (BNP) or N-terminal pro b-type natriuretic peptide 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 9 
(NT-proBNP), thyroid stimulating hormone (TSH), 
glycosylated 
hemoglobin 
(HgbA1c) 
and 
platelet 
bioenergetics [using Seahorse Extracellular Flux (XF) 
analysis]9, i.e., including glycolytic as well as basal and 
maximal respiratory rates.      
 
4. Additional secondary factors include Quality of life (QoL) 
and related indices:  questionnaires that include 
measures of sleep, depression, pain, cognitive function, 
physical activity, co-morbidities, cardiac self-care 
efficacy, and fatigability, HF QoL, via the Kansas City 
Cardiomyopathy Questionnaire (KCCQ), nutrient intake, 
and physician assessment of frailty. 
  
 
 
1. 
STUDY OBJECTIVE, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE 
 
1.1 OBJECTIVE 
 
Studies have demonstrated NO2 vs.placebo therapy increases ATP synthesis in skeletal muscle 
mitochondria concomitant with reduced whole-body oxygen cost during SS exercise7. Our own 
work has demonstrated safety and efficacy of a FDA-IND approved sodium nitrite (10 and 20 
mg) capsules, and its utility to upregulate the SIRT3-AMP pathway of skeletal muscle of younger 
HF patients8. It now seems exceptionally logical and opportune to apply these insights to older 
HF patients and to delineate mechanisms of disease and aging that respond to NO2 therapy. 
 
1.2 SPECIFIC AIMS 
 
Aim 1: To complete a randomized, placebo-controlled trial in HFpEF patients aged ≥70 years to 
assess benefits of 1 month of NO2 vs. placebo therapy to reduce perceived and performance 
fatigability and to improve aerobic functional capacity. 
 We hypothesize that NO2 therapy will reduce perceived and performance fatigability indices 
(rated perceived exertion) and improve peak oxygen uptake (VO2) in HFpEF patients  
 We hypothesize that decreased fatigability indices will correspond to increased daily activity 
as measured by accelerometry. 
Aim 2: To delineate factors which underlie fatigability and the utility of NO2 to modify them. 
 We hypothesize that the primary mechanism by which NO2 decreases fatigability is improved 
bioenergetics as measured by in vivo 31P MRS and in vitro mitochondrial respiration and ATP 
generation. 
 We hypothesize that among HFpEF patients with pulmonary hypertension, NO2 additionally 
mitigates fatigability by reducing pulmonary pressures and improving right ventricular-
pulmonary artery coupling.  
 
1.3 BACKGROUND AND RATIONALE 
HFpEF:  Today’s large and rapidly growing population of old adults is intrinsically susceptible to 
age-related HFpEF10. There are no effective treatments for what is becoming an epidemic10-12. 
High mortality and morbidity, exercise intolerance, fatigability, diminished QoL and spiraling 
healthcare costs are typical consequences12-13. Pharmacological interventions that targeted 
ventricular myocardial relaxation (lusitropic) abnormalities failed to show anticipated mortality 
and morbidity benefits14-15. More recent studies indicate that systemic inflammation16 and its 
downstream effects may be a more fundamental basis of HFpEF17. Thus, it stands out that 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 10 
several small exercise training (ExT) trials for HFpEF demonstrate improved aerobic exercise 
capacity18-20, as it infers that ExT may provide related therapeutic benefits21, 23. Associated 
analyses show that the ExT functional gains were achieved primarily by improvements in 
peripheral oxygen utilization20-22, and are substantiated by changes in skeletal muscle (i.e., 
increased proportions of type I relative to type II fibers)17, 22, analogous to exercise-induced shifts 
in gene expression that have been demonstrated in HF with reduced ejection fraction (HFrEF)24, 
25.  
 
Yet such compelling benefits of pilot ExT HFpEF trials were only demonstrated after months of 
moderate to high intensity exercise regimens, regimens that are unfeasible for most HFpEF 
patients. We propose there are intrinsic physiological components of HFpEF pathophysiology 
that predispose to “fatigability”, a concept we further delineate as “perceived fatigability” 
measured by rate of perceived exertion (RPE) during a steady-state walking stimulus and 
“performance fatigability” (deterioration in a self-selected walking speed over time)26 during the 
6 minute walk test (6MWT). Both perceived and performance fatigability indices are submaximal 
activity-based metrics, and are significantly more clear and reliable than “fatigue” assessed 
qualitatively. Overcoming fatigability is an important HFpEF therapeutic goal. 
 
The large randomized trial Heart Failure (HF): A Controlled Trial Investigating Outcomes of 
Exercise Training (HF-ACTION) provides critical perspective regarding relevance of fatigability26 
in subjects with HFrEF. Despite multiple steps to bolster adherence, poor compliance and 
attrition were substantial. High fatigability was a common barrier to adherence and compliance 
27, 28. Investigators of this proposal are at the forefront of the inorganic nitrate (NO3) and NO2 salts 
therapeutics field, and are pursuing pioneering studies to show safety and utility of oral NO2 29, 
30. Related studies have demonstrated acute benefits of NO3 and NO2 in respect to improving 
exercise efficiency; after ingesting NO3, VO2 lessens for an equivalent workload7, 31. Our pilot 
work shows safety and efficacy of chronic NO2 therapy. In a 1-year, randomized, controlled, 
double-blinded trial, we propose to study efficacy of 4 weeks of NO2 capsules to reduce 
fatigability and increase VO2 in older (≥70 years) HFpEF patients and to comprehensively 
delineate the mediating mechanisms by which these benefits are achieved. 
 
This proposal is significant and innovative in multiple respects. First, it focuses on HFpEF, a 
disease which is endemic with aging and which constitutes one of today’s foremost healthcare 
challenges for the growing population of older adults. Second, it focuses on fatigability as a 
relevant dimension of HFpEF management. Fatigability is a particularly important therapeutic 
concept as it is rooted in HFpEF pathophysiology, and it often determines a patient’s experience 
of his/her disease. No matter how compelling the rationale for ExT, implementation is unlikely 
unless intrinsic fatigability is mitigated. Third, it assesses the novel concept of NO2 therapy as a 
means to modify fatigability. The application of accelerometry to gauge changes in daily activity 
in relation to NO2 is also novel and formative. Fourth, the proposal is significant as it aims to 
clarify mechanisms that mediate NO2 benefits, including assessments of skeletal muscle as well 
as right and left heart vascular flow dynamics. Not only will such comprehensive methodology 
clarify NO2 physiologic utility, but it advances the principles of patient-centered care by clarifying 
mechanisms that determine a patient’s day-to-day experiences of their disease. Systemic, 
cellular, and subcellular mechanisms of fatigability will be delineated, providing insights that will 
help reorient care towards what a patient feels.   
 
1.4 SIGNIFICANCE 
 
Fatigability as a novel clinical metric:  While investigators have focused on HFpEF 
pathophysiology in terms of mortality, morbidity, and other traditional clinical endpoints, there 
has been negligible consideration of fatigability as an important clinical target. To some extent, 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 11 
this reflects inherent ambiguity and unreliability of “fatigue” as a qualitative index. This study is 
innovative in utilizing clearer-cut performance-based metrics of perceived and performance 
“fatigability”32, 33. This clarity of definition and assessment facilitates the application of fatigability 
as a benchmark with which to reliably assess physiological changes achieved using NO2. 
 
Eldahah34 emphasized the importance of fatigability as a vital dimension of care. High fatigability 
predicts reduced physical activity (i.e., people curtail or stop exercise if it is too tiring) 35 as well 
as increased disability and death36. High fatigability is also associated with slower walking 
speeds that entail relatively greater percentages of peak exercise capacity37. Co-I Glynn 
advanced fatigability as a reliable metric in healthy adults32, 38. In this study we apply these 
concepts to a disease state.   
 
Fatigability and HFpEF:  HFpEF is typically characterized in relation to cardiac, vascular, 
chronotropic abnormalities, and skeletal muscle abnormalities21. Fatigability may serve as an 
important integrative metric. Kitzman demonstrated HFpEF’s impact in skeletal muscle, including 
reduced type I aerobic fibers relative to type II39. Age-related sarcopenia also entails significant 
atrophy of type II fibers40 with aggregate effects of reduced muscle mass and abnormal muscle 
function41. Muscle contraction depends on adenosine triphosphate (ATP) generation from 
mitochondrial oxidative phosphorylation, a process that relies on nutrition and cellular respiratory 
capacity as well as oxygen (O2) delivery (contingent on vascular flow and the microcirculation). 
When aerobic metabolism is insufficient to meet energy demands for physical activity, aerobic 
capacity diminishes (phosphocreatine [PCr] depletion, adenosine diphosphate [ADP] 
accumulation) and anaerobic glycolysis is activated (with lactic acid accumulation). Diminished 
PCr and increased lactic acid have both been linked directly with muscle performance 
fatigability42 and to increased minute ventilation (VE) as the means to disperse increased skeletal 
muscle carbon dioxide (CO2) production. Fatigability may relate to both insufficient energy and/or 
the extra work of ventilation. Furthermore, fatty infiltration of muscle is also common among older 
healthy42 and HFpEF43 populations, with associated increases in exercise intolerance and 
fatigability. Related studies in healthy elderly show age-related decreases in the capacity for 
oxidative phosphorylation44-47. This may have important functional consequences as 
mitochondria produce over 90% of ATP needed for movement and lower mitochondrial function 
is related to higher fatigability47-50. 
 
Other HFpEF features compound fatigability risks. Central factors22, 51, 52 include insufficient 
contractile reserve, pulmonary hypertension (PH), chronotropic incompetence, and increased 
pulmonary dead space. Peripheral factors22, 53, 54 include endothelial dysfunction, reduced 
microvasculature, central vascular stiffening and ergoreceptor activation. Systemic factors 
include inflammation55-57, nutrition (diet and absorption), vitamin K, sleep (changes in sleep 
quality, increased nocturia and sleep apnea), and comorbidity (e.g., diabetes, chronic kidney 
disease, hypertension, anemia, depression). Relevant covariates are factored in this analysis. 
 
Oral NO2 salts as a novel therapy to treat HFpEF: This study is particularly innovative in applying 
inorganic NO2 salt to treat HFpEF. Novel factors include:  (i) Direct NO2 benefits on skeletal 
muscle in HFpEF; (ii) NO2 benefits on pulmonary and systemic vascular coupling and exercise 
responses in HFpEF; (iii) Utility of chronic NO2 therapy in a capsule form (as compared to NO3 
ingested in foods, inhaled as gas, or infused).  
 
Nitric oxide (NO) is known to regulate multiple physiological processes pertinent to exercise 
capacity and overall health29, 31, 58-60. Nitric oxide synthase (NOS) is an endogenous enzymatic 
source of NO. Dietary NO3 is an alternate source of NO. Leafy green vegetables and beetroot 
contain high content of NO3. NO3 is reduced to NO2 by bacteria in the saliva, absorbed into the 

Oral Nitrite for fatigability in HFpEF   
 
 
Sponsor: Mark T. Gladwin, MD 
_____________________________________________________________________________ 
 
Version 10.0, April 9, 2018 12 
blood, and then circulating NO2 is reduced to NO, particularly in hypoxic conditions, and functions 
in multiple vascular and metabolic controlling roles61.  
 
Studies in young healthy subjects have demonstrated benefits of NO3 in respect to improving 
exercise efficiency31, 60. VO2 is reduced for an equivalent workload31. Larsen compared healthy 
men before and after dietary NO3 supplements31. Gross efficiency increased from 19.7 ±1.6 to 
21.1±1.3%, with no differences in HR, lactate, VE or the respiratory exchange ratio (RER) 
indicating this was mediated purely as an intrinsic skeletal muscle change. These analyses also 
clarify the role of enhanced mitochondrial efficiency with NO2-mediated performance gains. 
Mechanistically, this may be due to decreased mitochondrial proton leak with the adenine 
nucleotide translocase being a potential protein target16. Gladwin has significantly advanced 
insights regarding NO2’s therapeutic potential29-30,61-62; in studies of patients with metabolic 
syndrome and hypertension, oral NO2 was well tolerated and upregulated skeletal muscle SIRT3, 
mitochondrial SIRT3 and AMP-activated protein kinase (AMPK) in HFpEF-PH8. Related benefits 
include improved glucose uptake (mediated by improved Glucose transporter type 4 [Glut4] 
expression)68. Related literature highlights disproportionate benefits of dietary NO3 in type II 
skeletal muscle fibers58, implying the conceptual utility for older adults prone to sarcopenia-
associated Type II fiber atrophy. Benefits are greater in those not already optimally fit (which 
seems well-suited to most older adults)58.   
 
Co-I Simon demonstrated utility of aerosolized NO2 in 6 HFpEF patients (age 65±8). All patients 
tolerated NO2 well. Acute hemodynamic effects of NO2 included significant decreases in the right 
atrium (RA), right ventricle (RV), pulmonary artery (PA) and pulmonary capillary wedge 
pressures (PCWP).* (Presented at American Thoracic Society, 2016 Meeting). These findings 
reinforce Borlaug’s recent study of infused NO2 in HFpEF patients aged 69±6 years 103. Here too, 
NO2 was well-tolerated and also showed significantly improved acute exercise hemodynamics. 
Borlaug concludes with a strong mandate to implement clinical trials incorporating chronic NO2 
therapy (such as we now propose). 
 
In order to clarify muscle vs. vascular-mediated NO2 benefits, this protocol includes thorough 
muscle and vascular assessments. NO2 effects on skeletal muscle mitochondrial bioenergetics 
will be measured using in vivo MRS and near infrared spectroscopy (NIRS) as well as ex vivo 
analysis of biopsy specimens using high-resolution respirometry and other analyses. In addition, 
a subset of the sample will undergo right and left heart vascular flow dynamics using invasive 
CPET (iCPET) and echocardiography.    
 
To date, most published studies have utilized NO3-rich beetroot juice, which provides short-
acting effects, including acute changes in exercise duration, shorter time to achieve a pre-
specified work level, and greater performance during intense intermittent exercise58, 64. However, 
the challenge of developing and studying more prolonged NO2 therapy remains. Co-I Hughan is 
leading formative pharmacokinetic (PK) studies to determine optimal safety and efficacy of NO2 
capsules, with studies of escalating dosing and chronic therapy65. Her expertise in human 
NO3/NO2 metabolism and application of NO2 therapy provides a critical dimension of innovation 
and rigor of the proposal. NO2 capsules may also help overcome inherent limitations to dietary 
NO3 in green vegetables, e.g., diet restrictions in the many older patients using warfarin, and/or 
abnormalities in mastication, salivation or achlorhydria that are also common in older adults.   
 
The PI, Dr. Forman has an ongoing pilot study of oral NO2 40 mg tid with healthy controls, HFpEF 
and HFrEF subjects. Ten control subjects have completed the pilot without any significant 
product related adverse events. Mild and transient symptoms consistent with what other 
investigators have observed have been present in some of the other subjects.  
 

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Experience thus far in the pilot with heart failure subjects has suggested a tendency for a 
vasoactive response to nitrite not seen in the controls. Subjects with both HFrEF and HFpEF 
enrolled thus far have all required dose reductions to 20 mg tid to prevent hypotension. Thus we 
have adopted the stratified dosing as described for this protocol.  
As the pilot is ongoing, data is incomplete. One preliminary analysis has been completed. In 
assessments of the 5 healthy controls, needle point data show 1 month of NO2 at 40 mg tid 
achieved reduced VO2 at the same submaximal workloads (Figure 3a), reduced VE/VCO2 
(Figure 3b) slopes at SS workloads, and significantly improved post-exercise recovery dynamics 
(Figure 3c) 
3a
.  3b 
 3c 
 
 
 
 
 
 
 
 
 
 
 
 
2. RESEARCH DESIGN AND METHODS 
 
 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGNS 
 
The study is a prospective, double blind, single-center, randomized drug treatment study of 4 
weeks of oral sodium NO2 (20 or 40 mg tid) in elderly subjects, 70 years of age or older with 
HFpEF. 
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN 
 
This is a prospective, randomized, double blind controlled trial of oral sodium NO2 vs. placebo 
in adults (n=18), ≥70 years of age with heart failure and preserved ejection fraction (“HFpEF”). 
Subjects who meet the Inclusion Criteria and none of the Exclusion Criteria will receive oral 
sodium NO2 or placebo for 4 weeks.  
We will stratify dosing as below: 
Drug:   

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a. oral formulation of sodium nitrite/placebo 40 mg three times daily (tid), once in the 
morning, in the early afternoon and again in the evening, approximately 6 hours apart in 
subjects age 70 to <80 years with low risk for hypotension 
 
b. oral formulation of sodium nitrite/placebo 20 mg tid, once in the morning, in the early 
afternoon and again in the evening, approximately 6 hours apart in subjects ≥80 years of 
age, and subjects age 70 to <80 years with risk factors for hypotension 
 
Subjects will undergo a screening visit, then pre-drug testing protocol in 3-4 visits, and then 
repeat those tests after 4 weeks on drug in 3-4 visits (number of visits dependent upon whether 
subjects consent to undergo right heart catheterization testing pre- and post-drug). 
 
On visit 4, subjects will be evaluated as outpatients with the first dose of study drug/placebo. BP, 
HR, RR, SpO2, MetHb levels and plasma NO2 will be measured at baseline and in the first two 
hours after dosing. Frequent safety monitoring will occur during the time subjects are on study 
drug/placebo by phone, through the post drug assessments in clinic to assess adverse events 
(AE), interval histories, medication compliance, symptoms and methemoglobin levels, and at the 
final visit, NO2 levels. 
 
Subjects will not be withdrawn from known effective therapy for the purposes of participating with 
the exception of: 
1. holding of anti-coagulant medications prior to the biopsies  
2. holding warfarin, dabigatran, rivaroxaban, apixaban or other novel anti-coagulants 
(NOACs) prior to right heart catheterization 
3. holding or reducing dose of hypoglycemic medications on an individualized basis for 
Visits 2, 3B, 4, 5, 6B, 7. 
 
Study Visits 
Visit 1  
The outpatient screening will take place in the UPMC Montefiore CTRC and will last 
approximately 2 hours. The following procedures and assessments will be completed: 
 
1. Obtain written informed consent using standardized procedures. The consent will include the 
option to participate in or decline the iCPET at visits 3B and 6B. 
2. Medical history obtained prior to the visit will be reviewed with subject. Subjects will be asked 
questions about any heart disease and related diseases in first degree relatives; no 
identifying information will be obtained. History and physical examination. Dr. Forman or 
study physician will evaluate subjects 70-<80 years of age for risk of hypotension and need 
for dose reduction. The study physician will also complete the Canadian Clinical Frailty Scale. 
Subjects will be reminded to inform study staff as soon as possible of any health or 
medication changes while they are in the study. 
3. Body weight and height. BP and other vital signs [temperature, pulse, respiratory rate (RR), 
HR, SpO2]. BP may be re-measured once if out of inclusion range. This is to allow for 
influences of medication activation, subject nervousness, lack of fluid intake, and known 
factors which contribute to clinical variation.  
Screening evaluation will include a hemoglobin and hematocrit (H/H) by the UPMC 
Presbyterian clinical automated testing lab and are to be covered by research funds. The 
total volume of blood drawn is approximately 1 teaspoon (4 mL). If subjects have a recent 
hemoglobin value, the physician will determine if this blood draw can be deferred. 
4. Subjects will be issued an ActiGraph GT3X+, non-invasive wrist monitoring device to be worn 
for a full 7 days during the first and approximately 9th weeks of the study to assess changes 
in daily activity, level/intensity and sleep. Subjects will receive instruction in the use of the 

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ActiGraphs and complete a physical activity/sleep diary for days wearing the device. Subjects 
will be reminded to return their ActiGraph at Visit 2/3A. 
5. Transthoracic Echocardiogram (performed by trained study staff). If subjects have had a 
recent echocardiogram, the records will be obtained prior to visit one and the test results will 
be reviewed and interpreted by a study physician in lieu of performing one. A trained 
sonographer will obtain the echocardiogram and a cardiology attending will be responsible 
for interpretation. The subject may be transported to a lab in the UPMC 
Presbyterian/Montefiore system for the echocardiogram. 
6. Subjects will be instructed in and provided forms to complete a three-day food record (3DFR) 
to observe subject’s dietary intake.  
Subjects who fail to meet applicable inclusion/exclusion criteria based upon the results of the 
screening assessment will be excluded from further study participation. Such subjects will be 
paid for the visit. 
Visit 2  
Subjects will be scheduled for an outpatient EMRC and/or Pepper Center/SMART Center visit 
within 28 days of Visit 1. The subject will be asked to refrain from caffeine the morning of this 
visit and to limit breakfast to a lighter meal prior to coming in. The visit will last approximately 3 
hours. For subjects who have diabetes, we will review their medication regimen and measures 
of diabetes control and advise subjects accordingly to prevent any exercise-induced 
hypoglycemia.  In most cases, no adjustment will be needed for this non fasting visit. Subjects 
will undergo: 
 
1. Interval history and brief physical exam. Measurement of body weight, BP and other vital 
signs (temperature, pulse, RR, HR, SpO2) 
 
Physical Function Battery: 
2. Non-invasive Cardiopulmonary Exercise Testing (nCPET):  A maximal graded exercise test 
in association with air-gas-exchange, an optimal gauge of aerobic capacity will be conducted 
by trained research exercise physiologists. Dr. Forman or another physician will be 
immediately available if needed. A computerized treadmill will be used to generate a 
symptom-limited exercise stimulus. ACSM criteria for starting and stopping will also be 
utilized. A lightweight mouthpiece or face mask will be positioned over the subject’s mouth 
and nose during the exercise for gas exchange assessments. VO2, VE/VCO2 slope, and RER 
will be measured as well as hemodynamics (max HR and BP), time, and ECG waveforms). 
Any unexpected abnormalities will be reported to the patient’s cardiologist; continued 
participation in the study will require physician clearance. Both the RPE and the Modified 
Borg Scale for Perceived Dyspnea (Shortness of Breath) will be completed during the 
nCPET. Dr. Forman has performed this test in more than 1,000 subjects ranging from elderly 
women greater than 80 years old to highly trained athletes. 
 
3. Fatigability assessment:  fatigability will be assessed based on RPE (6-20 scale) reported 
after a SS walking on a treadmill. Patients will walk 1.5 miles/hour (0.67 m/sec) for 5 minutes 
on a treadmill as per techniques validated by Simonsick, et al32. Those with RPE ≥10 will be 
categorized as highly fatigued. Our prior work in HF patients indicates this SS walking 
provocation is feasible for the patients we anticipate we will enroll, and that it will 
(appropriately) generate a wide range of perceived fatigability responses for meaningful 
analyses of underlying mechanisms. Subjects who are unable to complete 5 minutes of 
walking will be considered highly fatigued. 
 
4. A 6MWT will be performed. Subjects walk as quickly as possible on a 50 meter course66. 
Total distance and segment gait speed are assessed. Performance fatigability is assessed 

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as the ratio of gait speeds in the second vs. next-to-last segments. Based on Murphy, et al., 
a ≥6.5% decrease in speed is considered high performance fatigability106. 
 
5. Portable CPET during SS and 6MWT 
Subjects will wear a portable CPET device during the SS and 6MWT. Standard breath-by-
breath CPET ventilatory indices VO2, VE/VCO2, and RER are collected and used to 
characterize metabolic parameters in correlation with self-determined walking speeds over 
time67. Average VO2 per lap during steady-state (2 minutes of plateaued VO2) is used to 
calculate the energy cost of walking by normalizing the VO2 to lap gait speed (ml/kg/m). Using 
these data, performance fatigability can be assessed in proportion to the peak aerobic 
capacity (i.e., VO2 during 6MWT relative to peak VO2 assessed by nCPET-treadmill), as well 
as energy cost of walking (VO2 during 6MWT relative to gait speed). Mechanisms underlying 
NO2 benefit are also distinguished by comparing ventilatory vs. metabolic parameters, with 
the potential for metabolic performance to improve if fatigability is mitigated.  
 
6. Participants will complete the “Short Physical Performance Battery” which is a series of 3 
tests:  1. sitting and standing out of a chair; 2. walking over a 4 meter course (to measure 
walking speed); 3. standing with their feet in different positions to measure their balance. 
 
7. Participants will also wear ActiGraphs during pre- and post-intervention nCPET, SS treadmill 
test, and 6MWT. These accelerometry data are used to establish individualized cut points 
that facilitate analysis of free-living accelerometry assessments. Volume and pattern metrics 
are used to analyze the raw data 68, 69. We use the raw data and machine learning methods70, 
71 to explore patterns of activity that are most strongly associated with changes in fatigability. 
 
8. Near Infrared Spectroscopy (NIRS) 
NIRS will be performed during the nCEPT, SS and 6MWT. During these assessments, 
sensors will be placed on the skin of one leg to allow the non-ionizing, LED light source to 
assess tissue oxygenation and perfusion via measurements of the optical absorption of 
hemoglobin. Co-I Dr. Huppert will direct the training and data analyses of the NIRS 
measurements. 
 
9. Hand Grip Strength Assessment will be performed to measure muscle strength. Participants 
will be asked to sit in a chair without arms and squeeze a device as hard as they can with 
one hand at a time, rest and switch to the other hand with three trials. 
 
10. Questionnaires 
The following questionnaires will be administered: 
Sleep 
The Pittsburgh Sleep Quality Index 72
Depression 
PHQ-9: well-validated and responsive to change 73
Self-efficacy
Sullivan, a five-item summative score 74
Pain 
McGill pain questionnaire-15 descriptors (11 sensory, 4 
affective); pain rated on an intensity scale (0-3). Pain 
scores derived from the sum of the intensity rank values 
75 
Comorbidity 
The Charlson Comorbidity Index:  provides a convenient 
and well-validated assessment of aggregate morbidities 
76 
Frailty (done at Visit 
1 & 7) 
The Canadian Study of Health and Aging Clinical Frailty 
Scale is a 7 point frailty score that was developed for 
predictive sensitivity as well as its ease of use 77 

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We will ask participants to complete them interspersed among the physical function tests so as 
to reduce fatigue of both assessments. 
 
The 3DFR issued at Visit 1 will be reviewed with the subject for clarification and completion as 
well as the ActiGraph diary. 
 
Prior to departing, we will remind subjects to resume their usual medication regimen if changes 
were made with regard to diabetes. 
 
If study team or subject feel as though any of the study procedures at this visit are not safe and/or 
would be too burdensome to the subject, subject may not complete all of the procedures at this 
visit. 
 
Visit 3A  
Subjects will be scheduled for an outpatient visit at the University of Pittsburgh Magnetic 
Resonance Research Center (MRRC) in UPMC Presbyterian 5±2 days from Visit 2. This is a 
non-fasting visit (patients will be asked to eat a light meal) and will last approximately 2-3 hours. 
Subjects who are identified to have the presence of metal or non-MRI compatible device will not 
complete Visit 3A (or 6A). 
 
Subjects will undergo: 
1. Collection of interval history, measurement of BP and other vital signs (temperature, pulse, 
RR, HR, SpO2) by study or MRRC staff. 
Any concern for medical instability after vitals and interval history are collected will prompt 
contact with a study physician, and possible physical exam before proceeding. 
 
2. The study coordinator will clear the subject as safe for MRI prior to the enrollment. Then the 
safety protocol and procedures of the MRRC will be followed as well as a double check for 
safety. The MRS is an in-vivo test to measure maximal mitochondrial capacity to generate 
ATP. It will be performed on the subject’s leg as they complete a flexion-extension exercise 
within the bore of the magnet as described below.  
 
Prior to the exercise, the technician will collect a fully-relaxed (FRS), high resolution 31P 
spectrum of the resting muscle. Then the exercise protocol will be performed:  Participants 
lay supine with the non-dominant knee (unless contraindicated) elevated at ~30o and straps 
are placed over the legs. Participants perform voluntary isometric contractions (kicking) 
repeatedly as hard and as fast as they can for two bouts each followed by a  rest period. The 
protocol is designed to deplete PCr stores by 33-66% to ensure high signal to noise defining 
PCr recovery without inducing acidosis (pH <6.8), which inhibits oxidative phosphorylation. 
82, 83. 
Cognition 
Montreal Cognitive Assessment. A well validated tool to 
screen for dementia78 
Quality of Life 
Kansas City Cardiomyopathy Questionnaire (KCCQ). 23-
item instrument produces the following scores:  Physical 
limitation, symptoms, symptom stability, social limitation, 
self-efficacy, Quality of Life, and two summary 
measures, and is widely used as a standard quality of 
life measure for heart failure patients79 
Physical Activity 
CHAMPS (Community Healthy Activities Program for 
Seniors) Activities Questionnaire for Older Adults80 
Fatigability 
Pittsburgh Fatigability Scale81 

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3. Following completion of the MRS protocol, subjects will be provided parking and discharged. 
 
Note: Visit 3B is an optional visit. Subjects who choose to do the right heart catheterization visits 
will need to have blood drawn for a basic metabolic panel, PT/INR, and CBC/diff/platelets, 
preceding Visit 3B by no longer than 30 days, per the UPMC Cath. Lab protocol. We will 
coordinate this draw at one of the study or if the subjects have bloodwork prescribed for their 
ongoing medical care. Subjects will be provided parking for the blood draw visit, if it requires a 
separate visit. 
 
Visit 3B    
This visit will be completed by the subjects in the recruited sample who have consented to 
invasive cardiopulmonary exercise testing (iCPET). Subjects may decline this portion of the 
testing at consent. This fasting visit will take place in the UPMC Presbyterian Cardiac 
Catheterization lab (Cath. Lab) within 5±2 days from Visit 3A and last approximately 4-6 hours. 
Subjects with diabetes will receive advisement on issues related to blood sugar management 
per the standardized pre-catheterization protocol of the UPMC Cath. Lab. Subjects on warfarin 
or NOACs will be reminded by study staff to hold those medications 3 days prior and including 
the day of the catheterization. 
Upon arrival in the Cath. Lab, subjects will undergo: 
1. Collection of adverse events, interval history, measurement of BP and other vital signs 
(temperature, pulse, RR, HR, SpO2), as well as completing a breathing test, called 
spirometry, that assess subjects ability to breathe in and out. We will confirm appropriate 
medications were held per individual case. 
 
2. Brief physical exam 
 
3. Right heart catheterization 
 
Dr. Risbano, Co-Investigator, or a qualified pulmonologist or cardiologist investigator will lead 
the UPMC protocol for right heart catheterization, with strict adherence for safety and sterile 
conditions as indicated, including the use of fluoroscopy to guide the catheter placement. PA 
(internal jugular approach) and radial artery catheters are placed. Systemic and PA 
pressures (PAP) are measured continuously during exercise provocation (described below). 
PCWP is measured at baseline and each minute thereafter. End-expiratory assessments of 
right atrial pressure (RAP), RV pressure, PAP, and PCWP are assessed.  
 
Approximately 2 ml blood samples are drawn from the radial and PA catheters during rest 
and the last 15 seconds   every other minute of exercise for measurement of blood gases 
(PA blood gas will be used for assessments of Qt and peripheral blood gas for assessment 
of systemic O2 extraction i.e., PA oxygen (Ca) minus venous oxygen (vO2), as well as for pH 
and lactate. Systemic and pulmonary arterial blood are analyzed at 37°C for partial pressures 
of O2 and CO2 (PO2, PCO2), pH, hemoglobin (Hb), and O2 saturation. Blood O2 content is 
calculated from Hb and O2 saturation:  S(a)O2 x Hb x 1.34. The Fick principle is used to 
calculate Qt (i.e., Qt=VO2/(Ca-vO2)) during exercise. Predicted cardiac output (Qtmax) is 
calculated from the predicted VO2 max assuming a normal maximum exercise arterial-mixed 
venous O2 content difference (Ca-vO2) and Hb of 14 g/dL. The pulmonary vascular resistance 
(PVR) is calculated from (PAP−PCWP)/Qt. RPE and ratings of dyspnea are assessed during 
iCPET and analyzed in relation to simultaneously collected lactic acid levels as a further 
assessment of skeletal muscle metabolism underlying fatigability84. Furthermore, these 
relationships will be assessed in relation to concurrent ventilatory dynamics (VO2, VE/VCO2, 

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and RER) as determined by concurrent CPET. The total blood volume will depend on the 
total time exercising but is likely not more than approximately 32 ml (2 ml at baseline and 2 
ml per minute of exercise, maximum likely 15 minutes, from radial and pulmonary arteries) 
 
Cardiopulmonary Stress Exercise Testing to be performed during the right heart 
catheterization (invasive CPET):  Maximal graded exercise testing will be conducted in 
association with air-gas-exchange during the right heart catheterization. A qualified 
pulmonologist or cardiologist will be available throughout the exercise provocation. A 
computerized cycle ergometer using a ramp protocol will be used to generate a symptom-
limited exercise stimulus. A lightweight disposable pneumotach device face mask will be 
positioned over a subject’s mouth and nose during the exercise for gas exchange 
assessments; VO2, VE/VCO2 slope, and respiratory exchange ratio (RER) are ventilatory 
parameters that will be measured during exercise. Simultaneously, intra-cardiac 
assessments of pulmonary wedge pressure, right heart pressures, and cardiac output will be 
determined by the pulmonary arterial catheter.  HR and BP, time, and ECG waveforms will 
also be assessed.  Any unexpected abnormalities will be reported to the patient’s cardiologist 
or primary care physician; continued participation in the study will require physician 
clearance. Subjects will be asked for ratings of exertion and shortness of breath during the 
testing. ACSM criteria for starting and stopping will also be utilized. 
 
iCPET may be completed in combination with echocardiography depending on the quality of 
baseline images. Echocardiographic images may be obtained at rest at baseline, and then 
every 2 minutes during exercise and immediately post-exercise. Quantitative echo measures 
of LV size, and systolic and diastolic function are made at rest and exercise. Lusitropy is 
assessed based on the calculation of left ventricular end-diastolic volume (LVEDV) divided 
by PCWP. The net effect on cardiac function is a Starling curve, i.e., SV/LVEDV at rest and 
with exercise. 84 
 
4. Following completion of the testing, subjects will have catheters removed and monitoring for 
about 2 hours post-test. They will be given any needed education for post catheterization, 
including site care and resuming anti-coagulant or anti-diabetic medications that were held 
for the procedure. Prior to discharge, they will be confirmed medically stable by a study 
physician. Subjects will be provided a meal. 
 
Visit 4 
Subjects will be scheduled for an outpatient visit at the University of Pittsburgh CTRC in UPMC 
Montefiore within 10 days ±4 days from Visit 3A for subjects who did not complete iCPET or 
within 5 days ±2 days from Visit 3B for those who did complete iCPET.  
Subjects will be asked to observe 8 hours fasting prior to arrival. Upon arrival subjects will be 
served a limited standardized breakfast.  
This visit will last approximately 4-5 hours. To prevent acute effects of exercise on muscle 
mitochondrial function, this visit will occur no earlier than 2 days after Visit 3A/3B. Prior to the 
biopsy, study staff will call indicated subjects to hold their anti-platelet and anti-coagulation 
medications for 1-5 days (depending on the different medications and relative half-lives, as 
approved by their cardiologist). Subjects with diabetes who are on hypoglycemic medications 
will be advised if they need any adjustment to their usual regimen.  
 
While the standardized breakfast is being prepared, subjects will undergo: 
1. Interval history, measurement of BP and other vital signs (temperature, HR, body weight, 
RR, and SpO2)  
 

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2. Staff will also confirm subject is fasting and that the anti-coagulation regimen was withheld 
as prescribed. Nursing will place an IV line. 
 
3. A study physician will perform a brief physical exam. 
 
4. Skeletal Muscle Biopsies 
An important component of the protocol is the muscle biopsy. Subjects who refuse biopsy 
will not be enrolled. However, participants who complete the first biopsy and then state (prior 
to being given drug) that they will not complete a second biopsy at Visit 7 will be withdrawn 
from the study. Alternatively, patients who refuse the 2nd biopsy after they have completed 
the 4 weeks of NO2/placebo therapy will still complete the other post drug assessments. If 
other unique subject situations arise, they will be evaluated by the PI and a decision will be 
made with regard to completing the other post assessments or not (e.g., completion of 2 
weeks on drug, and then refusal to complete biopsy.) 
 
For the biopsy, patients will lie comfortably in the supine position for biopsy of the vastus 
lateralis muscle of the non-dominant leg. Biopsy sites will be prepped with chlorhexidine. 
Lidocaine will then be administered, superficially initially and then more deeply into the 
muscle, using a small gauge needle. Subjects who report a history of poor pain control or 
low threshold will be offered the use of a topical anesthetic cream prior to the lidocaine. 
Ultrasound may be used to enhance visualization of the site for the needle insertion. A small 
superficial incision will next be completed using a #11 blade scalpel. A 5 mm Bergstrom 
muscle biopsy needle will be inserted through the skin incision and advanced into the muscle. 
Suction will be generated using a syringe attached to the outside portion of the needle, to 
thereby suck skeletal muscle into a hole on the side of the needle positioned in the muscle 
tissue; this draws a small piece of muscle tissue (about 150 mg of muscle tissue) into the 
hollow shaft of the needle, which is then cut with a cutting trocar that slides through the shaft 
to cut the tissue drawn within its core. After harvesting the first sample, the needle will be 
rotated 90 degrees, and a second sample extracted to maximize yield for analysis. Additional 
passes of the needle may be necessary for an adequate muscle sample. The wound site will 
be closed with steri-strips and a sterile pressure bandage. 
 
Muscle specimens will be immediately processed, and stored. Muscle specimens are divided 
into portions. Fragment 1 is immediately frozen in liquid nitrogen in preparation for 
polymerase chain reaction (PCR) analysis for DNA /RNA isolation, protein homogenates 
used for enzymatic assays and Western analysis. Fragment 2 has the muscle fibers aligned 
for microscopy, placed in OCT, then cooled in a thawing isopentane slurry and placed in a 
cryotube and frozen in liquid nitrogen for histochemistry. Fragment 3 is prepared for electron 
microscopy. Fragment 4 is processed in the CTRC and EMRC immediately for bioenergetics 
analysis. Any extra tissue will be stored for future analyses.      
 
Subjects will rest for approximately 30 minutes post biopsy while wound is compressed and 
dressed and prior to baseline vitals prior to dosing. Study staff will record a baseline level of 
symptoms, i.e., dizziness, lightheadedness, etc. for use during weekly phone calls, prior to 
dosing.  
 
5. Research bloods 
After the standardized meal is completed, a blood sample via IV line will be drawn for 
inflammatory peptides (IL-1, IL-6, IL-15, TNFα), adipokines (leptin and adiponectin) vitamin 
D, GFR, BNP or NT-pro BNP, TSH, HbA1c. Samples for platelets and NO2 levels will also 
be drawn at baseline into a 1) heparinized syringe  and 2) a cell preparation tube (CPT). 
Additional collection of NO2 levels will be drawn at 30 minutes, 60 minutes and 120 minutes. 

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Heparinized blood will be immediately centrifuged at bedside to separate plasma and red 
cells and then flash frozen for later NO2 measurements (via reductive chemiluminescence 
to determine that NO2 levels were increased in the muscle as described. The second tube 
of blood (CP tube) will be immediately subjected to differential centrifugation in order to 
isolate platelets. Platelet bioenergetics will then be measured via the Seahorse Extracellular 
Flux Analyzer including basal, non-ATP producing, maximal and non-mitochondrial 
respiratory rates along with glycolytic rate. If subjects have had an HgbA1c, TSH or vitamin 
D level within approximately 3 months, or a BNP or GFR within approximately 1 month, (all 
per physician discretion), we may defer this blood draw. 
 
Extra aliquots will be stored for use in future studies. Total volume of blood drawn will be 
about 72 ml (about 4.9 tablespoons).  
 
6. pK 
CTRC nursing staff will measure BP, RR, HR, SpO2 and MetHb to ensure they are at a SS 
before administering the study drug/placebo (baseline). Subjects who are 80 years of age or 
older or <80 with risk factors for hypotension will receive 20 mg drug/placebo; all others will 
receive 40 mg drug/placebo. Subjects will receive their first dose of study drug/placebo as 
follows:  14N sodium NO2/placebo, from their day 1, AM dose, week 1 pill box, strength 20 
or 40 mg dependent upon parameters described in section “Study Design”, p. 6. Then, the 
following will be assessed:  BP (sitting and then standing after 3 minutes), HR and MetHb, 
RR and SpO2 every 15 minutes for the 2 hours post-drug administration period. If subjects 
experience high methemoglobin level, hypotension or symptoms, the PI will be informed and 
advise on plan for dosage or other management. See Dose Selection, subsection, Dose 
Reduction). 
 
7. Drug distribution 
Subjects will receive sufficient study drug/placebo supply to cover the period up to the next 
scheduled visit at Visit 5 and/or through Visit 7 as needed. We will instruct subjects to 
return all of the study drug packaging, including unused study drug and empty packaging at 
each study visit and/or at the time of discontinuation from treatment. 
 
Study staff will review medication administration and use of the drug diary card with all 
subjects. Subjects will be asked to record the time they take the drug each day and any 
symptoms with severity rating and/or health events during the dosing period.  
 
The study staff and/or CTRC RN will review biopsy site care with subjects and if indicated, 
remind subjects to resume their 
normal 
anti-coagulation/anti-platelet/anti-diabetic 
medications.  
 
The subject will receive payment for visits 1-4 and can then be discharged. 
 
Drug Dosing Period and Outpatient Monitoring 
Subjects will then complete no less than 28 (-3) days on drug. Subjects will continue on 
drug/placebo through the post testing period; the duration will vary with the visit windows and if 
subjects are doing both 6A and 6B.  During the 28-day period, subjects will be called on a weekly 
basis by study staff, beginning on the visit 4 day plus 7 days ±2 days, and continuing 
approximately weekly thereafter. Study staff will query subjects on any symptoms, home BP 
readings if applicable, health care, including medication changes, or adverse events, and 
compliance with study drug/placebo regimen. Any AEs or medical issues of concern will be 
promptly communicated to the PI or covering study physician by email or phone depending on 
urgency; and AEs will be documented in the study log which is reviewed with PI at least bi-

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weekly. Subjects will be again reminded to communicate any medication changes prescribed by 
their health care team to study staff as soon as possible, not to wait for the weekly call. (Serious 
adverse events will be reported as described in Section 6.4.5.) Management of hypotension or 
methemoglobinemia related symptomatology will be as described in 2.3.3 Dose Selection, sub-
section Dose Reduction.  
 
Post Assessments (continuing on NO2/placebo tid) 
Visit 5 
This visit will be scheduled 28 ±3 days from Visit 4, take place in the UPMC Montefiore EMRC 
and/or Pepper SMART Center and include all of the Functional testing described in Visit 2. 
 
1. Upon arrival subjects will have measurement of BP, temperature, RR, HR, SpO2 and MetHb 
and body weight. Study staff will check for interval history, adverse events, and complete a 
symptom assessment. The drug diary cards and empty drug boxes for completed weeks on 
study drug/placebo will be collected. Medication compliance will be reviewed. A study 
physician will perform a brief physical exam. 
Physical Function Battery 
a. nCPET  
b. Fatigability assessment on treadmill (SS).  
c. Self-determined walking (6MWT) 
d. NIRS will be collected during the nCPET, SS and 6MWT. 
e. Short Physical Performance Battery (SPPB). 
f. 
Participants again will be asked to wear the Actigraph during the post-intervention 
nCPET treadmill, 6MWT and steady-state treadmill. After testing, subjects will wear the 
ActiGraph for one week and complete a physical activity/sleep diary for days wearing 
the device. 
g. Hand Grip Strength Assessment 
2. The same questionnaires as at Visit 2 will be administered interspersed with functional 
testing. 
3. Subjects will be asked to complete another 3DFR as per Visit 2. 
4. Prior to discharge, subjects will be reminded to return the ActiGraph and 3DFR at visit 6A or 
6B, depending on testing schedule.  
 
If study team or subject feel as though any of the study procedures at this visit are not safe and/or 
would be too burdensome to the subject, subject may not complete all of the procedures at this 
visit. 
 
Visit 6A 
This visit will replicate the 31P Magnetic Resonance Spectroscopy protocol described at Visit 3A. 
The same safety precautions will be followed. This visit will be scheduled within 5 ±2 days of 
Visit 5. Following this visit, the subject will advance to either Visit 6B or 7 depending if consented 
to the iCPET. Pre- catheterization labs will be drawn as described per Visit 3A. 
 
1. Upon arrival subjects will have interval history and measurement of BP, temperature, RR, 
HR, SpO2, and MetHb. Study staff will check for interval history, adverse events, and 
complete a symptom assessment. The drug diary cards and empty drug boxes for completed 
weeks on study drug/placebo will be collected. Medication compliance will be reviewed.  
2. Any concern for medical instability after vitals and interval history are collected will prompt 
contact with a study physician, and possible physical exam clearing to proceed. 
3. If completion dates have passed, the 3DFR issued at Visit 5 will be reviewed with the subject 
for clarification and completion. 
 

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Visit 6B 
This visit will replicate the iCPET testing described in visit 3B for those subjects who elected to 
participate. This visit will be scheduled within 5 ±2 days of Visit 6A. The same safety protocol 
and procedures will be followed as in 3B. This visit will be completed by the subjects in the 
recruited sample who have consented to iCPET, Visit 3A. This fasting visit will take place in the 
UPMC Presbyterian Cardiac Catheterization lab and last approximately 4-6 hours. Subjects with 
diabetes who are on hypoglycemic medications will be advised on medications to hold the day 
before and morning of visit. 
 
iCPET 
Upon arrival in the Cath. Lab, subjects will undergo: 
1. Collection of adverse events, interval history, measurement of BP and other vital signs 
(temperature, pulse, RR, HR, SpO2 and MetHb) and symptom assessment. 
2. Brief physical exam 
3. UPMC protocol for right heart catheterization will be followed, with strict adherence for safety 
and sterile conditions as indicated. PA (internal jugular approach) and radial artery catheters 
are placed as described in 3B. Volume of blood as noted approximately 32 ml, maximum. 
4. Following catheterization, subjects will have catheters removed and be given any needed 
education for post catheterization site care. They will be confirmed medically stable by a 
physician, before leaving. Subjects will be provided a meal. 
 
Subjects will return their Actigraph and diary at this visit, or Visit 7 if they do not complete Visit 
6B with heart catheterization. 
 
Visit 7 (Completion Visit to be scheduled 10 days ±4 days for subjects only doing visit 6A or 5±2 
days following Visit 6B).  
 
This visit will be at UPMC Montefiore CTRC and will replicate the blood draw and muscle biopsy 
procedures described in Visit 4. The same precautions and procedures will be followed including 
reminding, withholding, and confirming of the anti-coagulant/anti-diabetic regimen per subject. 
Subjects will take their last morning dose of study drug/placebo on the day of the visit. Subjects 
will be asked to observe 8 hours fasting prior to arrival. Upon arrival subjects will be served a 
limited standardized breakfast.  Confirmation of last dose will be completed with subjects at the 
visit.. This visit will last approximately 2 hours.  
 
1. While the standardized breakfast is being prepared, interval history/AEs and temperature, 
RR, BP, HR, SpO2, MetHb, body weight, and symptom assessment will be collected. A brief 
physical exam will be completed. The Canadian Clinical Frailty Scale will be repeated. 
2. Medication compliance via drug diary cards, empty pill box compartments, and subject report 
will be assessed. Unused capsules will be counted and returned to IDS following the visit. 
3. The 3DFR issued at Visit 5 will be reviewed with the subject for clarification and completion. 
4. ActiGraph and diary will be collected if not at previous visit 
5. Blood draw to include:  Inflammatory peptides (IL-1, IL-6, IL-15, TNFα), adipokines (leptin, 
adiponectin). GFR, BNP or NT-pro BNP, platelet sample and NO2 levels. Additional NO2 
levels will be drawn at 30 minutes, 60 minutes and 120 minutes. Total volume of blood drawn 
will be about 64 ml (about 4.3 tablespoons). If subjects have had BNP or GFR within 
approximately the last month via their clinical care, the study physician can defer this blood 
draw. 
6. Skeletal Muscle Biopsies:  same as described in Visit 3. 
7. Transthoracic Echocardiogram. The subject may be transported to a UPMC 
Presbyterian/Montefiore lab for an echocardiogram. 
 

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Following the echocardiogram subjects will be discharged. They will receive payment for visits 
5-7 and study drug period. 
End of Trial and Follow-Up Period  
 
Post Study drug/placebo Telephone Assessment (about 7 days following Visit 7) 
 
Telephone assessment for interval histories, muscle biopsy follow up and AEs will be 
completed. Any remaining symptoms or AEs which are suspected to be related to the study 
drug/placebo will be evaluated by Dr. Forman and follow-up plan for care devised as 
needed. 
 
 

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*Unless Hgb within normal limits recently prior to Visit 1 and deferred by physician; †deferred if recent per physician discretion; 
**Platelets, nitrite/nitrates (time 0), + nitrites/nitrates ( time 30 mins, 60 mins, and 120 mins); inflammatory peptides (IL-1, IL-6, IL-15, 
TNFα); adipokines (leptin, adiponectin); vitamin D, GFR, BNP or NT-pro BNP, TSH, HgA1C (unless recently measured) 
‡Platelets, nitrite/nitrates; inflammatory peptides (IL-1, IL-6, IL-15, TNFα); adipokines (leptin, adiponectin); BNP, GFR, unless recently 
measured 
Table.2.2.1 Study Visits and Assessments 
Phase 
Visit
Assessments and Procedures
Screening 
Visit 1
Informed consent and physical exam; Hgb* 
Home accelerometry; Echo†; issue 3DFR, frailty assessment
Pre- Assessment 
Series 
Visit 2 (within 28 days of Visit 1) 
Non-invasive functional assessment battery:  Symptom-limited nCPET; 
Fatigability assessments (perceived [SS] and performance [6MWT]), 
NIRS during nCPET, SS, 6MWT, Accelerometry during nCPET, SS, 
6MWT and 1 week at home with diary; Hand grip, SPPB, Questionnaires 
 
Visit 3A (5 days ±2 days from V2)
31P MRS protocol; review 3DFR and return ActiGraph 3A or 3B per 
dates; pre-cath bloodwork if applicable
 
Visit 3B (5 days ±2 days from V3A)
Symptom limited iCPET with associated exercise echocardiography
 
Visit 4 (10 days ±4 days from V3A or 5 
days ±2 days from V3B)
Research bloods**; Skeletal muscle biopsy; Administration of first dose 
of NO2; PK assessment and BP assessment; Drug distribution
Drug dosing 
Weekly phone calls
Phone call monitoring of drug tolerance, symptom assessment, 
medication compliance, adverse events.
Post-Assessment 
Series 
Visit 5 (28 ±3 days from Visit 4)
Non-invasive functional assessment battery:  Symptom-limited nCPET; 
Fatigability assessments (perceived [SS] and performance [6MWT]), 
NIRS during nCPET, SS, 6MWT, accelerometry during nCPET, SS, 
6MWT and 1 week at home with diary; Hand grip, SPPB, 
Questionnaires; issue 3DFR
 
Visit 6A (5 days ±2 days from V5) 
31P MRS protocol; review 3DFR and return ActiGraph 6A or 6B per 
dates; pre-cath bloodwork if applicable 
 
Visit 6B (5 days ±2 days from V6A) 
Symptom limited iCPET with associated exercise echocardiography 
 
Visit 7 (10 days ±4 days from V6A or 5 
days ±2 days from V6B)
Frailty assessment, Skeletal muscle biopsy; Research bloods‡; echo 
after meal

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2.3 Study Drug (NITRITE) 
 
The study drugs are oral formulation of sodium nitrite and matching placebo. Sodium nitrite is 
available in dose strengths of 20 mg and 40 mg. Sodium nitrite of both dose strengths and 
matching placebo will be supplied as capsules for oral administration. All capsule formulations 
will be identical in appearance (size, shape, color) and smell. The packaging and labeling will be 
designed to maintain blinding to the Investigator’s team and to subjects. 
 
2.3.1 Study Drug Preparation and Dispensing 
The NO2 and placebo capsules will be prepared at and obtained from Triangle Compounding 
Pharmacy, Inc., Cary, NC and the University of Pittsburgh Medical Center Investigational Drug 
Pharmacy Service (UPMC-IDS) will be utilized for dispensing of the study drug/placebo. The 
labeling and packaging will be conducted according to Good Clinical Practice and regulatory 
requirements. The study drug is administered only to subjects enrolled in the study and in 
accordance with the protocol. 
Drug will be dispensed in four 7-day pill containers, with compartments for each dose, each 
labeled with subject name and directions for taking. Subjects will be given an additional capsule 
supply in a pill bottle as needed to maintain an adequate supply for the post-visit follow-up testing 
period. 
2.3.2 Drug Administration 
 
Standard 14N Sodium NO2 or placebo will be supplied as one capsule, three times daily oral 
administration at the dose strength of 20 or 40 mg (per subject as described in Study Design 
section), in four 7-day pillboxes for the first 28 days on drug, and then additional supplied for 
subjects to refill boxes dependent on their individual visit schedule. 
 
2.3.3 Dose Selection 
In the 2001 National Toxicology Program (NTP) Report summarizing 2-year rodent drinking water 
studies, there was no evidence of carcinogenic activity of NO2 in male or female F344/N rats 
exposed to up to 130 mg/kg/day in males and 150 mg/kg/day in females, or in male B6C3F1 mice 
exposed to up to 220 mg/kg/day. There was equivocal evidence of carcinogenic activity of sodium 
NO2 in the highest dose of 165 mg/kg/day in female B6C3F1 mice based on the positive trend in 
the incidences of squamous cell papilloma or carcinoma (combined) of the forestomach. 
Exposure to sodium NO2 in drinking water resulted in increased incidences of epithelial 
hyperplasia in the forestomach. However, no chromosomal damage (genetic toxicity) was 
observed in three studies conducted in rats and mice in vivo 85. Taken together, these findings 
suggest minimal carcinogenic NO2-mediated risk. 
 
Multiple studies now demonstrate the predominant safety of NO3 therapy in humans (most 
typically administered as beetroot juice)16, 64. At the University of Pittsburgh, several pilot studies 
show safety and efficacy of NO2, the active metabolite of NO3, and show strong physiological 
rationale for its use.  
 
Kara Hughan is a co-investigator on this proposal. She has been studying utility of oral NO2 
(administered as capsules) to improve insulin sensitivity over 12 weeks in adults aged 18-60 years 
with metabolic syndrome in an NHLBI K23 funded study. Hughan’s first subject (healthy) was 
started on NO3 1,000 mg once daily (with NO3 supplying a more prolonged source of plasma NO2 
across the day via enterosalivary circulation until next dosing) in combination with NO2 20 mg 
once daily.  

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During the first subject’s drug course, PK plasma NO3 and NO2 concentrations, MetHb and BP 
data were assessed every other week. Given that only minor increases in MetHb were detected 
(that remained well within the safety threshold) and that the plasma NO2 rise after ingestion then 
fell to baseline after ~3-6 hours with NO2 dosing, requests were made to the IRB to increase NO2 
dosing to 20 mg two times daily (bid).  
 
At the same time that IRB approval was being pursued for NO2 increases to 20 mg bid, a second 
subject was started on the original dosing regimen (NO3 1,000 mg once daily and NO2 20 mg 
once daily). At the 6-week time point, IRB approval was received to increase the NO2 dose to 20 
mg BID for the remaining 4 weeks. Thereafter Dr. Hughan recruited 3 subsequent subjects on 
sodium NO3 1,000 mg once daily and NO2 20 mg bid. 
 
At these latter 4 subjects’ biweekly safety visits, Hughan observed their morning NO3/NO2 dosing, 
measured BP and MetHb 30-40 minutes after dosing and plasma NO3/NO2 levels measured 
approximately 45-60 minutes after drug dosing. In all cases, none demonstrated 1) significant 
reductions in BP; 2) significant rise in MetHb (>5%); 3) excessive plasma NO3 and NO2 
concentrations in comparison to the single dose PK profile61. 
 
Given that there was only a brief and mild decrease in systolic BP with the single dose NO3 dosing 
and no BP reduction when 12 weeks of NO3 were combined with twice daily NO2, Hughan, 
Gladwin, and colleagues hypothesized that the moderate contribution of sodium in NO3 might 
mitigate potential hypotensive effects.  
 
In parallel, Mohler, ER86 reported results of oral NO2 40 mg bid vs. 80 mg bid vs. placebo for 10 
weeks in 55 adults with peripheral arterial disease, most with diabetes. The high NO2 doses were 
well-tolerated and were associated with relatively greater responses in flow mediated (endothelial) 
dilation. Given this report and the facts that 1.) Subjects at the University of Pittsburgh were 
similarly not approaching any safety stopping endpoints and, 2.) the NO2 doses were well 
tolerated, another dose modification to the IRB/FDA was completed in which the NO3 was 
removed from the regimen and NO2 was increased in dose (from 20 to 40 mg) and frequency 
(from bid to tid). 
 
Data below show results from 3 subjects (with metabolic syndrome and hypertension). 3 subjects 
(#6, 7, 8) were aged 51, 45, and 59 respectively; 2 were female (#6 and #8) and one was male 
(#7). NO2 was initiated at 20 mg po tid and then after 2 weeks to 40 mg tid. Each subject was 
assessed serially over 12 weeks. Bi-weekly serum NO2 levels (for individual subjects as well as 
the mean) are shown in Figure 1.  
 
Fig. 1: Serum NO2 levels  
 
 

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Figure 2:  BP responses for 3 subjects 
 
 
As figure 2 shows, there were mild drops in BP with 20 mg tid of NO2 that in 2 of the 3 cases 
increased on the higher NO2 dose thereafter. Subject #6 was also taking hydrochlorothiazide 
(HCTZ), subject #7 was also taking lisinopril, and subject #8 was also taking carvedilol. Most 
important, the NO2 doses of 40 mg tid were well tolerated in all, both in relation to the 
hemodynamics and overall well-being. Consistently, MetHb never increased above 5%.   
 
Dr. Hughan’s trials also track other dosing strategies of exogenous NO2 and NO3 therapy and her 
data provide a broad profile of safety and excellent medication tolerance. In her trial, there have 
been no serious adverse events (SAE). The most frequently reported adverse events (AE) were 
headache, dry mouth and flushing. Other AEs reported in this patient population include low back 
pain or back spasm, nausea, anxiety, sleep improvement, joint/muscle pain, swelling in arms/legs, 
mild chest pain, shortness of breath, and cold symptoms. All AEs were mild to moderate in 
severity, and resolved spontaneously or with minor treatments.  
 
Hughan’s analyses of NO2 capsules for patients with diabetes and hypertension is also linked to 
analyses of NO2-mediated changes in skeletal muscle gene expression. Patients taking NO2 
capsules demonstrated upregulated skeletal muscle NAD-dependent deacetylase sirtuin-3 
(SIRT3), mitochondrial SIRT3 and AMP-activated protein kinase (AMPK) in HFpEF-PH50. 
Related benefits include improved glucose uptake (mediated by improved Glucose transporter 
type 4 [Glut4] expression)8, 63. This research is ongoing. 
 
Co-Investigator Marc Simon has also been working on a NO2 project as part of the University of 
Pittsburgh Translational Program Project Grant (TPPG). He studied acute efficacy of inhaled NO2 
in 27 subjects (ages 56-77 years, mean 65 ±8) with pulmonary hypertension (PH), including 6 
subjects with PH associated with HFpEF. This was a prospective, open label safety and efficacy 
trial. The defined primary efficacy outcome was change in pulmonary vascular resistance. Safety 
endpoints included change in systemic BP and MetHb levels. All subjects underwent a standard 
right heart catheterization. Aerosolized NO2 was administered with an initial dose of 45 mg and 
then increased to 90 mg if the initial 45 mg dose was tolerated (i.e., no decrease in systolic BP 
>40 mm Hg, no decrease in peripheral oxygen saturation >10%, no increase in MetHb level >5%, 
no severe bronchospasm or dyspnea). All patients tolerated NO2 well and were asymptomatic. 
Only 1/27 patients did not receive the second dose of NO2 due to a transient asymptomatic 
decrease in systemic systolic BP (that returned to baseline within 5 minutes without intervention). 
Serum NO2 peaks at 15 min after inhalation of 45g was 7 μM and after 90 mg dose 15 uM. There 
were no significant decreases in peripheral oxygen saturation or increases in MetHb levels. 

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Hemodynamic effects of NO2 in the HFpEF subjects included significant decreases in RA, RV 
systolic and diastolic, PA systolic/diastolic/mean, and PCWP. PCWP and MPAP decreased by 
7.8 and 7.0 mm Hg, respectively (baseline median values 18 and 34 mm Hg, respectively). MetHb 
levels increased modestly but did not meet stopping criteria of the study. Dr. Simon presented his 
work at the American Thoracic Society Meeting in March, 2016. Overall, the data demonstrate 
feasibility and safety of aerosolized NO2 in HFpEF, and in many respects corroborate the recently 
published analysis by Borlaug in a study of HFpEF patients aged 69 ±6 years that showed infused 
NO2 benefits to acutely improve right heart hemodynamics during exercise1. None of Borlaug’s 
patients experienced hypotension or other adverse events. NO2 levels were undetectable at 
baseline, but then increased to 8.39 ±1.88 μM. PCWP was substantially improved by NO2 
compared with placebo (p<0.0003). Likewise, cardiac output reserve improved with exercise 
(p<0.002) and an increase in cardiac output relative to O2 consumption was normalized. NO2 also 
improved pulmonary artery pressure flow relationships in HFpEF and increased left ventricular 
stroke work with exercise versus placebo, indicating an improvement in ventricular performance 
with stress. 
 
As described in Section 1.4 Significance, the PI, Dr. Forman, has an ongoing pilot study of oral 
NO2 40 mg tid with healthy controls, HFpEF and HFrEF subjects. Ten control subjects have 
completed the pilot without any significant product related adverse events. Mild and transient 
symptoms consistent with what other investigators have observed have been present in some of 
the other subjects.  
 
Experience thus far in the pilot with heart failure subjects has suggested a tendency for a 
vasoactive response to nitrite not seen in the controls. Subjects with both HFrEF and HFpEF 
enrolled thus far have all required dose reductions to 20 mg tid to prevent hypotension. Thus we 
have adopted the stratified dosing as described for this protocol.  
 
Based on the combined efforts of Co-Is Hughan, Simon, and Gladwin, and PI Forman’s ongoing 
pilot, as well as the published work by Borlaug, we have demonstrated feasibility, physiological 
utility, and safety for older HFpEF patients, and a dosing rationale of NO2 20-40 mg tid for this 
study, with a dosing range for subject safety. Our subjects will complete 28 ±3 days dosing period, 
followed by a range of 9 to 21 days depending upon scheduling needs, and whether subjects 
consent for the pre- and post- iCPET testing, resulting in a possible maximum days on 
drug/placebo of 52 days. The dosing period in PI Forman’s NO2 pilot ranged 25-49 days, when 
combining 28-day dosing with up to 21 day post-testing period. Subjects are followed closing 
during this post drug testing period. Because of the physical limitations of HFpEF patients and 
the multiple staff and facilities needed for the post testing period, a more chronologically compact 
post drug testing period is not feasible. All data to date suggest this does not pose undue risk to 
subjects.  
 
Dose Reduction 
There is a rare possibility that subjects will have a high methemoglobin level or low BP from the 
study drug dose during Visit 4 or while they are on study drug. If this happens, they may 
experience lightheadedness or dizziness.   
 
High methemoglobin level 
In CTRC 
If this happens during visit 4 while they are in the CTRC, fluids and monitoring will be provided to 
ensure that the problem resolves and patient is stable. The physician investigator will factor 

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relevant subject symptoms and hemodynamics and then decide whether it is appropriate for the 
subject to proceed with study participation prior to discharge from CTRC as well as: 
1. Determine if unblinding is needed (if methemoglobin % >5) and, 
2. If subject is willing to do an interim visit with repeat drug dosing of 20 mg with 
follow up methemoglobin and BP pressure monitoring or 
3. Subject should be withdrawn 
 
During outpatient drug dosing period: 
If subjects report change in skin tone and dizziness or other signs of methemoglobinemia for >24 
hours, the need for an interim visit with the above described steps will be followed. The visit can 
be conducted in the UPMC Montefiore CTRC, EMRC or in the Pepper Center-SMART Center. 
 
At this visit, methemoglobin will be assessed. If greater than 5%, the PI will be unblinded to drug 
or placebo assignment. If subjects are on nitrite, subjects will be asked to repeat drug dosing of 
20 mg sodium nitrite with follow up methemoglobin and hemodynamic monitoring. A study 
physician or PI will evaluate data and make a determination regarding discharge on 20 mg tid. If 
discharged on 20 mg tid, subjects will be contacted via phone for the next 2 days for symptom re-
assessment. If symptoms remain resolved, subjects will continue on 20 mg tid. If symptoms 
persist, subjects will be requested to come in for repeat methemoglobin testing after 3 days.  If 
level persists above 5% on lower dose, participation in the study for the subject will be 
discontinued.  
 
Hypotension and related symptoms. 
In CTRC 
If this occurs while in the CTRC, care will be administered, including fluids and monitoring, to 
ensure that the problem resolves and patient is stable. The physician investigator will factor 
relevant subject symptoms and hemodynamics and then decide whether unblinding is indicated 
and if it is appropriate for the subject to proceed with study participation prior to discharge from 
CTRC. This will include what dose is appropriate and drug dispensing. The same process will be 
applied to subjects who are initially initiated on 20 mg; however, in these subjects there is 
relatively greater consideration of withdrawal as no lower dosing options are available. The PI 
may indicate the need for a repeat pK with 20 mg dose in subjects who started on 40 mg study 
drug/placebo. 
 
As outpatient: 
If subjects experience lightheadedness or dizziness after visit 4 while they are on the study 
drug/placebo, we request that they call the study coordinator who will inventory their symptoms, 
compare to baseline levels (measured at pK), and discuss need for interim visit with study 
physician. If a visit is indicated, subjects will be asked to repeat drug dosing of 20 mg with follow 
up methemoglobin and BP pressure monitoring. A study physician will factor relevant subject 
symptoms and hemodynamics and will confirm a management plan including the: 
-need for unblinding, 
-possibility if subject was on sodium nitrite to lower dose to 20 mg tid or 
-need to discontinue and drop from the trial.  
If subjects proceed on lower dose they will be reassessed via phone each day for the next 2 days 
thereafter to ensure that the patient is stable on the new dose.  If stable, subjects will continue 
with the half dose.  However if there are continued symptoms on lower doses, the physician 
investigator will reevaluate the patient’s suitability for continued participation based on symptoms 
and/or hemodynamics. 

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If subjects who were initiated on the 20 mg dose at Visit 4 report symptoms as described above, 
they will be asked to complete an interim visit, methemoglobin and BP pressure assessment; the 
physician may have to withdraw the subject since a dose lower than 20 mg tid is not an option. 
 
Other symptom 
The PI, via subject phone call monitoring, AE reports or at follow-up visits, will evaluate any other 
clinical presentation, sign or symptom in subjects which may warrant consideration of dose 
reduction. The procedure described for high methemoglobin or hypotension will be followed. 
 
2.3.4. Treatment Period 
Subjects will undergo study treatment duration of 4 weeks plus up to approximately 3 weeks of 
sodium NO2 or placebo three times daily. Three additional weeks are included to allow 
continuation of the study drug/placebo through the final outcome collection series of visits. This 
is considered appropriate to study benefits of oral NO2 therapy to improve physical function in 
patients with HFpEF. 
 
Randomization and Blinding 
We will use the high quality pseudo-random deviate generator in SAS® (SAS Institute, Inc., Cary, 
North Carolina) to randomize participants to NO2 or placebo in 1:1 ratio with a random block size. 
Block sizes will be revealed to the rest of the study team at the conclusion of the study to prevent 
educated guessing. Randomization will be stratified by the initial dose (20/40 mg) depending on 
whether age ≥80 or age 70-80 and hypotension risk to the extent possible within the small sample 
size. Study statistician will create schedules which pairwise link the randomization sequence 
number, participant identifier and treatment arm, and call in all prescriptions thereafter by the 
randomization sequence number. The independent research pharmacist at the institutional 
Investigational Drug Service will package all drugs/placebos to be similar in physical appearance. 
We have successfully employed the same process in other recently completed trials105. Therefore, 
all participants and study personnel involved in assessments will be blind to treatment 
assignment. 
 
2.3.5 Breaking the Blind 
Should the PI (or designated physician investigator) suspect that an adverse event (namely, 
methemoglobinemia, see Section 6.4.7) is related to the investigational product, he/she will 
contact one of three sources who will have access to the code:   
1. the UPMC IDS  
2. Dr. Perera, Biostatistician-Investigator 
3. Nydia Chien, MSN, RN, CCRC, Director of Regulatory Affairs, Translational Research 
Division of Pulmonary, Allergy, and Critical Care Medicine, Heart, Lung and Blood 
Vascular Medicine Institute 
They will be able to break the blind for that subject. An emergency unblinding/treatment would 
only be anticipated in the case of accidental overdose. In this protocol, signs and symptoms of 
methemoglobinemia are being monitored such that intervention would likely take place before 
reaching an emergent level if taken as prescribed. However, the PI and Coordinator will maintain 
quick access numbers to unblind quickly, if needed. Other signs and symptoms such as 
lightheadedness will be evaluated by the PI for determination if unblinding is indicated. 
 
2.3.6. Medication Compliance  
 
At Visit 4, subjects will self-administer their first dose of study medication under the supervision 
of the physician investigator or RN in the outpatient CTRC (Clinical Translational Research 

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Center) following completion of all baseline clinic assessments. After the pK (and subject is stable 
after first dose), subjects will be dispensed their study medication for home use, in, 4 prefilled 
seven-day labelled pill boxes to help with compliance and to help reduce burden or confusion. Pill 
counts will not be used during the first 28 days (phone call monitoring period) as having subjects 
remove the pills from the pill boxes to count would defeat the purpose of the pillboxes. On the 
weekly phone calls, the study coordinator will review the daily diary card (study drug/placebo and 
symptoms), inquire about missed doses and confirm empty pill boxes each week. Subjects will 
return empty pill boxes when they return for Visit 5, withholding those as needed for drug dosing 
during the final weeks. Medication compliance will be reviewed. Subjects will return any remaining 
capsules at their Visit 7 for final accountability. In the event that the compliance rate is <80% at 
any time reported by the subject, subjects will be re-educated on medication compliance. If 
medication compliance repeatedly falls outside of the acceptable range, the study investigators 
will discuss subject eligibility for continued participation in the study. Compliance during Dr. 
Forman pilot study has been excellent with no subject approaching the 80% level of concern. 
 
2.3.7. Medication Storage and Accountability 
The study investigators or the study coordinator will document the amount of study drug/placebo 
dispensed and/or administered to subjects, the amount returned by subjects, and the amount 
received from and returned to the UPMC-IDS. The study drug/placebo accountability records will 
be maintained throughout the course of the clinical trial. 
 
2.3.7 Concomitant Medications 
No changes are needed to the subject’s usual medication while taking sodium NO2/placebo. 
Subjects will hold their anti-coagulation regimen prior to the biopsies to reduce the risk of 
excessive bleeding. The concomitant medication list will be retrieved from the patient’s medical 
record, confirmed with the patient on the phone and at Visit 1 and then assessed for changes at 
study contacts thereafter. Any patient status or medication change during the study period 
advised by subjects’ health care team will be reviewed with the PI. 
 
2.3.8 Rescue Medications  
Not applicable 
 
2.4 STUDY ENDPOINTS 
Overall, study endpoints pre- and post-NO2 will include:  
1. Measures of physical function will include CPET indices: 
a. gait speed, 
b. strength (endurance, power), 
c. balance as measured by non-invasive CPET (nCPET) aerobic capacity, SS fatigability 
assessment, 6 minute walk test, non-infrared spectroscopy assessments of blood flow, 
Short Physical Performance Battery (SPPB), handgrips, and accelerometry  
And,  
d. pulmonary pressure and ventricular-pulmonary artery coupling, as measured by 
invasive CPET (iCPET) 
 
2. Skeletal muscle bioenergetics:  spectrophotometric, mitochondrial respiration, ribonucleic 
acid (RNA) isolation, protein isolation, electron microscopy  
Serology:  inflammatory peptides (interleukin (IL) 1, IL-6, IL-15, TNFα; adipokines (leptin 
and adiponectin); vitamin D, glomerular filtration rate (GFR), brain natriuretic protein (BNP) 
or N-terminal pro b-type natriuretic peptide (NT-proBNP), thyroid stimulating hormone 
(TSH), glycosylated hemoglobin (HgbA1c) and platelet bioenergetics [using Seahorse 

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Extracellular Flux (XF) analysis]9, i.e., including glycolytic as well as basal and maximal 
respiratory rates.   
3. Additional secondary factors include Quality of life (QoL) and related indices:  
questionnaires that include measures of sleep, depression, pain, cognitive function, 
physical activity, co-morbidities, frailty, nutrient intake, cardiac self-care efficacy, 
fatigability and HF, via the Kansas City Cardiomyopathy Questionnaire (KCCQ). 
 
2.5 STATISTICAL ANALYSIS 
 
2.5.1 Sample Size Determination 
Formal sample size justifications based on statistical power for detecting significant differences 
at a strict 0.05 level are not applicable for a pilot study such as ours. We will use the descriptive 
statistics obtained herein for formal sample size and power computations in planning the larger 
subsequent trial to obtain more definitive findings. We will base our sample size on preliminary 
data from the present project, published statistical methods for sample size and power for 
approaches as similar as possible to the proposed analyses, and commercially available 
specialized software (PASS 2012®; Number Cruncher Statistical Systems, LLC, Kaysville, Utah) 
in planning the future trial to be proposed under the R01 mechanism. 
 
 
2.5.2 Study Conduct Analysis 
We will clearly document participant flow from the number contacted to numbers screened, 
randomized and completed using a CONSORT diagram, with numbers and reasons for any 
exclusions, withdrawals, missed assessments and terminations at each stage. We will describe 
adherence to NO2 therapy as measured by queries to subjects, self-report of empty compartments 
on weekly phone calls and pill counts at the post visits using summary statistics as well as any 
post-randomization adjustments. Insights gleaned from study conduct analyses will be used to 
adjust protocol in the subsequent larger trial to increase its feasibility. 
 
2.5.3 Efficacy Analysis 
 
Overview-We will use SAS® version 9.3 (SAS Institute, Inc., Cary, North Carolina) for all 
analyses. The main focus of the analyses of the present pilot study will be to establish feasibility 
and obtain preliminary findings, and descriptive statistics needed for formal sample size and 
power computations for a subsequent larger randomized trial to be proposed under the R01 
mechanism to obtain more definitive conclusions. Therefore, we will pay greater attention to 
magnitudes of effects rather than statistical significance alone, and similar to published pilot 
studies in other settings, also employ graphical methods such as needle plots that focus on 
change at the individual level rather than on average 87, 88. First, we evaluate the distributional 
characteristics of the data set, the prevalence of missing values and general data quality using 
appropriate descriptive statistics and graphical summarizations for all variables for each arm for 
each time point, as well as baseline to follow-up change scores. Second, the baseline measures 
and participant characteristics will be compared between the two arms using differences in 
magnitudes of the descriptive statistics, and independent samples t-, Wilcoxon rank sum, Kruskal-
Wallis, chi-square or Fisher’s exact tests, as appropriate. Any variables found to be meaningfully 
different will be noted when interpreting the findings. Third, main analyses below will be 
performed.  
 
Aim 1-We will conduct Aim 1 analysis on an intention-to-treat basis. We will fit a series of linear 
mixed models using the SAS® MIXED procedure with baseline to follow-up change in primary 
outcome RPE, secondary outcome peak VO2, and each of the other secondary outcomes as the 

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dependent variable; and treatment (NO2/placebo), follow-up time (4 weeks) and their interaction 
as main factors of interest; baseline value of the dependent variable as the sole and a catch-all 
covariate; and a subject random effect to account for multiple follow-up measurements from each 
participant. We will construct appropriate means contrasts to compare the treatment groups at 
each of the follow-up time points. The magnitudes of the estimated means contrasts and graphical 
displays of individual change will be considered to support Aim 1 hypotheses rather than and in 
addition to statistical significance at the 0.05 level.  
 
Aim 2-We will use Pearson and Spearman rank correlation coefficients between outcomes (e.g., 
fatigability, daily activity) and potential mediators (e.g., bioenergetics, pulmonary pressures), both 
cross-sectionally and in terms of change from baseline, both combined and within arm, to simply 
quantify the influence of potential mediators on outcomes. We are aware of the formal methods 
for mediation analyses to elicit contributory role of NO2 and potential mechanism such as Baron 
and Kenny framework 89, reduction in treatment effect of NO after additionally adjusting for 
potential mediators, and mediation models using the SAS® %SOBEL and %INDIRECT macros 90-
92. However, our small sample size in the present pilot study does not afford the opportunity to 
reliably employ such methods. We plan to perform a rigorous mediation analysis using the said 
methods in the larger follow-up trial being planned with the results of the present study.  
 
Exploratory/Secondary/Sensitivity Analyses: We have proposed a main analysis treating 
RPE as a continuous variable for greatest statistical sensitivity in a small sample, but are aware 
that a clinically meaningful threshold may be 10 indicating fatigue33, 49. Therefore, we will 
consider additional descriptive summarizations based on the said dichotomous operational 
definition. We will also consider stratifying by or adjusting for (as a covariate) the initial dose 
(20/40mg). 
 
 
2.5.4 Safety Analysis 
We will describe and descriptively compare proportions of those with clinical endpoints/adverse 
events such as hospitalizations, urgent clinic/ER visits, and death between the treatment groups, 
as using chi-square and Fisher’s exact tests for dichotomous outcomes with such a small number 
of participants is unlikely to reliably demonstrate statistical significance. 
 
2.5.5 Handling Missing Data 
We will make every effort to prevent missing data by keeping the participants as engaged as 
possible with the study. The recommended approach for handling missing data in inferential 
analyses is multiple imputation 93-95. We will construct m=5 analytic data sets after imputing each 
value with multiple guesses to represent inherent uncertainty in imputing missing values, analyze 
each data set as though complete, and finally combine the results from the analyses to obtain 
overall estimates. We will use SAS® MI and MIANALYZE procedures. 
 
2.5.6 Data Management 
The study forms will be paper-based in that the data will be first recorded on paper forms at the 
time of the interview. A relational database will be constructed on a local server with daily backups 
where only select research team members will have access to the database. The database will 
include data entry forms with the same appearance as the paper forms to facilitate accurate data 
entry, routine data edit checks for consistency both within and between forms, and provisions for 
double data entry. After data entry, the paper forms will be archived in secure file cabinets. 
Database development and maintenance will occur with Microsoft Access®. All study subjects will 
be assigned unique study identifiers that will appear on all data collection instruments, tapes, 

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documents, and files used in the statistical analysis and manuscript preparation. The case report 
forms with study IDs will be housed in the same chart as personal identifier documents are while 
the subject is active in the protocol. Personal information documents will be stored separately 
once each subject’s participation is complete. No documents will contain both the study ID and 
personal information at the same time.  Only limited team members will have access to charts 
and database. No personal information concerning study participants will be released without their 
written consent. Other data quality assurance measures will include verifying the data, out of 
range data checks, and repeated evaluation of the data process. 
 
3.  HUMAN SUBJECTS 
 
3.1 SUBJECT POPULATION 
 
The anticipated age range for the study population will be >70 years of age based upon the target 
disease population. Eighteen subjects with any ethnic background who have a confirmed 
diagnosis of HFpEF will be eligible for enrollment. All subjects must provide written informed 
consent prior to participation. 
 
3.1.1 Inclusion of Women and Minority 
Women who meet the inclusion criteria, and have none of the exclusion criteria, will be enrolled 
without restriction as dictated by the study protocols. We will make efforts to enroll participants in 
this research in a distribution that mirrors the study population of the Pittsburgh area.  
 
3.1.2 Inclusion of Children 
This investigation will not enroll children based upon the target disease population and lack of safety 
data in adults. 
 
3.1.3 Inclusion of Prisoners 
 
Prisoners will not be recruited for this protocol given the lack of resources required to conduct study 
visits and manage logistics. 
 
3.2 INCLUSION CRITERIA 
 
Inclusion Criteria: 
 
Age ≥70 years 
 
Diagnosis of HFpEF adapted from the ESC Guidelines, to include: 
1. Prior diagnosis of HF via one of these: 
 medical record diagnosis by attending cardiologist 
 verbal confirmation of HFpEF with attending cardiologist 
 PI review of medical record to confirm HFpEF 
2. EF% ≥40 
 
Clinically stable (euvolemic; baseline HR <100 bpm) and without hospitalization or invasive 
cardiac procedure for 6 weeks 
 
Patients using 81 mg aspirin (ASA) will be eligible, but will be asked to hold the medication 
for 3 days prior to biopsy. This technique has previously been used with consistent safety. 
Patients will also be asked to avoid non-steroidal anti-inflammatory medications (NSAIDs) 
for 2 days prior to the biopsy. 

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 
Patients using anti-thrombin and anti-platelet therapy will plan to modify prior to muscle 
biopsies individually in coordination with the participant's primary cardiologist. 
 
3.3 EXCLUSION CRITERIA 
 
Exclusion Criteria:  
 
Allergy to lidocaine 
 
BP >180/95 or <100/60 mm Hg (either systolic or diastolic) 
 
Major cardiovascular event or procedure within the prior 6 weeks 
 
HF secondary to significant uncorrected primary valvular disease (except mitral regurgitation 
secondary to left ventricular dysfunction). If valve replacement has been performed, patient 
may not be enrolled for 12 months after this procedure. 
 
Severe uncorrected primary valvular heart disease (if valve replacement has been 
performed, patients will not be eligible for at least 12 months) 
 
Mechanical valve replacement requiring warfarin 
 
Severe peripheral or pulmonary artery disease 
 
Currently taking clopidogrel for a recent stent placement and/or a complex atherosclerotic 
lesion such that holding clopidogrel creates disproportionate risk.  
 
Current use of organic nitrates or phosphodiesterase type 5 inhibitors (PDE5s)  
 
Unable to hold warfarin or use bridging therapy, or to hold (81 mg) aspirin for 3 days or (325 
mg) aspirin for 3 days prior to muscle biopsy or thienopyridine medications for 5 days prior 
to muscle biopsy. 
 
Anemia:  Hgb <11.0 (♂),10.0 (♀) gm/dl 
 
Dementia or inability to give informed consent 
 
End-stage malignancy 
 
Severe Orthopedic exercise limitation 
 
Chronic use of oral corticosteroids or other medications that affect muscle function 
 
Chronic alcohol or drug dependency 
 
Any bleeding disorder that would contraindicate biopsy such as history of clinically 
significant bleeding diathesis (e.g., Hemophilia A or B, Von Willebrand's Disease or 
congenital Factor VII deficiency) 
 
Psychiatric hospitalization within the last 3 months 
 
Subjects with diabetes whose HgbA1c >10.0% 
 
Other chronic unstable disease such as active neoplasm, end stage chronic kidney, liver or 
other organ disease  
 
Relative Exclusions  
 
Subjects with a non-3T MRI compatible pacemaker, implantable cardio defibrillator, stent or 
other identified metal in their body will be excluded from the Magnetic Resonance 
Spectroscopy study. 
 
Subject who use PDE5s for erectile dysfunction and are willing to withhold use 24 hours prior 
and during entire dosing period may be enrolled. 
 
Subjects may opt not to do the pre- and post- right heart catheterization exercise test. 
 
4. IRB APPROVAL AND FDA AMENDMENTS 
 
The Investigator will obtain from the University of Pittsburgh HRPO, prospective approval of the 
clinical protocol and corresponding informed consent form(s); modifications to the clinical 

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protocol and corresponding informed consent forms, and advertisements (i.e., directed at 
potential research subjects) for study recruitment.    
 
The only circumstance in which a deviation from the current HRPO-approved clinical 
protocol/consent form(s) may be initiated in the absence of prospective HRPO approval is to 
eliminate an apparent immediate hazard to the research subject(s). In such circumstances, the 
Investigator will promptly notify the University of Pittsburgh HRPO of the deviation. The 
Investigator should also notify the sponsor of this event.   
 
The University of Pittsburgh HRPO operates in compliance with FDA regulations at 21 CFR Parts 
50 and 21 CFR 56, and in conformance with applicable International Conference on 
Harmonization (ICH) Guidelines on Good Clinical Practice (CGP).  
 
In the event that the University of Pittsburgh HRPO requires, as a condition of approval, 
substantial changes to a clinical protocol submitted under an FDA-accepted IND application, or 
in the event of the Investigator’s decision to modify the previously accepted clinical protocol: 
 
 
for Phase 2 and 3 clinical studies:  The Sponsor will submit (i.e., in advance of 
implementing the change) a Protocol Amendment to the IND describing any change to a 
Phase 2 or Phase 3 protocol that significantly affects the safety of subjects, the scope of 
the investigation, or the scientific quality of the study. Examples of Phase 2 and 3 clinical 
protocol changes requiring the submission of a Protocol Amendment include: 
 
o Any increase in drug dosage or duration of exposure of individual subjects to the 
investigational drug beyond that described in the current protocol, or any significant 
increase in the number of subjects under study. 
 
o Any significant change in the design of the protocol (such as the addition or deletion 
of a control group). 
 
o The addition of a new test or procedure that is intended to improve monitoring for, or 
reduce the risk of, a side effect or adverse event; or the dropping of a test intended to 
monitor the safety of the investigational drug. 
 
 
5. RECRUITMENT AND INFORMED CONSENT PROCEDURES 
 
 
5.1 RECRUITMENT METHODS 
 
1. A variety of mechanisms will be used to recruit subjects for this protocol.  
a. UPMC 
Heart failure patients can be referred by their physician and/or cardiologist from the UPMC 
Advanced Heart Failure Center, General Cardiology clinic, Benedum Geriatric Center, the 
Comprehensive Pulmonary Hypertension (PH) Program or other UPMC or community facility. 
For UPMC Presbyterian/Montefiore-based clinics, we will obtain physician and HRPO 
approval for screening of subject medical records for identification of eligible candidates. Dr. 
Forman currently already has, and will confirm continued approval from the Advanced Heart 
Failure physician group to screen their patients. He will engage with additional cardiologists 
and geriatricians for permission to screen from those clinics. Once identified as preliminarily 

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eligible, the study coordinator/staff will reaffirm with the subject's physician on the clinic visit 
day that recruitment of the subject is medically appropriate. Staff will also request direction 
from the attending physician as to the timing of speaking to the subject, i.e., before or after 
the subject has seen the physician for their visit. This will be done on a case by case basis. 
Study staff will speak with candidates at clinic visits or by telephone call to assess interest and 
review medical history to assess eligibility.  
 
Subjects referred from facilities outside the UPMC Epic medical record system will be provided 
a medical record release for their cardiologist/physician to release needed documents to 
confirm eligibility. 
 
Subjects can also be identified from the inpatient Cardiology service. The study coordinator 
will follow the same procedure as for outpatients by screening the inpatient service for subjects 
admitted with heart failure symptomatology, check medical history for heart failure diagnoses 
and engaging the attending cardiologist as to appropriateness for the study. Such subjects 
will need to be followed for 6 weeks disease stability before enrollment, but recruitment and 
ascertainment of interest can be initiated, with consideration of medical circumstances in each 
case. As noted, permission to speak with subject will be obtained from the Cardiology 
attending. 
 
b. Research Registries 
Two research registries are available to the PI. The HRPO submission will include the request 
to use the Clinical Translational Science Institute’s PittPlusMe Research Registry, which 
includes a database of over 90,000 individuals who have indicated their interest in 
participating in research studies. The PittPlusMe initiative includes the use of engaging 
subjects via social media. 
 
Second, the Claude D. Pepper Center has a Research Registry of over 3000 community 
dwelling subjects that are >60 years of age and is also available once HRPO and Pepper 
Registry approves.  
 
c. Advertisements 
An approved study flier and recruitment brochure will be placed in key places and/or be 
distributed to physician offices, related clinics, or on other occasions/venues that present as 
an opportunity to recruit (e.g., a PI speaking engagement or a community outreach event to 
reach minority subjects). Potential subjects can self-refer by contacting the study staff via a 
telephone number/email address that is provided on these advertisements. Study staff will 
utilize the approved phone screening script when responding to interested candidates. With 
subject permission, they will be screened on the phone to make a preliminary assessment of 
eligibility. We will obtain permission to access their medical records in the UPMC database 
or request records from their provider as needed to further document eligibility.  
 
Advertisement, such as on radio, television, or print copy in newspapers, or bus signs may 
also be utilized depending on recruitment rates.  
 
2. Once subjects have been determined to be eligible from medical record review and attending 
approval, recruitment and enrollment procedures will then follow, including: 
a. Confirmation of eligibility by PI 

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b. Updating subjects’ cardiologist and confirming management of holding anti-
coagulants medication pre-muscle biopsy and pre-catheterization per protocol, if 
needed.  
c. Setting dates for patient visits with subject and staff will also occur prior to screening 
visit to maintain timeliness of visits per protocol 
d. Scheduling outpatient screening visit (Visit 1) where study risks and potential benefits, 
and rights as a research subject will be described in detail, informed consent will be 
obtained and where final eligibility will be confirmed. 
 
5.2 INFORMED CONSENT PROCEDURES 
 
Subjects must provide informed consent. The information about this study will be given to the 
subject in language understandable to them. Either the physician investigator or a non-physician 
member of the research team will present the study. They will verbally present a general outline 
of the research plan, including inclusion and exclusion criteria, to the prospective participant. The 
consent form, outlining the design of the study, will include the risks and benefits of participating, 
and will be reviewed and the physician investigator and/or non-physician member of the research 
team will answer any questions. Prospective participants may take as much time as required to 
make an informed decision. Written informed consent will be obtained from each participant and 
the physician investigator prior to performing any research study procedures. 
 
 
6. POTENTIAL RISKS AND BENEFITS 
 
6.1 POTENTIAL RISKS 
6.1.1 Risk of Experimental Drug Intervention 
 
NO2:  
Numerous studies have evaluated acute, subacute and chronic drinking water exposures of NO3 
and NO2 in laboratory animals and drinking water and dietary exposures in humans. Recent 
studies are available using high doses of NO2 by oral route in the form of beet root juice. Recent 
studies have evaluated acute exposures of oral preparations of NO2 and NO3 on PK and BP and 
are characterized below. More extensive human data is available on parenteral sodium NO2 as it 
is currently available and approved by the FDA for use in the emergency treatment of cyanide 
poisoning 96, 97. It is also notable that neutrapharmaceutical preparations are currently being sold 
with levels of NO2 (12.7 mg per tablet) and NO3 (3.9 mg per tablet). 
 
Sodium NO2 has been used commercially as a food preservative, an anti-corrosive agent, a 
coloring agent, and an anti-anginal agent, with additional uses in laxatives, burn ointments, and 
liniments. Amyl NO2 has been inhaled or ingested as a euphoric stimulant. NO2 has also been 
found as a contaminant in well water. Literature searches generated case reports of nausea, 
vomiting, abdominal pain, dizziness, headache, flushing, cyanosis, tachypnea, dyspnea, 
hypotension and death attributed to excess NO2 (high-dose) exposure from these sources as a 
consequence of methemoglobinemia due to oxidation of heme-iron in oxyhemoglobin. Normal 
background methemoglobin production is 1-3%. If levels of methemoglobin rise above 
approximately 30% of total hemoglobin, a subject may appear cyanotic and experience dyspnea, 
due to the reduced oxygen carrying capacity of hemoglobin (methemoglobin cannot bind oxygen). 
Levels above 50% can cause seizures, hypotension, coma and death. Sodium NO2 administration 
for cyanide poisoning at the labeled dosage of 300 mg causes methemoglobinemia, a desirable 
effect, as methemoglobin binds to cyanide, thus protecting cellular mitochondria. A standard dose 

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of NO2 used for cyanide poisoning is 300 mg up to 600 mg. Note that methemoglobin levels have 
never risen higher than 3% at the currently used therapeutic doses (<75 mg) in 80 volunteers in 
phase I studies at the NIH. 
   
The Sponsor of this study proposal, Dr. Gladwin, has previously held an IND for sodium NO2 (IND 
# 70,411) for cardiovascular applications and currently has an approved IND for the use of sodium 
NO2 for lung transplant recipients (IND # 111,643). The cardiovascular IND involved the 
administration of sodium NO2 to 69 normal volunteers in 4 phase I-II clinical trials without observed 
adverse effects. He has also treated 11 subjects with sickle cell disease on this IND without 
observed adverse effects. The lower doses of NO2 used in these investigational treatment 
regimens – 60-120 mg daily or 20-40% of the dose (300 mg) used in the emergency treatment of 
cyanide poisoning–do not produce methemoglobin levels greater than 3% and have not been 
associated with clinically significant hypotension. There have been no adverse events noted in 
the 80 treated normal human volunteers and patients with sickle cell disease 96, 98, 99. In another 
study by Gladwin et al., 18 healthy adults received an infusion of sodium NO2 totaling 75 mg (15 
minutes each x 2 infusions). This was associated with a 7 mm Hg decrease in mean arterial 
pressure, a peak methemoglobin of less 3% and no other significant effects 96. Note this single 
dose is 1.9 times the larger single dose (tid) we plan to use in this trial.  
 
In an open-label three-way crossover study, 9 healthy adult subjects received two single high 
dose oral sodium NO2 aqueous solutions (0.12 and 0.06 mmol NaNo2/mmol Hb, equivalent to 
290-380 mg and 140-190 mg sodium NO2, respectively, depending on the total body hemoglobin 
level of the person) and one intravenous sodium NO2 dose (0.12 mmol NaNo2/mmol Hb100. Note 
that this is 1.2-3.2 times the daily dose we plan to use in this trial. There was a washout period of 
at least 7 days between each of the treatments. Mild headache occurred in 44-55% of subjects 
and was the most frequent complaint during each treatment session, which the authors ascribed 
to the sodium NO2, not methemoglobinemia, as the percentage of methemoglobinemia stayed 
below clinically toxic levels (<15%). By report, up to 22% experienced nausea, which subsided 
within 30 minutes. The pharmacokinetic analysis of this study indicated similar bioavailability of 
oral and IV delivery of NO2, as well as similar side effect and safety profiles. 
 
A recent study determined the safety and feasibility of prolonged intravenous NO2 infusion. 
Twelve adult volunteers received increasing starting doses of sodium NO2, 4.2 to 533.8 μg/kg/hr 
for 48 hours. Dose limiting toxicity occurred at 445.7 μg/kg/hr (10.6 mg/kg/day) and was limited 
to asymptomatic transient decreases of arterial BP of up to 20 mmHg and asymptomatic 
increases of methemoglobin levels above 5%. No tolerance or clinically significant rebound was 
observed 101. Note, this is 8.2 times the daily dose we plan to use in this trial (based on an adult 
body weight of 95 kg). 
 
For NO2, two retrospective case-control studies have shown that high maternal dietary NO2 intake 
from cured meat or drinking water during pregnancy might be associated with risk of childhood 
brain tumors and possibly gastric and esophageal cancer. This evidence is only based on 
retrospective case-control studies; cohort studies have found no significantly increased risks 102. 
Note that in this protocol, no subjects will be of childbearing age. 
 
In the 2001 National Toxicology Program (NTP) Report summarizing 2-year rodent drinking water 
studies, there was no evidence of carcinogenic activity of sodium NO2 in male or female F344/N 
rats exposed to up to 130 mg/kg/day in males and 150 mg/kg/day in females, or in male B6C3F1 
mice exposed to up to 220 mg/kg/day. There was equivocal evidence of carcinogenic activity of 
sodium NO2 in the highest dose of 165 mg/kg/day in female B6C3F1 mice based on the positive 

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trend in the incidences of squamous cell papilloma or carcinoma (combined) of the forestomach. 
Exposure to sodium NO2 in drinking water resulted in increased incidences of epithelial 
hyperplasia in the forestomach. However, no chromosomal damage (genetic toxicity) was 
observed in three studies conducted in rats and mice in vivo 103. Taken together, these findings 
suggest minimal carcinogenic NO2-mediated risk. 
 
In PI Forman’s current pilot study, we are using 120 mg tid, lower or similar to doses of NO2 cited 
and used safely in any of these studies. Our higher NO2 dose of 40 mg three times daily is <40% 
of the dose for cyanide poisoning.  
 
To summarize, we anticipate the following symptoms by organ system and likely frequency of 
risk: 
Gastrointestinal 
 
Common:  none 
 
Frequent:  none 
 
Infrequent:  dry mouth 
 
Rare:  nausea, abdominal pain and vomiting 
Hematologic 
 
Common:  none 
 
Frequent:  none 
 
Rare:  methemoglobinemia 
Cardiovascular 
 
Common:  none 
 
Frequent:  hypotension in heart failure subjects on higher nitrite doses 
 
Rare:  flushing, tachycardia, hypotension in healthy controls 
Neurologic 
 
Common:  none 
 
Frequent: none 
 
Rare:  headache, dizziness, seizure, coma 
Respiratory 
 
Common:  none 
 
Frequent:  none 
 
Rare:  tachypnea, dyspnea, cyanosis 
Renal 
 
Nocturia:  infrequent 
 
 
6.1.2 Risk of Study Procedures 
 
Cardiopulmonary and Functional Assessments:  
Both non-invasive CPET (nCPET) and invasive CPET (iCPET) entail symptom limited (maximal) 
exercise provocation. The exercise stimulus is associated with a 1 in 10,000 chance of significant 
untoward outcome (e.g., myocardial infarction, arrhythmia), including the possibility of death. 
However, all those enrolled will have a physical exam immediately before the test to best insure 
they are stable; the nCPET will have the cardiologist or physician present or immediately 
available; the iCPET will have the study physician doing the right heart catheterization procedure 
immediately available, and who will ensure they are maximally safe and well-cared for if any 
problems develop. All physicians and study exercise testing personnel are all ACLS trained and 

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a code cart is in the immediate vicinity. In addition, patients’ cardiologists will be notified before 
patients are enrolled, and will be asked to approve only those patients they deem to be stable. 
 
During the other physical activity assessments (e.g., 6MWT, 5 minute steady-state treadmill walk, 
SPPB, and hand grip) there are also inherent exercise-associated risks, yet since nCPET will be 
completed first, that initial assessment provides some certainty that subjects are stable for the 
walking based assessments thereafter.  
 
 
Other problems that might also develop from a CPET include skin reactions to the electrode 
leads. Men may also need to have parts of their chest hair shaved in order to attach 
electrodes; this hair grows back over the next few weeks.   
 
iCPET facilitates delineation of the central vs. peripheral mechanisms underlying exercise 
performance and fatigability. As an exercise provocation, iCPET entails the same (nominal) 
exercise-related risk as nCPET. In addition, iCPET entails risks related to right heart and 
arterial line catheterizations with added risks. 
o Right heart and A-line catheter placement entail risks of infection, bleeding, and local pain. 
Therefore, meticulous efforts to keep the catheter site free of germs help to minimize these 
risks. Xylocaine numbing medicine is used to reduce any possibility of pain, but sometimes 
the Xylocaine itself produces a brief burning feeling as it enters through the skin. 
o For right heart catheterization, there are added risks of arrhythmia and perforation 
associated with intra-cardiac central line placement. The catheter is placed in the right 
heart through an internal jugular vein. This procedure is completed in the heart 
catheterization suite, providing state-of-the art fluoroscopy guidance as well as benefits of 
a top nursing staff, ACLS code teams, and optimal standards of sterility. Serious risks with 
right heart catheterization (arrhythmia, bleeding, puncture, mortality) are <1% in the 
literature, and <.01% at UPMC. Of note, right heart catheter is only left in place during the 
procedure and removed immediately thereafter, thereby minimizing risks (infection, 
migration, thrombosis) that are associated with prolonged right heart catheterizations125.  
Less serious risks (skin infection and/or bleeding) are also <1%.  
o Fluoroscopy also entails risks associated with cumulative radiation exposure, but since 
average fluoroscopic imaging time for a right heart catheterization is less than 30 seconds, 
radiation dose is minuscule. In pregnant women, exposure to radiation can cause birth 
defects, however pregnant patients are not eligible for this study.   
o Risks associated with radial line placement are primarily oriented to the possibility of 
residual occlusion 104. However, this has occurred in <.05% of cases at UPMC, and all 
have been temporary, resolving with conservative management. Other risks include 
pseudo-aneurysm, infection, and hematoma, which are <.05% cumulatively. In this 
protocol, radial artery catheterization will be completed in the hospital’s catheterization 
suite, which will help minimize risks overall, as compared to hospitals where A-lines are 
placed at bedside with much less controlled environmental circumstances. 
 Subjects are asked to hold anti thrombin agents (warfarin, NOACs) prior to right heart 
catheterization. Holding the anti-thrombin regimen s theoretically increases the risk of 
formation of thrombus and cardiovascular events, albeit a very small amount. Subjects will 
be reminded to resume their regimen post procedure 
 
o Echocardiograms present no direct risks. A gel is also applied to increase transmission 
of sound waves for optimal imaging quality. In rare instances, some people may find this 
to be anxiety producing. Dr. Gorcsan, who has advanced training in echocardiography, 
will oversee staff performing echocardiograms to assure these are reliably completed 
with a minimum of patient distress. 

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Skeletal Muscle biopsy 
 Bruising. Muscle biopsies are associated with a chance of bruising (about 1 in 100). Subjects 
are instructed in what signs or symptoms at the biopsy site warrant contacting the study 
coordinator post biopsy. 
 Muscle biopsies are also associated with a small chance of infection (less than one in 1000) 
and pain.  
Infection. Careful steps to keep the area free of germs will minimize infection risks. At the 
biopsy visit, subjects will be given instructions for care and signs or symptoms of infection 
for which they should contact study staff.  
Pain. Xylocaine numbing medicine is used to reduce any possibility of pain, but 
sometimes the Xylocaine itself produces a brief burning feeling as it enters through the 
skin. Additional numbing cream may be used in subjects who report lower pain thresholds 
or inadequate response to numbing in the past. The physician will evaluate the need and 
safety for this in select cases as needed. 
 Holding of anti-coagulant regimen. With the permission of the attending cardiologist, blood-
thinning medicines such as aspirin (81 mg - 3 days) as well as warfarin (3 days) or others will 
be held before the muscle biopsy, and restarted post muscle biopsy. However, if the primary 
cardiologist prefers, bridging therapy using a different blood thinning medication (enoxaparin) 
will be used while the aspirin and warfarin are on hold. Holding the anti-coagulant regimen 
which is in place for prevention of cardiovascular events theoretically increases the risk of 
cardiovascular events, albeit a very small amount.  
 
Risks of withholding anti-diabetic medication. 
Subjects may be asked to hold their anti-diabetic medication before or on the day of 
the exercise tests, biopsies and right heart catheterization visits. It will depend on the 
type of medication (and for the exercise test, current glycemic control), anticipated 
time of test and next meal. This may result in a transient increase in blood glucose 
which will self-resolve and does not carry long term risk. We will use Dr. Risbano and 
the UPMC Cardiac Catheterization lab care standard for guidance on this.  
 
Genetic testing 
 As a broad concept, results of genetic testing on muscle and blood are thought to influence 
future employment or insurability for subjects or their blood relatives if new diagnoses are 
identified. The results of these genetic tests are de-identified, subject to privacy laws and 
are not available to future employers or insurers. Further, because the results of the genetic 
tests performed in this study will only be linked to a disease condition that is already known, 
it is unlikely that the results of these genetic tests will have any significant impact on 
subjects’ current health profile. The usefulness of these results in providing treatment for 
medical conditions has not been determined because they are research tests. 
 
Magnetic resonance spectroscopy  
 The MR Research Center (MRRC) has extensive experience with MRS studies in diverse 
populations and is well-trained to reassure subjects. During these MRS sessions, even with 
previously scanned subjects, measures are taken to insure subject comfort. If any participant 
exhibits any significant clinical findings on any of the exams (e.g., tumor on MRI), they will be 
referred to appropriate clinicians. In the case of an emergency during participation in the study, 
the staff will initiate the appropriate emergency procedures as per standard operating 

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procedure at UPMC. All staff at UPMC involved in MRI/MRS scanning are trained yearly in 
safety and emergency protocols.  
 MRS is not associated with any known adverse effect except for people with metal or magnetic 
implants (such as metallic clips in the brain or cardiac pacemakers).  
o Metal objects within the body can heat to potentially dangerous temperatures 
or possibly move in the patient’s body.  
o Some types of tattoos (home-made) can also heat and cause discomfort. Any 
subjects with the possibility of these risk factors will not be tested in the 
MRI/MRS studies. 
o Metal objects can also become projectile when placed near the magnetic field. 
This has been reported, but it is a very rare occurrence. Protection from 
magnetic objects can be safeguarded by the usual safety techniques that are 
practiced in MRI/MRS sessions, including having subjects and researchers 
take all metal objects off of the subject before entering the environment. 
 
Thus any candidate who meets inclusion/exclusion criteria with the exception of a metal or 
possibility of metal in their body will be invited to participate, but will forego the MRI testing. 
 
Double screening of participants, by the study coordinator and again on the day of testing will 
be completed for thorough assurance that the subject is safe for MRI. 
 
 Another potential risk is claustrophobia caused by being in the enclosed space of the 
MRI/MRS scanners. Subjects who may become anxious or uncomfortable during any part of 
the procedure will be immediately taken out of the scanner and not tested. Subjects are 
instructed to talk to the experimenter if they feel uncomfortable for any reason during the 
testing procedures. If subjects feel uncomfortable they are immediately taken out of the 
scanner. 
 Noise levels in the magnet can be uncomfortable for subjects and they will be wearing 
earplugs through all MRI/MRS procedures. There is also microphone in the bore of the 
magnet. As above, subjects who feel uncomfortable are taken out of the scanner. 
 
NIRS 
 
Subjects may feel uncomfortable during the placement of the NIRS device onto their legs 
although it is not painful. There are some risks for minor skin irritation, redness, and chafing 
associated with the use of the doubled-sided tape to attach the NIRS sensors to the thighs. 
Though no other adverse effects from non-ionizing LED light source have been reported, it 
is possible that effects not yet reported may occur. 
 
Actigraphy 
 
Subjects will be instructed on wearing an ActiGraph GT3X+, non-invasive activity monitor for 
one week at the beginning and end of the study and during the treadmill nCPET and all 
walking testing performed. There may be the inconvenience of having to wear a device 
around the wrist, and a small chance of causing a rash or irritation of the skin but should be 
no more than that of watch wristband. It has a small flashing light that may slightly bother 
some people.  
 
Questionnaires  
 
Patients will answer questionnaires as part of the study assessments which require 
responses about daily activities, diet, quality of life, medication, health history, cognitive 

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ability and sleep which in some cases may be a source of emotional distress. Efforts will be 
made to keep the environment and support by staff to be reassuring and pleasant.  
 
Peripheral Blood sample 
 
Blood samples for NO2 monitoring as well as serological assessments for the study entail 
needle sticks and/or IV placement.. There is a small chance of infection and pain. Careful 
steps to keep the area free of germs will minimize infection risks. Furthermore, since only 
small gauge needles are used, pain is minimal.  
 
A risk of drawing blood is anemia, from a lower hemoglobin level. A common symptom of 
anemia is fatigue (feeling tired or weak). The amount of blood to be drawn over the course 
of this investigation is an approximate maximum of 224 ml over approximately 12 weeks, if 
subjects complete both pre- and post- right heart catheterizations (using an overestimation 
of HFpEF candidates cycling for 15 minutes during each catheterization.) This is equivalent 
to about 1 cup. For subjects who choose not to complete the right heart catheterization 
visits, the maximum volume for the study is about 168 ml, or about ¾ cup. Neither of these 
amounts are likely to cause anemia, especially with blood draws extended over 6-8 weeks. 
Also note that we are screening and excluding for anemia at Visit 1. 
 
Risks of fasting 
8 hours for visits 4 and 7 
Fasting for blood work is common and carries little risk. Subjects may feel tired, hungry or 
irritable until they are served the standardized breakfast upon arrival.  
For visits 3B and 6B 
These visits occur after an overnight fast and subjects will receive a meal after the testing, 
which may last 4-6 hours. Although there is no risk to a longer fast, subjects may feel tired, 
hungry or irritable until then.  
 
Overall subject burden  
 
Subject burden is a possibility due to the number of visits and particularly during the 
comprehensive physical assessment battery that is completed as part of the protocol. 
However, as noted in the Methods, precautions have been integrated with the assessments 
to minimize risk to the subjects. Rest periods are provided after each active functional 
assessment, moreover, active functional assessments alternate with ones that are completed 
at rest (questionnaires). Furthermore, at the end of each assessment and before the next 
functional assessment and the rate of perceived exertion (RPE) is reassessed to evaluate 
fatigability and to make sure that the subject has returned to baseline of physiological and 
subjective parameters prior to the next test. The iCPET option allows the subject to decide if 
they are willing to undergo that portion of testing. The consent language includes that subjects 
may withdraw at any time. The consent form includes language and Dr. Forman and the study 
coordinator will be sure that subjects understand the number, duration and activity for each 
visit, so as to fully inform potential subjects on this issue. 
 
6.2 ALTERNATIVE TREATMENTS 
 
The alternative treatments for the subjects participating in this investigation are to continue their 
medical care under the direction of their primary physicians. 
 
6.3 POTENTIAL BENEFITS 
 

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There may be no direct medical benefit to subjects in the study. It is hoped that researchers will 
learn more about the effectiveness of using oral NO2 to improve strength, gait speed and 
balance. Oral NO2 may also increase efficiency of work by reducing their oxygen uptake 
required for the same work intensity. We hope the information learned from this study will 
benefit other heart failure patients in the future. 
 
6.4 DATA SAFETY MONITORING 
 
6.4.1 Data Safety Monitoring Board (Safety Officer) 
 
In lieu of a Data Safety Monitoring Board, the funding agency, the National Institute on Aging, 
(NIA) has approved the use of a Safety Officer for this small sample, Phase II trial. Dr. Forman 
has appointed Dr. Saul Silver, UPMC Shadyside-Cardiology as the Safety Officer. The NIA has 
approved the appointment. The PI will provide Dr. Silver a detailed written summary of all adverse 
events on a monthly basis; any that are serious, unexpected, and potentially treatment-related 
adverse events (SAEs) will be immediately (within 24 hours) reported to the Human Research 
Protection Office, the Safety Officer, NIA Program Officer, and the Sponsor. In the monthly SO 
report, we will compare and contrast any SAE with prior events (or more frequently as indicated). 
Dr. Silver will review accumulating data from research activities to assure the continued safety of 
human subjects, relevance and appropriateness of the study, and the integrity of research data.  
See also sections 6.4.5 and 6.4.6 regarding reporting of SAEs and/or fatalities. 
To remain objective, Dr. Silver will maintain independent from the study. Accordingly, he will not 
be directly involved in the conduct of the study and will not have scientific, proprietary, financial 
or other interests that may affect independent decision-making. Signed documents confirming no 
conflict of interest will be provided with the application for approval to the NIA and maintained in 
the study regulatory binder. 
In addition, the report to Dr. Silver will address the following information as it is submitted to the 
University Of Pittsburgh Human Research Protection Office (HRPO) at the time of annual review 
or more often as required: 
• A list of the research personnel who participated in the data and safety monitoring.  
• The frequency of monitoring that took place during the renewal intervals and/or the  dates that 
data and safety monitoring was conducted.  
• A summary of cumulative data related to unanticipated problems (including adverse events) 
including a determination of causality and whether the risk to benefit assessment has 
changed.  
• If appropriate, a summary of pertinent scientific literature reports, therapeutic developments, or 
results of related studies that may have an impact on the safety of study participants or 
the ethics of the research study.  
• A summary of the outcome of reviews conducted to ensure subject privacy and research data 
confidentiality.  
• Final conclusions regarding changes to the anticipated benefit-to-risk assessment of the study 

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participation and final recommendations related to continuing, changing, or terminating 
the study.  
6.4.2 Data Safety Monitoring Plan  
 
Monitoring of safety and data quality in the proposed study will be the responsibility of all 
personnel on the project, with primary responsibility and supervision by the PI. The University of 
Pittsburgh HRPO will approve the informed consent document for the study and provide 
institutional oversight of data and safety issues. The study protocol will be approved prior to 
recruiting or obtaining consent from any participants. Moreover, the study will be reviewed at a 
minimum of an annual basis by the HRPO committee. Each participant will sign the informed 
consent document described above prior to participating in the study. To ensure participant safety, 
once participants are enrolled in the study, study staff will immediately report all adverse and 
serious adverse events to one of the physician investigators or PI. The PI will, per standardized 
procedures, report serious adverse events to the HRPO, Safety Officer, FDA, and NIA Program 
Officer for their review and to the sponsor as indicated. With regard to monitoring of data quality 
and protected health information, all required personnel proposed for this project will have the 
required human subjects and confidentiality training, which includes information about 
maintaining data integrity and security. Confidentiality will be guarded using established 
procedures such as double locked files and facilities for storing research charts, coding by study 
identification numbers rather than any personally identifying information to avoid revealing the 
identity of subjects, and aggregating data across participants. The key linking names and study 
identification numbers will be kept separately from the data sets with limited access by study 
personnel. Only study personnel will have access to the data sets on protected servers. In order 
to maintain the highest standard of data entry quality, all data will be double-entered, with 
discrepancies highlighted so that they can be reviewed by the project coordinator. Oversight of 
all aspects of data management will occur by the PI. 
 
Data Monitoring Plan. The proposed study will use the FDA definition of adverse events (AE) and 
serious adverse events (SAE). Any SAE, which is unexpected and related to study intervention, 
will be reported immediately to the HRPO, Safety Officer, FDA, and NIA Program Officer and will 
be followed by an additional letter detailing the nature of the SAE. In the event that a participant 
either withdraws from the study or the investigators decide to discontinue a participant due to a 
SAE, the participant will be monitored by the PI physician investigator until (a) a resolution is 
reached (e.g., the problem has resolved or stabilized with no further change expected), (b) the 
SAE is determined to be clearly unrelated to the study intervention, or (c) the SAE results in death. 
Outcomes of SAEs will be regularly reported to the HRPO and the sponsor. A summary of the 
SAEs that occurred during the previous year will be included in the annual progress report as well 
as in the annual HRPO renewal. 
 
6.4.3 Parameters to be monitored 
 
The following progress will be monitored throughout the course of the research to ensure the 
safety of subjects as well as the integrity and confidentiality of their data. 
 
 
An evaluation of the progress of the research study, including subject recruitment 
and retention, and an assessment of the timeliness and quality of the data. 
 
 
A review of collected data (including adverse events, unanticipated problems, and 
subject withdrawals) to determine whether there is a change to the anticipated 

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benefit-to-risk assessment of study participation and whether the study should 
continue as originally designed, should be changed, or should be terminated. 
 
 
An assessment of external factors or relevant information (e.g. pertinent scientific 
literature reports or therapeutic development, results of related studies) that may 
have an impact on the safety and study participants or the ethics of the research 
study. 
 
 
A review of study procedures designed to protect the privacy of the research subjects 
and the confidentiality of their research data. 
 
The severity of adverse changes in physical signs or symptoms will be classified as follows: 
 
 
Grade 1 (Mild): asymptomatic or mild symptoms; clinical or diagnostic observation only; 
intervention not indicated. 
 
 
Grade 2 (Moderate): minimal, local or noninvasive intervention indicated; limiting age-
appropriate ADL (Activities of Daily Living). 
 
 
Grade 3 (Severe): medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care 
ADL. 
 
 
Grade 4 (Life-threatening): consequences; urgent intervention indicated. 
 
 
Grade 5 (Death): event is a direct cause of death. 
 
6.4.4 Frequency of Monitoring 
Dr. Forman will review subject safety data as it is generated. Dr. Forman and the research staff 
will meet at least bi-weekly to re-evaluate study goals, subject recruitment, data coding and 
retention, documentation and identification of adverse events, complaints and confidentiality of 
subjects. There will be an evaluation of the progress of the research study, including assessments 
of data quality, time lines, participant recruitment, accrual, and retention. Dr. Forman will also 
review the outcome and adverse event data to determine whether there is any change to the 
anticipated benefit-to-risk ratio of study participation and whether the study should continue as 
originally designed or should it be re- evaluated and changed. Dr. Forman will meet with the entire 
investigatory team at regular intervals for broader discussion of relevant details. Simultaneously, 
the Safety Officer will receive monthly reports of all AEs as described in section 6.4.1. 
6.4.5 Reportable Adverse Event 
For this study, a serious adverse event is any untoward clinical event that is thought by Dr. 
Forman, PI, to be study-related, and results in any of the following outcomes: 
1. Death 
2. A life threatening adverse event  
3. Inpatient hospitalization or prolongation of an existing hospitalization   

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4. A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
5. A congenital anomaly or birth defect 
6. Important medical events (that may not result in death, be life threatening, or require 
hospitalization) may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the patient, or subject, and may require medical, or surgical 
intervention to prevent one of the serious outcomes listed above. 
 
If clinically important and unexpected adverse experiences or clinically important study-related 
adverse experiences occur, they will be recorded on the adverse event case report form. 
6.4.6. Adverse events reporting timeline 
Life-threatening or fatal unexpected adverse events associated with the use of the study drug or 
procedures must be reported to the HRPO within 24 hours of discovery of the incident with 
subsequent follow-up submission of a detailed written report. The FDA must be notified by 
telephone or facsimile transmission of a human adverse event that is fatal or life-threatening no 
later than 7 calendar days after receiving the respective human adverse event information, 
followed by the subsequent submission of a written IND Safety Report. 
Serious and unexpected adverse events associated with the use of the study drug or procedures 
must be reported to the HRPO with subsequent follow-up submission of a detailed written report 
in accordance with the respective policies and procedures of the HRPO. Written IND Safety 
Reports will be submitted to the FDA as soon as possible and, in no event, later than 15 calendar 
days following the investigator-sponsor’s receipt of the respective adverse event information.  
All reports noted in section 6.4.6 will also be provided to the NIA Program Officer and the protocol 
Safety Officer. 
6.4.7. Withdrawal of Subjects and Stopping Criteria 
Withdrawal of Subjects due to Adverse Events: 
As described in Section 2.3.3. Dose Selection, sub-section Dose Reduction, we propose to 
discontinue participation for any subject who experiences any of the following: 
 
a. Continued MetHB level >5% or continued lightheadedness and/or dizziness after protocol 
described in Section 2.3.3 for dose reduction is followed. 
 
Other symptoms 
b. The PI, via subject phone call monitoring, AE reports or at follow-up visits, will evaluate 
any other clinical presentation, sign or symptom in subjects which may warrant 
consideration of interim visit and dose reduction. The procedure described in Section 
2.3.3. Dose Selection, sub-section Dose Reduction (regarding drug discontinuation and 
determination or need to withdraw the subject) will be followed. 
 
Other Reasons for Withdrawal 
Medication compliance 
c. In the event that the medication compliance rate is <80%, subjects will be re-educated on 
medication compliance. If medication compliance repeatedly falls outside of the 

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acceptable range, the study investigators will discuss subject eligibility for continued 
participation in the study. 
d. Participants who complete the first biopsy and then state (prior to being given drug) that 
they will not complete a second biopsy at Visit 7 will be withdrawn from the study. 
Alternatively, patients who refuse the 2nd biopsy after they have completed the 4 weeks 
of NO2/placebo therapy will still complete the other post drug assessments. If other unique 
subject situations arise, they will be evaluated by the PI and a decision will be made with 
regard to completing the other post assessments or not (e.g., completion of 2 weeks on 
drug, and then refusal to complete biopsy.) 
 
e. The PI will evaluate any other clinical presentation, sign, symptom or behavior in subjects 
which may warrant consideration of withdrawal. 
 
Discontinuation of the Clinical Trial 
 
Stopping Rule: 
Extensive published literature and considerable local experience at University of Pittsburgh 
indicates NO2 is not inherently dangerous at the doses targeted in this trial. Certain side effects 
of NO2 are idiosyncratic in older HFpEF patients and it is anticipated that there will be subjects 
who do not tolerate the treatment who may need to be withdrawn from the study as described for 
symptoms in 6.4.7. Nonetheless, if a subject enrolled in the study experiences a fatal event that 
is directly attributable to the NO2, the overall study treatment will be discontinued.  
 
6.5 RISKS MANAGEMENT PROCEDURES 
 
6.5.1 Protection Against Risks  
 
General Risks of Study Protocol and Procedures 
All research interventions/activities will be conducted in private patient care areas. The collection 
of sensitive information about subjects is limited to the amount necessary to achieve the aims of 
the research, so that no unneeded sensitive information is being collected. 
 
All demographic and clinical information about the subject during recruitment will be stored on an 
electronic password-guarded study database under the supervision of the Investigator for this 
protocol. The electronic database that is being used for the purpose of this study has not been 
fully validated to be in compliance with the FDA regulations at 21 CFR Part 11; i.e., due to the 
limited scope of this clinical investigation. The case report forms will be stripped of individual 
identifiers and stored anonymously with a subject number. Information linking subject identifiers 
with the coded subject number will be stored under password protection on computers in locked 
areas, with access only to the database manager. Maintaining records in locked files in locked 
offices will protect confidentiality of subjects. All staff will sign confidentiality statements. Access 
to the database will be limited to the data manager and staff under the supervision of the 
Investigator. Minimal identifying information will be stored in the subject research chart while 
active in the protocol. No research chart will contain documents with personal identifiers and their 
study subject number. Hard copy charts are stored in locked files in a locked office. 
 
Research blood and muscle specimens will be collected in vials with only subject identifiers and 
stored according to a similar coding protocol as described above. These specimens will be stored 
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will limit future access to any remaining samples to only those investigators with prior HRPO 
approval for their studies. 
 
All staff involved in this study are properly credentialed and instructed in the areas human subject 
research, testing, confidentiality, and safety.  
 
The Investigator will retain the data for the entire period of this study and will retain the specified 
records and reports for up to two years after the marketing application is approved for the 
investigational drug; or, if a marketing application is not submitted or approved for the 
investigational drug, until two years after investigations under the IND have been discontinued 
and the FDA so notified. The Investigator may continue to use and disclose subjects’ de-
identified information for the purpose of this study for a minimum of seven years after final 
reporting or publication of the study. If the subject and/or legal representative decide to withdraw 
or be withdrawn from study participation, they may request that the study data and samples be 
destroyed. Subject names or other directly identifiable information will not appear on any 
reports, publications, or other disclosures of clinical study outcomes. 
 
Source data to be included in the clinical study data, either on the original form or recorded by 
staff include: 
a. Information taken from or copies of electronic medical records to confirm 
inclusion/exclusion criteria 
b. Echocardiogram testing and interpretation  
c. All progress notes 
d. Communications from referring physicians 
e. De-identified emails communicating subject clinical information to PI or study physician 
f. 
Assessments of vitals, body weight at all visits 
g. Data generated from the nCPET, iCPET, MRS and all functional assessments,  
h. Questionnaires completed by subjects 
i. 
CTRC records of patient visits  
j. 
Analyses of screening and outcome assessments of blood and muscle tissue from internal 
and external investigator lab analyses  
k. Data collected from subjects’ drug and diary cards 
 
Missing data points that are anticipated will be documented in the progress notes and evaluated 
with HRPO input regarding the need for an Exception Report, i.e., unanticipated absence of 
staff or investigator to perform test, unanticipated absence of subject. Unintentional missing 
data due to subject inability will be noted. These will be reported to the PI for evaluation and 
corrective action plan implemented as needed. 
 
All staff performing assessments that are witness to data collection which may be spurious are 
advised to communicate to the PI. As most of the measurements are objective, this is not 
anticipated to be an issue.  
 
6.5.2 Protection Against Potential Risks of Experimental Intervention   
 
 
Involvement by trained staff/investigators with experience in the administration of the study 
drug: 
Dr. Forman, PI, and physician Co-Is, Drs. Hughan and Simon are principally responsible 
for monitoring and protecting the safety of subjects’ use of sodium NO2. Their combined 

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experience in the use of the study drug is adequate to protect against (the relatively low) 
risks. 
 
 
Continuous monitoring by the Data and Safety Monitoring Board: 
 
A Safety Officer, as noted in section 6.4.1, will function in lieu of a DSMB for this protocol. 
The monitoring plan is described there as well. 
 
 
Required Education in the Protection of Human Research Participants: 
 
The Principal Investigator and all sub-investigators listed on the University of Pittsburgh 
HRPO approved protocol are required to complete the Collaborative Institutional Training 
Initiative (CITI) courses in research fundamentals, including Human Subjects Research 
and Responsible Conduct of Research. Staff will complete these courses as well, prior to 
any subject contact. Investigators and staff with subject contact will also complete the 
Good Clinical Practice module. Investigators with an identified Conflict of Interest (COI) 
will complete the COI module. These web-based tutorials are a requirement of the HRPO 
for investigators prior to protocol submission.  
 

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7.  COSTS AND PAYMENTS 
 
7.1 COSTS 
 
Study drug/placebo and all research testing will be supported by ongoing research grants. All 
medications, lab tests, and any procedures described will not be billed to the subjects and/or their 
health insurance company.  
 
7.2 PAYMENTS 
Subjects will be reimbursed $325 or $475 for the baseline study visits after completion of Visit 4 
[Visit 1 ($75), Visit 2 ($75), Visit 3A ($75), Visit 3B ($150)-optional, Visit 4, ($100)]. Subjects will 
be reimbursed an additional $350 or $500 following the completion of Visit 7 [Visit 5, ($75), Visit 
6A ($75), Visit 6B ($150)-optional and Visit 7 ($100) and study drug/placebo compensation 
($100)]. The total compensation will be $675 or $975 paid. Subjects will also be provided a ticket 
for outpatient parking costs at all visits. In the event that a subject must be withdrawn for any 
reason, they will be reimbursed for the individual study visits that were completed to date per the 
above reimbursement schedule. 
 
No heart 
cath visits
Doing heart 
cath visits
Participant Reimbursement V1 (Informed consent, physical, 
Hemoglobin, 3DFR, echo) 
$75
$75
Participant Reimbursement V2 (nCPET, SS, 6MWT, NIRS, SPPB, 
HG, Actigraphy, Questionnaires) 
$75
$75
Participant Reimbursement V3A (MRS) 
$75
$75
Participant Reimbursement V3B (iCPET, w/echo) 
$150
Participant Reimbursement V4 (Biopsy, research bloods, pK, Drug)  $100
$100
Payment #1 
$325 
$475 
Participant Reimbursement V5 - Post  (nCPET, SS, 6MWT, NIRS, 
SPPB, HG, Actigraphy, Questionnaires), 3DFR
$75
$75
Participant Reimbursement V6A- Post (MRS)  
$75 
$75 
Participant Reimbursement V6B- Post (iCPET w/ echo)  
 
$150 
Participant Reimbursement V7- Post (biopsy and echo)  
$100 
$100 
Study drug/placebo 
$100 
$100 
Payment #2
$350
$500
TOTAL:
$675
$975
Subjects who require an interim visit will be compensated $40 and provided parking. 
 
 
 
8.  QUALIFICATIONS AND SOURCE OF SUPPORT 

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8.1 QUALIFICATIONS OF THE INVESTIGATORS 
Sponsor: 
 
Mark Gladwin, MD, is Professor of Medicine, University of Pittsburgh School of Medicine. Dr. 
Gladwin is the Chairman of the Department of Medicine and Director of the Vascular Medicine 
Institute (VMI) at the University Of Pittsburgh School Of Medicine. He is an internationally 
recognized authority in the field of sodium NO2 including both the basic science and a broad range 
of clinical applications in cardiovascular disease. He is a current IND holder for the investigation 
of sodium NO2 in lung transplant. 
 
Investigators: 
PI:  Daniel Forman, MD, is a Professor (Pending) of Medicine at the University of Pittsburgh 
School of Medicine, Department of Medicine and Chair, Geriatric Cardiology Section, Divisions of 
Geriatrics and Cardiology. Dr. Forman has a well-established track record of translational work 
focused on the interplay between skeletal muscle and physical function in older HF patients. 
Moreover, he is an expert in functional assessments in older HF patients. He is responsible for all 
aspects of this investigation, and will work directly with research staff to screen patients, 
coordinate data collection and quality, and he will personally complete all muscle biopsies and 
supervise all functional evaluations. He will play a primary role in data analysis and publications. 
Sub-Investigators: 
Mark Gladwin, MD is Professor of Medicine, University of Pittsburgh. Dr. Gladwin is the Chairman 
of Medicine, Department of Medicine, former Chief, Pulmonary, Allergy, and Critical Care 
Medicine and Director of the VMI at the University of Pittsburgh. He is an internationally 
recognized authority in the field of sodium NO2 including both the basic science and a broad range 
of clinical applications in cardiovascular disease. He is a current IND holder for the investigation 
of sodium NO2 in lung transplant. He will be responsible for data interpretation. 
  
Sruti Shiva, PhD is an Associate Professor in the Department of Pharmacology and Chemical 
Biology and VMI at the University of Pittsburgh. Her research lab focuses on the mechanisms by 
which reactive nitrogen species (particularly NO2 and NO) regulate mitochondrial function, the 
factors that influence this regulation and the implications of this regulation on pathology. 
 
John Gorcsan, III, MD is a Professor of Medicine and Director of Echocardiography Laboratory. 
He will provide his expertise and two-decade experience in quantification of left ventricular 
function by echocardiographic methods. He has extensive experience in whole organ ventricular 
physiology and quantitative echocardiography in patients with both depressed and preserved 
ejection fraction, having directed echocardiography core labs for multi-center studies in the past.  
 
Nancy Glynn, PhD is Assistant Professor, Epidemiology, who completed formative work that 
established and validated indices of performance and perceived indices of fatigability, i.e., 
performance fatigability which refers to the mechanistic property of declining function during a 
constant physical activity stimulus, and perceived fatigability as a perception of tiredness 
associated with a physical activity stimulus. 
 
Bret Goodpaster, PhD is Director of the Exercise Metabolism Core and Professor at the Sanford 
Burnham Medical Research Institute, Orlando, Florida. In his prior work at the University of 

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Pittsburgh he developed expertise regarding in vivo assessment of skeletal muscle bioenergetics 
with 31P magnetic resonance spectroscopy as a means to assess oxidative ATP synthesis. He 
will oversee the processes used to analyze muscle biopsy samples and assist with interpretation 
of de-identified data. 
 
Hoby Hetherington, PhD, MS is Director of the Magnetic Resonance Research Center at UPMC 
Presbyterian and Professor, Department of Radiology, University of Pittsburgh School of 
Medicine. He will facilitate acquisition of MRS spectroscopy as well as quality assessment of all 
data collected. Dr. Hetherington will also work directly with Drs. Goodpaster and Santanasto to 
facilitate analysis and interpretation of de-identified MRS data as part of an ongoing series of 
protocols that include multiple study cohorts.  
 
Subashan Perera, PhD is an Associate Professor, Division of Geriatric Medicine and 
Department of Biostatistics at the University of Pittsburgh and has over 14 years of experience 
in providing statistical support to health science research at major academic medical institutions. 
He is a Senior Statistician and Co-Leader of Data Management, Analysis and Informatics Core of 
the NIA-funded Pittsburgh Claude D. Pepper Older Americans Independence Center. In addition, 
he is the principal source of statistical support to numerous clinical trials and intervention studies 
funded by NIH, PCORI, AHRQ, CMS and VA. Dr. Perera will play a key role in randomization, 
data analyses and publications. 
 
Kara Hughan, MD is an Assistant Professor in the University of Pittsburgh School of Medicine, 
Department of Pediatrics, Division of Pediatric Endocrinology and Diabetes. Dr. Hughan has 
designed and led the Phase I and early Phase II human subject trials with our IND-approved oral 
NO2 at the University of Pittsburgh. She has extensive experience with evaluation of pK/pD, safety 
and efficacy of oral NO2. As Co-Investigator, she will play a role in data collected on oral NO2 drug 
metabolism (plasma NO3/NO2 concentrations, RBC NO concentrations), safety (BP, MetHb) and 
efficacy (platelet and muscle mitochondrial function) and perform related statistical analyses on 
the data gained on these outcomes. Dr. Hughan will also assist with related trial regulatory/DSMB 
reporting. Dr. Hughan will work with Dr. Forman and his team on implementing the NO2 therapy 
in this study. Dr. Hughan will provide leadership in protocol refinement, manuscript writing and 
publications. 
 
Marc Simon, MD, MS is an Associate Professor of Medicine in the Department of Medicine-
Cardiology and Director, Heart Failure Research/Clinical Hemodynamics Core Facility of the VMI 
who will oversee the iCPET cardiac catheterization. His work brings added insights regarding RV 
adaptation to elevated PA pressures. Using right heart catheterization and imaging techniques 
during iCPET, he has developed novel techniques to characterize RV contractility (end-systolic 
elastance, [Ees]) and its relationship to pulmonary artery (PA)-dependent afterload (arterial 
elastance, [Ea]). He will be available for call coverage for 24 hour physician investigator 
availability to subjects. 
 
Anne Newman, MD, MPH is Chairperson of the Department of Epidemiology, Graduate School 
of Public Health, University of Pittsburgh and has been a seminal leader in understanding the 
relationship between lean body mass, relative proportions and configuration of fat and related 
impact on strength and health. Dr. Newman will interpret related data from the study and 
participate in manuscript preparation. 
 
Theodore Huppert, PhD is an Associate Professor in the Department of Radiology and has 
extensive experience in the area of non-invasive optical imaging (NIRS) both within the area of 

Oral Nitrite for Fatigability in HFpEF 
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the human brain as well as muscle. He is the director of the NIRS Brain Imaging Laboratory at 
University of Pittsburgh Dr. Huppert is an biomedical engineer and will work with Dr. Forman and 
his team on implementing the NIRS measures in this study. Dr. Huppert will provide leadership in 
protocol refinement, manuscript writing and publications, and will participate on other study 
committees as needed. Dr. Huppert will also work closely with Dr. Forman on the collection and 
novel examination of NIRS data in association to fatigability.   
 
Adam Santanasto, MPH, PhD is a Research Assistant in the Department of Epidemiology with 
particular expertise in the conduct of MRS spectroscopy in older adults. Dr. Santanasto will 
administer the MRI testing protocol. Dr. Santanasto will also participate in data analyses and 
manuscript preparation. 
 
Frederico Toledo, MD is an Endocrinologist and Associate Professor of Medicine at the 
University of Pittsburgh and the Director of the EMRC. He has specific expertise in evaluation of 
the relationship of mitochondria impact on the human body. As part of this project, Dr. Toledo will 
advise on withholding of hypoglycemic medications, provide backup for performing muscle 
biopsies and overseeing exercise tests as needed and provide insight on data analyses and 
publications.  
 
Michael G. Risbano, MD is an Assistant Professor of Medicine in Division of Pulmonary Allergy 
and Critical Care Medicine in the Department of Medicine at the University of Pittsburgh. He is an 
attending physician in the Comprehensive Pulmonary Hypertension Program and performs right 
heart catheterizations to diagnose and follow the hemodynamic progression of patients with 
pulmonary hypertension. He will perform the right heart catheterizations of the subset of subjects 
who choose to undergo the procedure and contribute to the data analysis. 
 
Manisha Jhamb, MD, MPH is an Assistant Professor in the Department of Medicine, Division of 
Renal-Electrolyte. Her research focuses on exercise benefits for CKD patients, including 
translational focus on gene expression changes in skeletal muscle that are induced by exercise 
routines. Dr. Forman is acting in a mentoring role to Dr. Jhamb as she learns skeletal muscle 
biopsy and analytic techniques. She will also assist in this protocol with patient visits and call 
coverage for 24 hour physician investigator availability to subjects. 
 
Gavin Hickey, MD an Assistant Professor of Medicine in the Department of Medicine-Cardiology. 
He will assist with oversight of nCPET testing, medical review of patients, patient visits and call 
coverage for 24 hour physician investigator availability to subjects. 
 
Jessica Bon Field, MD, MS is an Assistant Professor of Medicine in Division of Pulmonary 
Allergy and Critical Care Medicine in the Department of Medicine at the University of Pittsburgh. 
Dr. Field’s work emphasizes the impact of COPD on function. She will assist with oversight of 
nCPET testing, medical review of patients, performance of muscle biopsies, patient visits and call 
coverage for 24 hour physician investigator availability to subjects. 
 
Andrea Levine, MD is a fellow in Pulmonary Medicine. Her research focuses on changes in 
protein expression in muscle in cardiovascular disease. She will be doing analysis of samples 
and also can provide assistance for coverage of functional testing and other study visits. 
 
Consultants 
Greg Lewis, MD is Assistant Professor of Medicine, Harvard Medical School, Director, 
Massachusetts General Hospital Cardiopulmonary Exercise Laboratory, and Director, 

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Massachusetts General Hospital Cardiology Intensive Care Unit. Dr. Lewis is a world expert in 
iCPET with particular insight in the application of iCPET to distinguish between central (cardiac; 
pulmonary) vs. peripheral (arterial oxygen utilization) mechanisms that determine exercise 
performance. His insights and skills for data analysis will complement those of Dr. Forman in 
nCPET. 
 
Rebecca Vanderpool, PhD is formerly a Postdoctoral Associate at the University of Pittsburgh, 
now faculty at the University of Arizona Department of Medicine, Division of Pulmonary, Allergy 
and Critical Care Medicine and Pulmonary Hypertension Program. Dr. Vanderpool brings 
extensive expertise in the analysis of invasive cardiopulmonary stress tests. She will work in 
conjunction with Drs. Simon and Lewis in the analysis of the iCPET data, particularly in regard to 
assessments of RV-PA coupling.  
 
 
 
8.2 SOURCES OF SUPPORT 
National Institutes of Aging Grant 1R56AG051637-01A1 
 
 
 

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