Systemic Sclerosis (SSc) Vasculopathy: Improved Clinical Monitoring and Treatment
Phase: PHASE1
Conditions: Rheumatologic Disease
Sponsor: VA Office of Research and Development

 
1
Protocol Summary 
Version 9: March 2015 
 
“Vascular and Autonomic Maladaptations in Patients with Vascular Dysfunction”. 
 
Principal Investigator: 
Russell S. Richardson, Ph.D., Professor of Medicine 
Department of Medicine, Division of Geriatrics 
University of Utah and GRECC. 
 George E. Whalen VA Medical Center 
500 Foothill Drive, Salt Lake City, UT 84148 
(760) 207-4570 
(801) 584-5640 (fax) 
r.richardson@hsc.utah.edu 
 
Co-Investigators: 
Josef Stehlik M.D., MPH 
Department of Medicine, Cardiology, University of Utah 
Cardiovascular Center, University Hospital 
50 North Medical Drive, Salt Lake City, UT 84132 
(801) 585-5122; Josef.Stehlik@hsc.utah.edu 
 
Mark A. Supiano, M.D. 
Department of Medicine, Division of Geriatrics, University of Utah and GRECC. 
 George E. Whalen VA Medical Center 
500 Foothill Drive, Salt Lake City, UT 84148 
 (801) 587-9103; mark.supiano@hsc.utah.edu 
 
Phillip Kithas, M.D. 
Department of Medicine, Internal Medicine 
George E. Whalen VA Medical Center 
500 Foothill Drive, Salt Lake City, UT 84148 
(801) 587-1565 ext. 2657 
 
Jose N. Nativi Nicolau, M.D. 
Department of Medicine, Cardiology, University of Utah 
Cardiovascular Center, University Hospital 
50 North Medical Drive, Salt Lake City, UT 84132 
(801) 339-1147; jose.nativi@hsc.utah.edu 
 
 
David Morgan, M.D. 
30 N 1900 E RM 3C444, SLC, UT 84132-2101 
801-581-6393 
david.morgan@hsc.utah.edu 
 
Amber Bledsoe, M.D. 
30 N. 1900 E RM 3C444, SLC, UT 84132-2101 
801-581-6393 
Amber.bledsoe@hsc.utah.edu 
 
 

 
2
Van Reese, M.S.  
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4335; Van.reese@hsc.utah.edu 
 
Markus Amann, Ph.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4335; markus.amann@utah.edu 
 
Joel D. Trinity, Ph.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4335; joeltrinity@mail.utexas.edu 
 
D. Walter Wray, Ph.D. 
Department of Medicine, Division of Geriatrics, University of Utah and GRECC 
 George E. Whalen VA Medical Center 
500 Foothill Drive, Salt Lake City, UT 84148 
(858) 534-6967   
walter.wray@hsc.utah.edu 
 
Melissa A. H. Witman, Ph.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
melissa.hayman@utah.edu 
 
Ryan Garten, Ph.D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
Rsgarten@gmail.com 
 
Jon Groot, B.S. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
wadodude@hotmail.com 
 
Matt Rossman, B.S. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
Matt.Rossman@utah.edu 
 
Song Young Park, B.S. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
Song.Park@utah.edu 
 
Jayson Gifford, B.S. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
jayson.gifford@utah.edu 
 
 
 
 

 
3
Maydeen Ogara, A.S., CRA, AGTS (Clinical Research Coordinator) 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 1495 
maydeen.ogara@hsc.utah.edu 
 
Simran Sidhu, Ph.D. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
(801)582-1565 ext. 4313 
Simran.Sidhu@utah.edu 
 
Joshua Weavil, M.S. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
(801)582-1565 ext. 4313 
j.weavil@utah.edu 
 
Ashley Nelson, M.D. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
(801)582-1565 ext. 4313 
Ash.Nelson@hsc.utah.edu 
 
Tyler Mangum, B.S. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 4313 
T.mangum@utah.edu 
 
Josh Lee, Ph. D. 
VA Medical Center Bldg 2, 500 Foothill Drive, SLC UT 84148 
801-584-5658 
J.F.Lee@utah.edu 
 
Heather Clifton, Ph. D. 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
582-1565 ext. 1949 
heather.clifton@utah.edu 
 
Daniel Machin, Ph. D. 
Internal Medicine 
VA Medical Center Bldg. 2, 500 Foothill Drive, SLC UT 84148 
801-582-1565 Ext. 4334 
d.machin@utah.edu 
 
 
Tracy Frech, M.D. 
Rheumatology 
4B200 School of Medicine, 30 N. 1900 E. 
Salt Lake City, UT  84132 
801-548-4837 
Tracy.Frech@hsc.utah.edu 
 
 
 
 

 
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Mark Sievert, M.D.  
School of Medicine, 50 N. Medical Drive 
Salt Lake City, UT  84132 
608-215-2310 
Mark.sievert@hsc.utah.edu 
 
Anthony Donato, M.S. Ph.D. 
Geriatrics, VAMC 500 Foothill Drive 
Building 2, Room 1D30 
Salt Lake City, UT  84148 
801-582-1565 Ext 4237 
Tony.donato@utah.edu 
 
 
 
 
 
 
 

 
5
BACKGROUND AND INTRODUCTION: 
 
 
Heart failure (HF) is a serious condition where the heart is unable to fill or pump 
blood efficiently throughout the body in order to meet the body’s needs.  Typically, the 
heart becomes weakened as a result of progressive coronary artery disease or 
hypertension.  There are several different types of HF, including chronic and acute heart 
failure.  Common symptoms seen in patients with HF are fatigue, weakness, rapid or 
irregular heartbeat, dyspnea, persistent cough or wheezing, lack of appetite, and chronic 
or acute swelling of feet, ankles, legs, or abdomen.  HF most often effects older 
individuals that have one or more of the following risk factors: high blood pressure, 
coronary artery disease, myocardial infarction, irregular heartbeats, diabetes, congenital 
heart defects, sleep apnea, certain viruses, alcohol use, or some kidney conditions. 
There is no cure for HF and in the most severe stage; heart transplantation (HTx) is 
usually the best option. 
  
One of the primary criteria in diagnosis and severity of HF is the response to a 6-
min walk test, emphasizing the clear detrimental effect of this disease on the capacity for 
physical activity. Surprisingly, it is now well established that this cannot be attributed to a 
cardiac limitation, but rather is related to peripheral adaptations characteristic of the 
disease. Together, several recent studies have advanced the “muscle hypothesis”, 
which proposes that disease-related changes in ergoreceptor (metaboreceptor and 
mechanoreceptor) sensitivity in skeletal muscle collectively result in elevated muscle 
afferent activity that in turn further elevates sympathetic nerve activity (SNA) [1]. While 
the mechanism responsible for the adaptation is poorly understood, there is new 
evidence from animal studies that skeletal muscle oxidative stress may contribute to 
heightened ergoreceptor sensitivity [2]. Nonetheless, relative importance of metabolic 
versus mechanical receptors to this maladaptive process remains somewhat uncertain.  
As HF progresses, poor contractile function and the subsequent reduction in 
arterial blood pressure is corrected by the carotid and pulmonary baroreflexes. There is 
evidence both for [3] and against [4] disease-related changes in carotid baroreflex 
control of heart rate (vagus nerve) and of muscle sympathetic nerve activity (MSNA) in 
HF, by mechanisms which remain poorly understood. The arterial baroreflex typically 
buffers the muscle metaboreflex by limiting peripheral vasodilation, but in HF the loss of 
baroreflex sensitivity could contribute to the apparent metaborefelx sensitivity simply 
through decreased buffering capacity [5]. To our knowledge, the combined effects of 
altered baroreflex, chemoreflex, and mechanoreflex responses in HF have not been 
systematically studied in humans. 
Heightened SNA has been identified as a primary predictor of mortality in HF [6]. 
The consequence of this well described sympathoexcitation during exercise in HF 
patients may be a level of vasoconstriction in the exercising muscle that cannot be 
overcome, i.e. an impaired “functional sympatholysis”. However, to date, no attempt has 
been made to evaluate disease-related changes in alpha adrenergic sensitivity in this 
cohort. Further, if this disease does in fact infer a level of vasoconstriction that is 
restraining muscle blood flow during exercise, interventions that may limit this 
maladaptive process are of clear interest. One such intervention is exercise training, 
which reduces SNA in many experimental models of HF [7, 8]. Recent studies in HF 
patients have supported earlier animal work, demonstrating the ability of aerobic 
exercise training to restore SNA to that of young healthy controls [9]. To date, very little 
is known regarding the mechanism(s) and time course for the well-documented training-
induced improvements in SNA  
 
Vascular dysfunction has been documented in patients with HF [10], which 
includes the inability of vessels to respond to physiological stimuli such as increased 
blood flow and endothelium-dependent vasodilation [11]. Heart transplantation (HTx) 

 
6
results in a denervated heart. As a result the changes in cardiac output and heart rate 
normally triggered by changes in the sympathetic and parasympathetic tone are 
abolished or blunted.  
There has been increasing interest in vascular function as the health and 
elasticity of major arteries is a risk factor for various types of cardiovascular disease and 
has been suggested to “naturally” deteriorate with age.  In addition, there has also been 
increasing interest in isolating the physiological and mechanical contributors to the blood 
flow response, especially during exercise (exercise hyperaemia).  The examination of 
vascular function in patients with HF and following HTx maybe important with regards to 
the prevalence of cardiovascular disease in these populations. While the abnormal 
hemodynamic responses common in HF and HTx patients compared with healthy 
individuals may help to further explain the mechanisms behind vessel vasodilation and 
changes in blood flow at the onset of exercise. 
 
Oxidative stress is determined by the level of free radicals and the body’s 
antioxidant capacity.  High levels of vascular free radicals have been associated with 
many chronic diseases including heart failure, hypertension, atherosclerosis, diabetes, 
and the normal aging process [12].  With the presence of heart failure and the invasive 
nature of a heart transplantation it seems reasonable that ingesting an exogenous 
antioxidant cocktail (vitamins C, E, and alpha lipoic acid), used with efficacy by our group 
in the past [13-17], may decrease circulating levels of free radicals and may actually 
improve vascular function but the exact effects and potential mechanisms of action in 
this population are unknown. 
One mechanism by which the antioxidant cocktail improves vascular function, 
may be through preventing oxidation of tetrahydrobiopterin (BH4), a critical co-factor for 
the endothelial nitric oxide synthase enzyme, allowing greater NO bioavailability and less 
oxidative stress production [18]. Some suggest that loss of BH4 may be one of the 
precipitating factors inducing free radical production [19], and acute administration of 
BH4 alone has been demonstrated to improve vascular function in aged [20] and those 
with HF [21]. Although, evidence from the in vitro literature indicates there is a strong 
potential for synergy between BH4 and antioxidants [22], this has yet to be tested in 
humans if BH4, antioxidants, or BH4 + antioxidants are capable of improving vascular 
function in HF patients and age matched controls over placebo. 
 
  
OBJECTIVES: 
The objectives of this study are to provide greater insight into changes in muscle and 
cardiovascular function and exercise-induced hyperaemia associated with congestive 
heart failure (HF) and post-heart transplantation (HTx). 
 
 
Specific Aim 1: 
To examine vascular function and the subsequent exercise-induced hyperaemia in both 
healthy controls and patients having congestive heart failure and post-cardiac transplant. 
 
Hypothesis: There will be progressively reduced vascular function from the time of HTx 
through 10 years post-HTx. 
 
Hypothesis: HTx patients we will not see an increase in cardiac output with the initiation 
of passive exercise. 
 
Hypothesis: With the isolated limb passive movement model exercise induced 
hyperaemia will be significantly attenuated in the HTx patients and little to no 
hyperaemia will occur in the non-moving control leg. 

 
7
Hypothesis: The antioxidant cocktail will attenuate circulating levels of free radicals and 
may improve vascular function, both as assessed by flow-mediated vasodilation (FMD) 
and the passive movement protocol, but will be further improved with co-ingestion of 
BH4, over placebo, antioxidants, or BH4 alone. 
 
Specific Aim 2:  
To determine the extent to which muscle ergoreceptor (mechanoreceptor and 
metaboreceptor) and arterial baroreflex dysfunction is present in HF, to evaluate the 
interaction of these reflexes, and to identify which cardiovascular parameters are most 
affected by potential ergoreceptor and baroreceptor adaptations. 
 
Hypothesis: HF patients will exhibit greater mechanoreceptor and metaboreceptor 
sensitivity than age-matched controls, while carotid baroreflex sensitivity will be blunted 
in HF. This heightened ergoreceptor sensitivity in HF is due, in part, to blunted baroreflex 
sensitivity.     
 
Hypothesis: A reduction in oxidative stress, induced by either acute or chronic ingestion 
of the antioxidant cocktail, will reduce mechanoreceptor and metaboreceptor sensitivity 
in HF patients and not age-matched controls.  
 
Hypothesis: Chronic ingestion of and antioxidant cocktail, combined with the eNOS 
cofactor tetrahydrobiopterin (BH4), will reduce mechanoreceptor and metaboreceptor 
sensitivity in HF patients and not age-matched controls. 
 
Specific Aim 3:  
To determine whether exercise training can alter muscle ergoreceptor and carotid 
baroreceptor sensitivity in HF patients, and to characterize the time course for 
ergoreceptor- and baroreceptor-related changes.   
 
Hypothesis: Exercise training will reduce muscle ergoreceptor sensitivity and increase 
carotid baroreflex sensitivity if HF but not age-matched controls.         
 
Specific Aim 4: 
To determine the degree to which elevated SNS activity in expressed in the exercising 
muscle at rest and during aerobic exercise. 
 
Hypothesis: HF patients will exhibit a sustained response to infused adrenergic drugs 
during exercise compared to healthy controls, demonstrating “impaired functional 
sympatholysis” in the cohort. 
 
 
PARTICIPANT SELECTION CRITERIA: 
 
Inclusion criteria: 
 
Patients treated for heart failure (NYHA class I, II, or III) 
 
Patients who have undergone heart transplantation (HTx) 
 
Patients with Hypertension (HTN) 
 
Patients with Chronic Obstructive Pulmonary Disorder (COPD) 
 
Patients with Scleroderma 
 
Healthy controls of all ages 
 
 

 
8
Exclusion criteria: 
 
Age less than 18 years 
 
Patients unwilling or unable to give consent 
 
Subjects in whom the study procedures would not be recommended for medical 
reasons. This determination will be made by Dr. Stehlik, the physician directly 
responsible for the patient’s care. 
 
It is anticipated that patients who are currently hospitalized would not participate until 
released to outpatient care. 
 
NYHA class IV heart failure patients. 
 
Women who are pregnant.  
 
 
DESIGN: 
Procedures that are part of this study are strictly research-related and are not 
considered standard of care. This is a cross-sectional and short-term longitudinal study. 
Patients may be eligible to participate in the study several times (e.g. participation before 
and after transplant, participation at different time intervals after heart transplantation, 
participation in a training protocol), but are not required to do so. Patients may enroll in 
protocols 2, 3, or 4 any time after completion of protocol 1. However, after completing 
protocol 2, 3, or 4, patients must wait two months before enrolling in an additional 
protocol. Patients may also participate in protocol 3 (pre-training) or protocol 4 without 
agreeing to the exercise training portion of the study. The study procedures will include 
multiple visits to the laboratory in a fasted and rested state, as outlined below. 
Comparisons will be made between the healthy controls, HF, and HTx patients.   
 
STUDY PROCEDURES: 
The protocols that patients may choose to participate are outlined on the diagram below; 
   
 
 
 
 

 
9
STUDY PROCEDURES (continued): An overview of each visit to the laboratory, 
including chronology of each protocol, is listed on the following pages. This is followed 
by separate headings that provide details for the underlined techniques and 
measurements identified in each protocol, listed alphabetically. 
 
Preliminary Day 
A signed consent form will be obtained prior to any research procedures. Upon arrival to 
the lab (VA Bldg. 2, Room 1D21), a screening questionnaire will be filled out that will 
include questions concerning risks such as: age, family health history, smoking, 
hypertension, hypercholesterolemia, sleep-related breathing disorders, physical activity 
and allergies (iodine, latex, drugs, etc). A venous blood sample (4 ml) will be drawn from 
which a Metabolic Panel, Complete Blood Count (CBC), and a Lipid Panel will be 
assessed. Women of childbearing age will provide a urine sample for pregnancy testing, 
and will provide information concerning method of birth control. 
Following health history and blood sample, all subjects may be asked to perform 
several tasks to assess cognitive abilities and fitness levels and to wear a pedometer for 
a week, which will measure activity levels. All subjects will complete a series of exercise 
tests. Subjects will complete one or more of the following: a graded exercise test to 
volitional exhaustion (bike or treadmill, < 15 min), maximal handgrip test (1 min), 
maximal leg extension test (< 15 min), and maximal plantar flexion test (< 15 min). 
Sufficient recovery will be allowed between tests, an ECG will be assessed at all times. 
The PI will be present for all exercise tests, and a physician will be immediately available 
if needed.  
The handgrip, leg extension and plantar flexion graded exercise tests are 
considered low cardiovascular risk tests due to the recruitment of an isolated small 
muscle mass that results in minor cardiovascular stress. For all subjects these exercise 
tests will be conducted in the presence of the PI. In addition, a board certified physician 
(Dr. Kithas or Supiano) will be immediately available if needed.  
For the graded test to volitional exhaustion, exercise will be completed under 
physician supervision, and the subject will be monitored by an  ECG throughout the 
exercise test. Any abnormal ECG responses during the exercise test (overt arrhythmia, 
severe [3 mm] ST segment depression, elevated [1mm] ST segment in non-Q wave 
lead, frequent extra-ventricular systoles) or adverse responses such as chest pain, leg 
pain, or unsteadiness will serve as criteria to stop the test, in accordance with American 
Heart Association guidelines. Questionable findings will be referred to a cardiologist for 
inspection/diagnosis prior to further involvement in the protocol.  
 
PROTOCOL 1 (Acute Supplementation, 4 visits): 
 
On four separate visits, patients will take one of the following: placebo, antioxidants, BH4 
(10mg/kg), or BH4 + antioxidants, 2 hours prior to arrival in the lab in a balanced, double-
blind fashion. A member of the investigative team will provide these pills to subjects in 
bags labeled “A”, “B”, “C”, or “D” during the preliminary visit described above. These four 
visits will take place at least 48 hrs. apart, but are expected to be completed within 2 
weeks.Protocol 1 procedures: Subjects will report to the laboratory (VA bldg. 2 room 
1D21) at 0800 in a fasted state, having abstained from caffeine and exercise for the past 
12 hours. Upon arriving in the lab a venous blood sample (4 ml) will be drawn from 
which measures of oxidative stress and antioxidant capacity will be assessed. Following 
the blood sample, all subjects will have their height, weight, skin fold thickness and 
resting arterial blood pressure (ABP) recorded. Women of childbearing age will be asked 
to submit to a urine pregnancy test provided by the research site. Next, arterial stiffness 
measures will be completed. After lying quietly for an additional 20 minutes, the subject 

 
10
will undergo the flow mediated vasodilation (FMD) test in their arm (brachial artery). 
Nitroglycerin (NTG) will then be administered to evaluate endothelium-independent 
vasodilation. Two 
oral 
nitroglycerin 
tablets 
(0.8mg) 
will 
be 
administered 
sublinguilly.  Measurements will be made for 5 minutes.  Subjects will then rest 20-min to 
allow drug washout and restoration of steady-state hemodynamics. Following NTG, limb 
blood flow will be measured during four exercise protocols that utilize a single arm and 
single leg. First, the arm and leg will be passively moved through a normal range of 
motion (the participant will stay relaxed and try not to engage their muscles). Qualified 
personnel of the Vascular Laboratory will be supporting and moving the arm or leg for 
the participant. Next, an active exercise test will be performed, which requires that the 
participants give maximum effort for either the arm or leg by moving that appendage 
through a normal range of motion against some resistance. The arm or leg will be the 
only part of the body moving or producing any effort.  
 
PROTOCOL 2 (Chronic Supplementation, 7 visits): 
 
For this protocol, patients will be divided into four groups taking one of the following: (1) 
placebo, (2) antioxidants, (3) BH4 (10mg/kg), or (4) BH4 + antioxidants, one time daily for 
30 days. A member of the investigative team will provide these pills to subjects in bags 
labeled “A”, “B”, “C”, or “D” during the preliminary visit described above. Assignment to 
group will be balanced and both investigator and subject will be blinded to the group. 
Compliance for pill consumption will be monitored by an online database to which 
subjects will log in each day or a phone call if internet access is not available. 
 
Day 0: Subjects will report to the laboratory (VA bldg. 2 room 1D21) at 0800 in a fasted 
state, having abstained from caffeine and exercise for the past 12 hours. Upon arrival, 
women of childbearing age will submit toa urine pregnancy test, provided by the site, as 
part of the preliminary study process. Catheters will be placed in the arm (brachial artery 
and vein) or leg (common femoral artery and femoral vein) by a licensed physician using 
standard 
sterile 
technique. 
After 
instrumentation, 
an 
Arterial 
Elasticity 
Measurements/Pulse Contour Analysis (PCA) test will be performed immediately 
followed by a flow mediated vasodilation (FMD) test in the arm (brachial artery) or leg 
(popliteal artery). Following the FMD test, a muscle biopsy will be taken from the vastus 
lateralis of one leg. Next, two microelectrodes will be placed in the contralateral leg 
(popliteal fossa) for direct recording of muscle sympathetic nerve activity (MSNA). 
Subjects will rest for approximately 10 minutes, followed by resting measurements of 
arterial blood pressure (ABP), heart rate (HR), limb blood flow (LBF) (ultrasound 
Doppler), muscle sympathetic nerve activity (MSNA), and collection of resting blood 
samples.  
After resting measurements, baroreflex sensitivity will be assessed using both 
pharmacologic and direct techniques. Using the modified oxford technique, a series of 
I.V. pressor and depressor bolus drug infusions will be administered, and changes in 
ABP, HR, LBF and MSNA will be measured. After a 15-min recovery, carotid baroreflex 
sensitivity will be assessed directly using a neck chamber that provides localized 
distension or compression of the carotid artery, provoking small changes in carotid sinus 
pressure. This neck pressure/neck suction (NP/NS) technique will alter afferent carotid 
baroreflex activity, and again changes in ABP, HR, LBF, and MSNA will be measured. 
This will be followed by an additional 15-min recovery for return to a hemodynamic 
steady-state. 
Following baroreflex testing, muscle ergoreceptor sensitivity will be determined. 
Muscle mechanoreceptor sensitivity will be determined during 3-min of passive leg 
movement. This will be followed by muscle metaboreceptor sensitivity testing in both the 

 
11
arm and leg utilizing post-exercise circulatory occlusion (PECO). Three levels of arm 
(handgrip exercise) and leg (knee-extensor exercise) will be performed at 20, 40, and 
60% of maximal effort (3-min bout), with 2-min of PECO at the cessation of each 
exercise bout. During each of these exercise bouts, ABP, HR, LBF, and MSNA will be 
measured, and blood samples will be taken at each exercise intensity for blood gas 
analysis, and to assess the balance between antioxidants and oxidative stress across 
the leg.   
After a 15-min recovery, the PECO protocol described above will again be 
performed with superimposed carotid baroreflex testing (modified oxford technique) to 
assess potential metaboreceptor-baroreceptor interaction. 1-2 doses of both PE and 
SNP will be administered during the post-exercise cuff occlusion depending on the rate 
of blood pressure change during drug infusion. 
 
Days 10, 20, 30, 33 and 37: Following the baseline visits, subject will Patients will return 
to the lab five times after enrolling in the 30-day supplementation: days 10, 20, and 30, 
and two visits following discontinuation of the supplementation (days 33 and 37).  
 
On Day 30, subjects will repeat the “Day 0” protocol outlined above.  
 
Days 10, 20, 33, and 37 are non-invasive study days, with the following protocol;  
Subjects will report to the laboratory (VA bldg. 2 room 1D21) at 0800 in a fasted state, 
having abstained from caffeine and exercise for the past 12 hours. Upon arriving in the 
lab a venous blood sample (4 ml) will be drawn from which measures of oxidative stress 
and antioxidant capacity will be assessed. Following the blood sample, all subjects will 
have their height, weight, skin fold thickness and resting arterial blood pressure (ABP) 
recorded. Women of childbearing age will take a urine pregnancy test. Next, arterial 
stiffness measures will be completed. After lying quietly for an additional 20 minutes, the 
subject will undergo the flow mediated vasodilation (FMD) test in their arm (brachial 
artery). Following the FMD test, limb blood flow will be measured during four exercise 
protocols that utilize a single arm and single leg. First, the arm and leg will be passively 
moved through a normal range of motion (the participant will stay relaxed and try not to 
engage their muscles). Qualified personnel of the Vascular Laboratory will be supporting 
and moving the arm or leg for the participant. Next, an active exercise test will be 
performed, which requires that the participants give maximum effort for either the arm or 
leg by moving that appendage through a normal range of motion against some 
resistance. The arm or leg will be the only part of the body moving or producing any 
effort.  
 
 
PROTOCOL 3  
Pre-Training Protocol: 
Subjects will report to the laboratory (VA bldg. 2 room 1D21) at 0800 in a fasted state, 
having abstained from caffeine and exercise for the past 12 hours, and catheters will be 
placed in the arm (brachial artery and vein) or leg (common femoral artery and femoral 
vein) by a licensed physician using standard sterile technique. After instrumentation, an 
Arterial Elasticity Measurements/Pulse Contour Analysis (PCA) test will be performed, 
immediately followed by a flow mediated vasodilation (FMD) test in the arm (brachial 
artery) or leg (popliteal artery). Next, two microelectrodes will be placed in the 
contralateral leg (popliteal fossa) for direct recording of muscle sympathetic nerve 
activity (MSNA). Subjects will rest for approximately 10 minutes, followed by resting 
measurements of arterial blood pressure (ABP), heart rate (HR), limb blood flow (LBF) 

 
12
(ultrasound Doppler), muscle sympathetic nerve activity (MSNA), and collection of 
resting blood samples.  
After resting measurements, baroreflex sensitivity will be assessed using both 
pharmacologic and direct techniques. Using the modified oxford technique, a series of 
I.V. pressor and depressor bolus drug infusions will be administered, and changes in 
ABP, HR, LBF and MSNA will be measured. After a 15-min recovery, carotid baroreflex 
sensitivity will be assessed directly using a neck chamber that provides localized 
distension or compression of the carotid artery, provoking small changes in carotid sinus 
pressure. This neck pressure/neck suction (NP/NS) technique will alter afferent carotid 
baroreflex activity, and again changes in ABP, HR, LBF, and MSNA will be measured. 
This will be followed by an additional 15-min recovery for return to a hemodynamic 
steady-state. 
Following baroreflex testing, muscle ergoreceptor sensitivity will be determined. 
Muscle mechanoreceptor sensitivity will be determined during 3-min of passive leg 
movement. This will be followed by muscle metaboreceptor sensitivity testing in both the 
arm and leg utilizing post-exercise circulatory occlusion (PECO). Three levels of arm 
(handgrip exercise) and leg (knee-extensor exercise) will be performed at 20, 40, and 
60% of maximal effort (3-min bout), with 2-min of PECO at the cessation of each 
exercise bout. During each of these exercise bouts, ABP, HR, LBF, and MSNA will be 
measured, and blood samples will be taken (at each exercise intensity) for blood gas 
analysis and to assess the balance between antioxidants and oxidative stress across the 
leg.   
After a 15-min recovery, the PECO protocol described above will again be 
performed with superimposed carotid baroreflex testing (modified oxford technique) to 
assess potential metaboreceptor-baroreceptor interaction. 1-2 doses of both PE and 
SNP will be administered during the post-exercise cuff occlusion depending on the rate 
of blood pressure change during drug infusion. 
 
Exercise Training (12 weeks): 
Patients who agree to participate in the training portion of the study will report to the 
laboratory (VA Building 2 room 1D23) for 12 consecutive weeks (2-3 days per week, 1-2 
hours per visit) for supervised exercise training. Small muscle mass exercise will be 
performed so that high intensity training can be carried out without reaching 
cardiopulmonary limits to O2 transport, which is of critical importance when studying HF 
patients. Single-leg training will thus be utilized, using the knee-extensor exercise 
paradigm described below. Exercise intensity will be customized to the ability of each 
patient, and will be increased as tolerated every two weeks by re-assessing maximal 
power output in a conventional incremental test, to gain the greatest effect. A member of 
the research team will supervise all training sessions, and a physician will be 
immediately available if needed. Drs. Wray and Richardson have extensive experience 
with this exercise training protocol, including studies in the elderly, COPD and HF 
patients [13, 14, 23-33].     
 
 
 
Intra-training and Post-training Protocol (weeks 2, 4, 6, 8, 10, and 12): 
Patients will undergo bi-weekly assessment arterial elasticity, flow mediated vasodilation 
(FMD), muscle ergoreceptor sensitivity, and baroreflex sensitivity to track training-related 
improvements in muscle and cardiovascular parameters. The sequence and 
instrumentation will be identical to the pre-training protocol described above, with the 
following two exceptions; MSNA measurements will be made only at weeks 4, 8, and 12, 
and femoral catheters will be placed only at weeks 6 and 12. For experimental days that 

 
13
do not include femoral arterial and venous catheters, the antecubital vein will be 
catheterized. 
 
PROTOCOL 4 (6-8 hours per visit, 2 visits) 
 
Day 1 (Arm Protocol)  
Resting measurements (4 hours): Upon arrival to the lab, catheters will be placed in 
the non-dominant arm (brachial artery and basilica or cephalic vein) by a licensed 
physician. Following placement of catheters, intra-arterial infusion of Phenylephrine 
(PE), Dexmedetomidine (DEX), Tyramine (TYR), Phentolamine (PHEN), and Saline 
(control) will be performed in a randomized fashion. During the PHEN infusion, 
propranolol will be co-administered (I.V.) to prevent beta adrenergic tachycardia (control 
subjects only). Each drug infusion will last for approximately 15-min, and 20-min will be 
allotted between each infusion to allow drug washout and restoration of steady-state 
hemodynamic values.  
 
Exercising measurements (4 hours): Subjects will perform five bouts of incremental 
handgrip exercise at three intensities (30%, 45%, and 60% of MVC, four minutes per 
stage). During each exercise bout, either PE, DEX, TYR, PHEN, or Saline (control) will 
be administered in a randomized fashion. During the PHEN infusion, propranolol will be 
co-administered (I.V.) to prevent beta adrenergic tachycardia (control subjects only). For 
PE, DEX, and TYR, exercise will be performed for 3 min to reach steady-state, followed 
by 1-min of drug infusion. For the PHEN trial, drug will be administered continuously 
throughout the exercise protocol and compared to continuous Saline infusion. Each 
exercise bout will last approximately 12 minutes, with 30 minutes rest between exercise 
bouts.  
 
During both rest and exercise, arterial blood pressure, venous blood pressure, heart 
rate, and limb blood flow (ultrasound Doppler) will be measured continuously. An 
independent observer not involved in data collection will watch the electrocardiogram 
(EKG) and blood pressure monitors at all times to track the cardiovascular responses to 
exercise and drug infusions.  
 
Day 2 (Leg Protocol) 
Resting measurements (4 hours): Upon arrival to the lab, catheters will be placed in 
the femoral artery and vein by a licensed physician. Following placement of catheters, 
intra-arterial infusion of Phenylephrine (PE), Dexmedetomidine (DEX), Tyramine (TYR), 
Phentolamine (PHEN), and Saline (control) will be performed in a randomized fashion. 
During the PHEN infusion, propranolol will be co-administered (I.V.) to prevent beta 
adrenergic tachycardia. Each drug infusion will last for approximately 15-min, and 20-
min will be allotted between each infusion to allow drug washout and restoration of 
steady-state hemodynamic values.  
 
Exercising measurements (4 hours): Subjects will perform five bouts of incremental 
knee-extensor (KE) exercise at three intensities (25%, 50%, and 75% of KE max, four 
minutes per stage). During each exercise bout, either PE, DEX, TYR, PHEN, or Saline 
(control) will be administered in a randomized fashion. During the PHEN infusion, 
propranolol will be co-administered (I.V.) to prevent beta adrenergic tachycardia. For PE, 
DEX, and TYR, exercise will be performed for 3 min to reach steady-state, followed by 1-
min of drug infusion. For the PHEN trial, drug will be administered continuously 
throughout the exercise protocol and compared to continuous Saline infusion. Each 

 
14
exercise bout will last approximately 12 minutes, with 30 minutes rest between exercise 
bouts.  
 
During both rest and exercise, arterial blood pressure, venous blood pressure, heart 
rate, and limb blood flow (ultrasound Doppler) will be measured continuously. An 
independent observer not involved in data collection will watch the electrocardiogram 
(EKG) and blood pressure monitors at all times to track the cardiovascular responses to 
exercise and drug infusions.  
 
TECHNIQUES AND MEASUREMENTS 
 
Antioxidant Supplementation: Antioxidant or placebo will be consumed in two doses 
approximately 120 minutes and 90 minutes prior to the other procedures.  The first 
antioxidant dose at 120 minutes prior will include: Vitamin E 200 IU (1 tablet), Vitamin C 
500 mg (1 tablet), and Alpha-lipoic Acid 300 mg (3 tablets).  The second antioxidant 
dose approximately 90 minutes prior will include: Vitamin E 400 IU (water dispersable) (1 
tablet), Vitamin C 500 mg (1 tablet), and Alpha-lipoic Acid 300 mg (3 tablets).  The PI’s 
and Co-I’s have previously utilized this supplementation protocol in studies of both aging 
and disease and a very similar protocol when testing the ability of antioxidant 
supplementation to prevent free radical-mediated damage [14-16, 34-36]. 
 
Arterial Elasticity Measurements/Pulse Contour Analysis (PCA) test: Arterial 
elasticity will be measured using the HDI/Pulsewave™CR-2000 CardioVascular Profiling 
System (Hypertension Diagnostic, Inc., Eagan, Minnesota, USA).  The DO-2020 System 
records and analyzes the blood pressure waveform data from the Arterial PulseWave 
Sensor.  PCA measurements will be obtained in the morning following an overnight fast 
of at least eight hours and prior to engaging in any strenuous physical activity.  Following 
10 minutes of supine rest, large and small artery elasticity indices will be obtained.  A 
blood pressure cuff will be wrapped around the subject’s upper left arm, and a rigid 
plastic wrist stabilizer to minimize movement of the right wrist will be used during the 
PCA test.  With the right forearm in a supinated position, an Arterial PulseWave™Sensor 
will be placed on the skin directly over the radial artery at the point of the strongest 
pulse.  The sensor will be adjusted to the highest relative signal strength, and the large 
and small arterial elasticity measurements will be obtained during 30 seconds of blood 
pressure waveform collections.  In addition, other cardiovascular parameters will be 
recorded during this waveform collection, including pulse rate, systolic blood pressure, 
diastolic blood pressure, systemic vascular resistance, and total vascular impedance.  
Measurement will be obtained and averaged over three consecutive 30-second trials.  
This part of the procedures will last approximately 30 minutes.   
 
Blood sampling: As part of the preliminary screening, a venous blood sample (4 ml) will 
be drawn from all subjects for assessment of overall health (metabolic panel, complete 
blood chemistry, and lipid panel). On the experimental days, arterial and venous blood 
will be collected anaerobically with sterile syringes from the above-mentioned catheters 
at rest and during each exercise level for assessment of metabolic and aerobic status, 
and to test for markers of oxidative stress. For protocol 1, 15 ml of venous blood will be 
obtained. For protocol 2, one sample of arterial (4 ml) and venous (4 ml) blood will be 
taken at rest and during each level of arm and leg exercise. 
 
Catheter placement:  Prior to catheter placement, the subject will first be connected to 
a 3-lead ECG monitor. At each catheter insertion site, local anesthesia with lidocaine will 

 
15
be applied (1% lidocaine; 1.33mg/kg assuming 75 kg body weight, with an upper limit of 
10 ml). Sterile technique will be applied to minimize risk of infection. 1.0 mg of atropine 
will be available in case of sudden vagal reactions. The leg (femoral artery and vein, 
18G Cook Catheter or equivalent) will be cannulated by a qualified and privileged 
physician who will assume responsibility for the placement and removal of these 
catheters. Following catheter removal, wounds will be dressed and the subjects 
educated as to proper care. No subject will be released until hemostasis is clearly 
present.  Follow-up contact with the subject will continue over the following two days. It 
should be noted that the PI and Co-I’s of this proposal have significant experience with 
catheter-related studies [29, 30, 37-46], having coordinated the use and insertion of 
these exact catheters over a hundred times in the last ten years without incidence of 
bleeding, infection or tissue damage. In fact, several subjects have performed multiple 
studies with the PI, indicating the benign nature and positive outcome of the procedures. 
The PI and Co-I’s of this proposal have current University of Utah IRB approval for this 
procedure in elderly (IRB# 00030810) and COPD patients (IRB# 00032404).   
 
Flow-mediated vasodilation (FMD) test: A blood pressure cuff will be placed on the 
upper arm and inflated to >250 mmHg for five minutes to partially occlude the forearm. 
Ultrasound Doppler measurements will be made in the brachial artery to evaluate FMD 
upon cuff release. Dr. Richardson’s laboratory has direct experience with this technique 
[23].  
 
Limb Blood Flow: An ultrasound Doppler system (Logiq 7, GE Medical Systems, 
Milwaukee, Wisconsin, USA) equipped with two linear array transducers operating at an 
imaging frequency of 7-8 MHz and 12-14 MHz will be used for the leg and arm, 
respectively. Vessel diameter will be determined at a perpendicular angle along the 
central axis of the scanned area, where the best spatial resolution is achieved.  The 
brachial artery (BA) of the right arm will be insonated approximately midway between the 
antecubital and axillary regions, medial to the biceps brachii muscle. The blood velocity 
profile will be obtained simultaneously using the same transducers with a Doppler 
frequency of 4.0-5.0 MHz, operated in the high-pulsed repetition frequency mode (2-25 
kHz) with a sample depth of 1.5-3.5 cm  and the probe maintained at an insonation 
angle of 60° or less. Sample volume will be maximized according to vessel size and 
centered, verified by real-time ultrasound visualization of the vessel. Using artery 
diameter and Vmean, blood flow is calculated as: blood flow (ml/min) = Vmean · π · (Vessel 
Diameter/2)2 · 60. At all sample points, arterial diameter and angle-corrected, time-
averaged, and intensity-weighted mean blood velocity (Vmean) values can be calculated 
using commercially available software (Logiq 7). Ultrasound digital images and velocity 
spectra segments of five minutes will be recorded and saved to the GE Logiq 7 hard 
drive for off-line image and waveform analysis. Dr. Richardson’s laboratory has 
extensive experience with this methodology [23, 28, 40, 47, 48]. 
 
Modified Oxford Technique: Carotid baroreflex sensitivity will be determined by 
assessing HR responses to an increase or decrease in arterial blood pressure evoke by 
I.V. phenylephrine (PE) and sodium nitroprusside (SNP), respectively. Drugs will be 
administered sequentially intended to provoke a 10-15 mmHg change in blood pressure 
(PE 100-150 µg; SNP 50-150 µg). These drugs are considered to be investigational by 
the FDA, and thus an investigational new drug (IND) number has been issued to the PI 
(IND#108367). This technique has been utilized extensively in a research setting to 
determine baroreflex sensitivity in HF [49]. 
 

 
16
Muscle biopsy: All biopsies will be taken from the vastus lateralis approximately 3.5 cm 
deep, 15 cm proximal to the knee and slightly distal to the ventral mid-line of the muscle.  
The 5 mm diameter biopsy needle (Bergstrom) is attached to sterile tubing and a syringe 
to apply a negative pressure to assist in the muscle sample collection [50]. Lidocaine 
(2%) will be used as local anesthetic. The muscle samples from each biopsy will be 
immediately frozen in liquid nitrogen and stored at -80oC for future microscopy, EPR 
spectroscopy, molecular and biochemical assays or immersion-fixed in gluteraldehyde 
for EM analysis. With our current technique and experience, utilizing 2-3 passes of the 
needle and double cut, we are able to attain as much as 300-400 mg of muscle which 
will permit the performance of all proposed analyses. The Principal Investigator has 
been involved in over 100 of these procedures with no negative outcomes [31-33, 46, 
51], and currently holds VA credentialing and IRB approval for this procedure in both 
COPD patients and control subjects (IRB#00032404, “Mechanisms of Adaptation to 
Exercise in Health and COPD”). Additionally, in support of this technique, a series of 
subjects recently underwent 5 biopsies each over a period of time, illustrating that the 
biopsy experience is acceptable to the subjects.  
 
Muscle sympathetic nerve activity: Postganglionic muscle sympathetic nerve activity 
(MSNA) will be recorded with standard microneurographic techniques. In the right leg, 
low-voltage (3-4 Volts) stimuli will be applied to the skin behind the knee using a pencil-
shaped electrode. Stimuli will be applied intermittently (i.e. 1 stimulus every 15 seconds) 
for approximately two minutes to map the path of the nerve. This procedure will cause 
light muscle twitching of the foot. Once the nerve path is identified, two very small 
tungsten microelectrodes (1-4 micrometers) will be inserted into the skin. The first 
electrode will be inserted 2-3 cm distal to the nerve map and acts as a reference, and 
the second electrode will be inserted near the fibular head and advanced until it reaches 
the peroneal nerve (1-2 cm). This electrode will be used for recording only. MSNA 
recordings display a pulse-synchronous burst pattern and an increase in burst frequency 
with end-expiratory breath hold and Valsalva maneuver, but with no response to startle 
or skin stroking. These characteristics will be used to determine proper electrode 
placement for the MSNA recordings. One of the co-investigators (Dr. Wray) is trained in 
microneurography and routinely performs this procedure under IRB # 00030810, 
“Vascular Function in Health and Disease”. 
 
Neck Pressure/Neck Suction (NP/NS): Graded levels of pressure and suction will be 
generated by a variable pressure source and delivered through solenoid valves to a 
custom-made neck chamber that encompasses the anterior 2/3 of the neck, creating an 
air-tight seal around the neck. The application of neck pressure (NP) increases pressure 
within the neck chamber that compresses the carotid baroreceptors and causes a 
decrease in carotid sinus transmural pressure, thereby exposing the carotid 
baroreceptors to a mild hypotensive stimulus. Neck suction (NS) stretches the carotid 
baroreceptors and causes an increase in carotid sinus transmural pressure that 
effectively delivers a mild hypertensive stimulus to the carotid baroreceptors. Baroreflex 
sensitivity will be assessed by measuring changes in HR, ABP and MSNA during 2–4 
trials of random ordered single 5 s pulses of NP (+40 and +20 Torr) and NS (-20, -40, -
60, and -80 Torr), or through the rapid pulse train NP/NS protocol (12 consecutive, ECG-
triggered 500 ms pulses of NP and NS ranging from +40 to -80 Torr). Dr. Wray has 
extensive training using the neck collar and NP/NS protocol [52-56].  
 
Non-invasive arterial blood pressure: Systemic arterial blood pressure (ABP) will be 
measured non-invasively on a continuous basis using finger photoplethysmography 
(Finometer, Ohmeda, Madison, WI, USA). The finometer cuff is placed on the middle 

 
17
finger of the right hand and is supported on a modified surgical stand adjusted to 
position the finger cuff at heart level. 
 
Exercise: Passive leg movement will be performed by a member of the research team 
moving the subject’s leg through a normal range of motion (90°- 180°).  
 
Active leg exercise will be performed using a custom-built knee-extensor ergometer with 
the subject moving the leg between 90°- 180°.  
 
Plantar flexion exercises will be performed using an ergometer specifically designed to 
isolate the gastrocnemius and the soleus complex.  
 
Arm exercise will involve rhythmic handgrip using a computer-interfaced dynamometer.   
 
Each of these exercise modalities are small muscle mass and therefore only minimally 
tax the heart and lungs, even at maximal efforts [57].  All exercise testing will be 
completed in the VA Medical Center Building 2 room 1D23. 
 
Post-Exercise Circulatory Occlusion (PECO): To active afferent muscle fibers that are 
sensitive to exercise-induced metabolites, supra-systolic (220-240 mmHg) circulatory 
occlusion of the exercised limb will be applied upon cessation of the 3-min exercise bout. 
This will be achieved through inflation of a modified blood pressure cuff placed on either 
the arm (upper arm, immediately proximal to elbow) or leg (upper thigh) for 2-min. This 
procedure is used routinely in a clinical research setting, including studies involving HF 
[58].  
 
STUDY DRUGS: The overall objective of all drug infusions for this protocol is to achieve 
maximal receptor occupancy in the limb while avoiding spillover of the drug into the 
systemic circulation. All drug doses have been determined using previously published 
ranges, and are calculated per 100 ml (e.g. dl) limb volume. All drug preparation, 
dispensing, and disposal will be handled by the VAMC SLC Inpatient Pharmacy. All 
drugs have been approved by the FDA for use in the manner described herein 
(IND#113,067). 
 
1. Phenylephrine (PE): At rest, PE will be administered in five doubling doses (0.03-
0.06-0.12-0.24-0.48 mcg/dl/min, 2-min per dose) to establish a dose-response for 
PE-induced vasoconstriction. During exercise, PE will be administered at 0.24 
mcg/dl/min, and adjusted for the exercise-induced increase in limb blood flow. 
The method for determining this dose range is based on previously published 
studies [40, 59].    
 
2. Dexmedetomidine (DEX): At rest, DEX will be administered in five doubling 
doses (1.5-3-6-12-24 ng/dl/min, 2-min per dose) to establish a dose-response for 
DEX-induced vasoconstriction. During exercise, DEX will be administered at 6 
ng/dl/min, and adjusted for the exercise-induced increase in limb blood flow. The 
method for determining this dose range is based on previously published studies 
[60, 61]. 
 
3. Tyramine (TYR): At rest, TYR will be administered in five doubling doses (2-4-8-
16-32 mcg/dl/min, 2-min per dose) to establish a dose-response for DEX-induced 
vasoconstriction. During exercise, TYR will be administered at 8 mcg/dl/min, and 

 
18
adjusted for the exercise-induced increase in limb blood flow. The method for 
determining this dose range is based on previously published studies [62-65].    
 
4. Phentolamine (PHEN): At rest, PHEN will be administered in three doubling 
doses (5-10-20 mcg/dl/min) to establish complete alpha adrenergic blockade. 
During exercise, PHEN will be administered at 5 mcg/dl/min, and adjusted for the 
exercise-induced increase in limb blood flow. The method for determining this 
dose range is based on previously published studies [66-70]. 
 
5. Propranalol: At rest and during exercise, propranolol (a non-selective beta 
adrenergic antagonist) will be administered intravenously for 5-min (0.15 mg/kg, 
max 10 mg) prior to PHEN infusion to limit the possibility of beta adrenergic 
tachycardia during PHEN administration. This infusion will be administered only 
in control subjects, as the HF patients all receive beta-blocking drugs as part of 
their optimized pharmacotherapy. 
 
6. Kuvan (BH4): will be administered at a dose of 10 mg/kg body weight. 
 
7. 
Nitroglycerin (NTG): will be administered to evaluate endothelium-independent 
vasodilation in the  arm and leg.  Two oral nitroglycerin tablets (0.8mg) will be 
administered sublinguilly.  Measurements will be made for 5 minutes.  Subjects 
will then rest 20-min to allow drug washout and restoration of steady-state 
hemodynamics. 
 
 
 
 
POTENTIAL RISKS 
Catheter-related risks.  There is also the possibility of fainting, dizziness, and possible 
pain and bruising as a result of catheter insertion. There is a risk of infection, swelling, and 
discomfort at the insertion sites, and a risk that bleeding may occur after catheters 
removal. A clot or excessive bleeding at the puncture site could result in a partial 
blockage of the blood flow to the arm or leg, which in extreme cases could lead to loss of 
the limb. These risks are minimized by careful patient selection, catheter insertion by a 
physician with experience using proper equipment and sterile methods, adequate 
pressure for >30 minutes following catheter removal, and appropriate patient education 
and follow-up. The PI and several CO-I’s named herein have significant cumulative 
experience in this areas, having been involved in several hundred successful catheter-
based studies with no instances of bleeding, infection, femoral vein thrombosis or soft 
tissue damage as a result of the procedures proposed. 
 
Drug Infusion Risks: At higher intra-arterial doses, the risk exists for significant spillover 
of the drug into the systemic circulation resulting in acute changes in arterial blood 
pressure. Thus, during the higher limb blood flow which occurs during exercise, blood 
pressure will be closely monitored, and infusions will be discontinued if acute 
hypertension is observed (>15 mmHg rise in systolic or diastolic pressure). The 
proposed doses for all drugs included in this protocol have been demonstrated to act 
locally, with minimal systemic effects. The PI and CO-I’s on this protocol have extensive 
experience with intra-arterial drug infusions, and routinely perform infusion studies 
identical to those described above in a concurrent IRB-approved project (IRB#30810, 
“Vascular Function in Health & Disease”).    
 

 
19
Phenylephrine (PE): Systemic doses of PE may cause headache, reflex bradycardia, 
excitability, restlessness and rarely arrhythmias, and is contraindicated in patients with 
severe hypertension, ventricular tachycardia, or in patients who are hypersensitive to it 
or to any of the components. If used in conjunction with oxytocic drugs, the pressor 
effect of sympathomimetic pressor amines is potentiated. The obstetrician should be 
warned that some oxytocic drugs may cause severe persistent hypertension and that 
even a rupture of a cerebral blood vessel may occur during the postpartum period. PE 
contains sodium metabisulfite, a sulfite that may cause allergic-type reactions including 
anaphylactic symptoms and life-threatening or less severe asthmatic episodes in certain 
susceptible people. The overall prevalence of sulfite sensitivity in the general population 
is unknown and probably low. Sulfite sensitivity is seen more frequently in asthmatic 
than in nonasthmatic people. Phenylephrine hydrochloride should be employed only with 
extreme caution in elderly patients or in patients with hyperthyroidism, bradycardia, 
partial heart block, myocardial disease or severe arteriosclerosis. The pressor effect of 
sympathomimetic pressor amines is markedly potentiated in patients receiving 
monoamine oxidase inhibitors (MAOI). Therefore, when initiating pressor therapy in 
these patients, the initial dose should be small and used with due caution. The pressor 
response of adrenergic agents may also be potentiated by tricyclic antidepressants. 
 
Dexmedetomidine (DEX): Systemic doses of DEX may cause acute hypotension and 
bradycardia. Because systemic DEX decreases sympathetic nervous system activity, 
hypotension and/or bradycardia may be expected to be more pronounced in patients 
with hypovolemia, diabetes mellitus, or chronic hypertension and in elderly patients. 
There are no published contraindications. 
 
Tyramine (TYR): Systemic doses of TYR may produce acute hypertension and a reflex 
bradycardia. There are no published contraindications. 
 
Phentolamine (PHEN): Systemic doses of PHEN may cause acute and prolonged 
hypotensive episodes, tachycardia, and cardiac arrhythmias. In addition, weakness, 
dizziness, flushing, orthostatic hypotension, nasal stuffiness, nausea, vomiting, and 
diarrhea may occur. Myocardial infarction, cerebrovascular spasm, and cerebrovascular 
occlusion have been reported to occur following the administration of phentolamine, 
usually in association with marked hypotensive episodes. Systemic PHEN administration 
is contraindicated in patients with myocardial infarction, coronary insufficiency, angina, 
or other evidence suggestive of coronary artery disease; hypersensitivity to 
phentolamine or related compounds. 
 
Propranolol: Contraindicated in patients with cardiogenic shock, sinus bradycardia and 
greater than first degree block, bronchial asthma, and in patients with known 
hypersensitivity to the drug. Systemic doses of propranolol may cause bradycardia and 
hypotension.   
 
Nitroglycerin (NTG): is contraindicated in patients with early myocardial infarction, 
severe anemia, increased intracranial pressure, and those with a known hypersensitivity 
to nitroglycerin. Sublingual NTG is also contraindicated in patients who are using a 
phosphodiesterase-5 (PDE-5) inhibitor (e.g., sildenafil citrate, tadalafil, vardenafil 
hydrochloride).  
 
Myocardial ischemia, infarction, arrhythmia, and death during exercise.  This should be 
uncovered in the non-invasive initial testing of each subject, and if found the subject will 
be excluded and referred to his/her physician.  The responses to absolute and relative 

 
20
indications for the termination of an exercise test are variable depending upon the 
finding. However, an incident of ischemia presented as chest pain, shortness of breath, 
acute hypotension (<80/40 mmHg), or abnormal ECG findings (>2-3 mm ST depression 
or appearance of S3 or S4 heart sounds) would result in the immediate termination of 
exercise. The subject would be placed in a supine position, administered oxygen and 
assessed by the on site physician and research staff. If the condition did not improve or 
worsened either 911 or the Code Team would be called dependent upon the location. In 
all cases this occurrence would be documented and the subject would ultimately be 
referred to their physician. Cardiac function will be monitored during all exercise studies 
with an ECG monitor. 
 
Microneurography: The nerve recording procedure occasionally may result in the leg 
muscles feeling tired one or two days after the experiment, a pins-and-needles feeling 
(paresthesia), or a greater sensitivity to touch in the leg.  However, since 1979, 
microneurography has been performed on several thousand subjects in the U.S. and 
abroad without any lasting complications. Dr. Wray has performed microneurography in 
many subjects without persistent complications. Many subjects volunteer repeatedly for 
microneurographic studies, indicative of the benign nature of this technique. 
 
Muscle biospy risks: Using sterile technique and a minimal skin incision (about 1/8”) over 
the anterolateral mid-thigh, the standard needle aspiration method for muscle biopsy has 
not produced bleeding nor infectious complications to date. There is the possibility of a 
small scar the size of the incision (1/8”). No subject is released until hemostasis has 
been secured, and the skin site dressed. Subjects are always carefully instructed in the 
routine prevention of infection and bleeding of biopsy sites. As lidocaine is used as the 
anesthetic there is the risk of toxicity, however the low concentration 0.5% and the use 
of less than 10-15 ml greatly reduces this concern. 
 
Neck Pressure/Neck Suction (NP/NS): There are no reports in the medical literature 
evaluating the risks of NP/NS in either patients or healthy control subjects. However, 
there are over 100 studies indexed in the NLM PubMed database (keyword search 
“neck”; “suction”; “heart rate”) that have used the NP/NS technique safely and 
effectively, with no report of catastrophic events such aortic dissection or stroke. 
Included among these are eight published studies from four independent labs which 
have utilized the technique in heart failure patients [71-78]. The nature of the NP/NS 
technique is somewhat similar to carotid sinus massage, providing some framework for 
risk assessment. Contraindications for carotid sinus massage are the presence of a 
carotid bruit, a known carotid stenosis >50%, and a history of myocardial infarction or 
stroke within the preceding 3 months. These contraindications will serve as exclusion 
criteria for participation in the NP/NS portion of the study, and in these patients, only the 
modified oxford technique will be used to assess baroreflex sensitivity. Further, 
ultrasound Doppler of the carotid artery and physical exam will be performed in all 
patients during the preliminary visit to the laboratory to identify as-yet undiagnosed 
carotid stenoses and bruits, respectively. If any significant plaque is identified during 
these exams, only the modified Oxford test will be performed for assessment of 
baroreflex function.  
 
Pregnancy Risks: The risks for some of the procedures included in protocols 2 and 3 to 
an unborn fetus are not known. Thus, if participant is pregnant, they may not participate 
in these protocols. If participant is not pregnant, but of childbearing age, they must use 
appropriate contraceptives to prevent pregnancy while participating in these protocols. 
Participants will be asked to submit to a urine pregnancy test provided by the research 

 
21
site. Should participants become pregnant while enrolled, they will be withdrawn from 
the study. 
 
Participation in multiple protocols: Each protocol is associated with the risks outlined 
above. However, there is no known accumulative risk associated with participation in 
multiple protocols.   
 
STATISTICAL METHODS, DATA ANALYSIS AND INTERPRETATION: 
 
Protocol 1: The data collected in this study will examine the effects of cardiac 
transplantation on vascular function and exercise hyperaemia. The cross-sectional data 
will be analyzed using student’s t-tests and analysis of variance (ANOVA). A repeated-
measure ANOVA will be performed on all longitudinal data collected. Correlations will be 
performed on all the data. A priori power analysis will be performed, but previous studies 
suggest that approximately 20 subjects per study group will be necessary to achieve 
adequate power with the expected effect size.  
 
Protocols 2, 3 and 4: A total of 80 subjects (40 with heart failure NYHA class I-III and 
40 age-matched controls) will be recruited for the protocols outlined above. With the 
stringent restrictions and pre-screening processes outlined above, we estimate a 6:1 
ratio of those interested in participating to those who are ultimately deemed eligible. 
Further, we estimate a dropout rate of 10%, which may increase for subjects who agree 
to participate in multiple protocols. We have taken these issues of subject recruitment 
and adherence into account in the overall experimental design, such that the protocols 
with the proposed sample size are attainable within the timeline of the proposed project.  
 
The experimental design and statistical analyses have been developed with an 
emphasis upon the focused and well powered testing of the primary hypotheses, but 
guided by the parallel intent to minimize subject burden and study costs. The proposed 
sample size (n = 80) is based upon power analyses of preliminary data for primary 
measurements such as HR, ABP, MSNA, and blood flow. To ensure the recognition of 
even smaller differences than those observed in preliminary data, we will study 40 
subjects per group, resulting in >90% power. Statistical analyses will include 
independent statistics (ANOVA and non-paired t-tests) to examine the differences in the 
cross-sectional comparisons (i.e. HF vs. age-matched controls). Two or three-way 
repeated measures ANOVA will be utilized to identify between group differences in these 
outcome measurements during multiple levels of exercise (i.e. PECO test) and during 
multiple levels of baroreflex stimulation. In addition, each specific aim will routinely utilize 
regression analyses to assist in the interpretation of the data collected. Although some 
multivariate measurements are being made and such an approach will likely require the 
characterization of covariation patterns, our initial approach will be predominantly 
univariate. This design will be aided by the detailed subject screening and subject 
selection which minimizes the number of covariates by, where possible, equally 
distributing variables across groups (e.g. gender, activity level, etc.). However, 
secondary covariate analyses will be performed that determine the role of measured 
variables that are not driven by age or disease state, but do exert an influence upon the 
major dependent variables.  
 
Clinical Records: Patient demographics (DOB, gender, height, weight), past medical 
history, medication use, and results of cardiovascular tests (echocardiogram, 
catheterization, stress test) will be collected. 
 

 
22
The following procedures are in place in the Vascular Laboratory to assure safety 
of research participants: 
1. 
Properly trained personnel will monitor all exercises to ensure they are 
performed correctly. 
2. 
The blood draws will be performed by a certified phlebotomist/registered 
nurse, who is familiar with all disease transmission safeguards and emergency 
procedures, should a problem arise. 
3. 
Blood pressure cuffs will only be inflated to obtain the necessary 
measurements, and will be deflated immediately afterwards. 
4. 
Immediate emergency – call 911, as typically employed at the Salt Lake 
City VA Medical Center that are not covered by the code team from the hospital. 
5. 
A crash cart and defibrillator are maintained in the laboratory. 
 
The following procedures are in place in the Vascular Laboratory to protect 
sensitive subject information: 
1. 
The laboratory is kept locked when there are no lab personnel present. 
2. 
The Logic 7 is turned off when not in use for vascular analyses.  The 
computer is password protected and only the investigators related to this project 
know the password. 
3. 
Hard copies of subject information will be kept in a locked file cabinet in 
Dr. Richardson’s office. Any electronic copies will be kept on a password secured 
computer. 
4. 
Subjects are given copies of their measurements upon request. 
5. 
Each subject will be identified by code numbers.  These numbers will be 
used in a statistical spreadsheet while all statistical analyses are performed.  The 
maximal length of storing this information is 5 years, upon which it will be 
destroyed.  
 
ADMINISTRATIVE RESPONSIBILITIES:  
 
Facilities and Equipment: All protocols will be conducted in the state-of-the-art Human 
Vascular Laboratory at the VA Medical Center, Building B, room 1D23. The laboratory 
measures 600 sq ft in size, and is more than adequate to house these studies. This 
laboratory contains all equipment necessary to complete the proposed work, including: 
Three adjustable hydraulic gurneys; Three ultrasound Doppler system (Logiq 7 and 
Logiq I, GE Medical Systems, Milwaukee, Wisconsin, USA) for determination of limb 
blood flow and vasodilation; A Finger photoplethysmograph (Finometer, Ohmeda, 
Madison, WI, USA) for measurements of arterial blood pressure; A metabolic cart (O2 
consumption) with spirometer (pulmonary function testing); (A 12-lead ECG; A “crash-
cart” and defibrillator; Several custom-built ergometers specifically designed for knee-
extensor, plantar flexion, handgrip, and cycling exercise; and several PC-based work 
stations for data acquisition and analysis. All research records and collected samples will 
be housed in a locked structure in this laboratory.  
 
Recruitment: All cardiac transplant subjects in this study will be volunteers recruited 
through the VA hospital and University of Utah Cardiac Transplant Program, though 
printed advertisement or physician referral. In the case of physician referral, patients 
who are deemed eligible for the study will be referred to the study coordinator to gather  
further information – the physician will not provide patients with a detailed description of 
the study. The healthy control subjects will be recruited by printed advertisement, 
physician referral, and word of mouth around the VA hospital and the University of Utah.  
 

 
23
Subject payment will be as follows: 
 
Preliminary Day: Subjects will be paid $20 upon completion.   
 
Protocol 1: Subjects will be paid $100 upon completion of the four visits, $15/hr pro-rate.  
 
Protocol 2: Subjects will be paid $300 ($15/hr pro-rate) upon completion of the chronic 
supplementation regimen. 
 
Protocol 3: Subjects may choose to participate only in the pre-training portion of the 
study, or enroll in the exercise training program.  
 
Subjects who do not enroll in the exercise training portion of the protocol will 
receive a total of $100 upon completion of the “pre-training” visit, $15/hr pro-rate. 
 
Subjects who enroll in the exercise training portion of the protocol will be paid 
$500 ($15/hr. pro-rate) upon completion of the exercise training  
 
Protocol 4: Subjects will be paid $150 per visit, two visits required.  
 
Data Safety & Monitoring: 
 
Although there are risks associated with the proposed studies, these risks are 
considered minimal. Nonetheless, in the interest of participant safety and to ensure 
compliance with IRB and FDA regulations, we propose the implementation of a data 
safety and monitoring group. Four members of the Utah Vascular Research Lab faculty 
(Drs. Richardson, Amann, Trinity and Wray), Mark Supiano, M.D., Micah Drummond, 
Ph.D., Alan Light, Ph.D., Jacob Jessop, D.O. and Dorothea Rosenberger, M.D. will 
make up the monitoring group, all of whom are affiliated with research at the University 
of Utah and the SLC VA. These members will meet on a quarterly basis, and, in the 
interim, the board will convene in response to any adverse events or unanticipated 
problems associated with the proposed studies.  
In order to meet the regulatory requirements of 21 CFR 312.56 regarding 
monitoring for compliance with federal regulations, thereby ensuring data quality and 
that the rights, safety and welfare of study subjects are adequately protected, this study 
will include a provision for internal monitoring by an independent qualified reviewer. The 
PI will identify a qualified reviewer and present the name of the proposed reviewer to the 
DSMB for approval. The reviewer will monitor all aspects of the study for compliance 
with federal regulations and Good Clinical Practices after the first three (3) subjects have 
been enrolled.  
The reviewer will provide a written summary of findings which will be presented 
to the DSMB at the next meeting. Corrective actions indicated will be followed by the 
DSMB at subsequent meetings and documented in the minutes. If indicated, the DSMB 
will require additional monitoring. Also, if indicated, any protocol deviations or violations 
will be reported the IRB with appropriate corrective action described and a preventative 
action plan outlined to avoid recurrence.  
If follow-up monitoring is not indicated, there will be a final monitoring visit after 
the last subject has been enrolled but prior to study close-out. As above, a written 
summary of findings will be provided to the DSMB for any appropriate action prior to 
DSMB authorization of study closure. This final monitoring report will be provided to the 
IRB as part of the Final Report in ERICA. 
 
All subjects participate on a strictly voluntary basis, and thus any protocol may be 
stopped at any time and for any reason at the subject’s request. Additional criteria to 

 
24
discontinue a protocol will be any evidence that the subject’s health is at risk, as 
identified by a member of the investigative team. Subject safety is at the forefront of all 
studies, and for this reason all investigators are trained to identify overt (i.e. abnormal 
ECG patterns, acute change in blood pressure, etc.) and more subtle (i.e. skin pallor or 
redness, changes in subject demeanor, etc.) signs that a subject may be adversely 
affected by the experimental interventions. Adverse events and unanticipated problems 
are broadly defined as any instance of a medical complication arising from catheter 
insertion (i.e. excessive bleeding and swelling at insertion site) or drug infusion (i.e. 
acute hypertension, bradycardia, etc.) as listed under the “potential risks” section below.  
In the instance of an adverse event, the protocol will be discontinued and the 
study will be temporarily suspended while DSMB members will review the cause of the 
event. When appropriate, unanticipated adverse events will be reported within 10 
working days, in accordance with the University of Utah IRB Reporting Policy.  
 
 
IND Regulations for Data Safety & Monitoring: 
As a sponsor of an IND, we are additionally responsible for reporting protocol deviations, 
violations, and adverse events to the FDA, which will be implemented in compliance with 
the Code of Federal Regulation (21CFR312.32, b – e), as follows; 
(b) Review of safety information. The sponsor shall promptly review all information 
relevant to the safety of the drug obtained or otherwise received by the sponsor from any 
source, foreign or domestic, including information derived from any clinical or 
epidemiological investigations, animal investigations, commercial marketing experience, 
reports in the scientific literature, and unpublished scientific papers, as well as reports 
from foreign regulatory authorities that have not already been previously reported to the 
agency by the sponsor. 
(c) IND safety reports --(1)Written reports --(i) The sponsor shall notify FDA and all 
participating investigators in a written IND safety report of: 
(A) Any adverse experience associated with the use of the drug that is both 
serious and unexpected; or 
(B) Any finding from tests in laboratory animals that suggests a significant risk for 
human 
subjects 
including 
reports 
of 
mutagenicity, 
teratogenicity, 
or 
carcinogenicity. Each notification shall be made as soon as possible and in no 
event later than 15 calendar days after the sponsor's initial receipt of the 
information. Each written notification may be submitted on FDA Form 3500A or in 
a narrative format (foreign events may be submitted either on an FDA Form 
3500A or, if preferred, on a CIOMS I form; reports from animal or epidemiological 
studies shall be submitted in a narrative format) and shall bear prominent 
identification of its contents, i.e. , "IND Safety Report." Each written notification to 
FDA shall be transmitted to the FDA new drug review division in the Center for 
Drug Evaluation and Research or the product review division in the Center for 
Biologics Evaluation and Research that has responsibility for review of the IND. If 
FDA determines that additional data are needed, the agency may require further 
data to be submitted. 
(ii) In each written IND safety report, the sponsor shall identify all safety 
reports previously filed with the IND concerning a similar adverse 

 
25
experience, and shall analyze the significance of the adverse experience 
in light of the previous, similar reports. 
(2)Telephone and facsimile transmission safety reports. The 
sponsor shall also notify FDA by telephone or by facsimile 
transmission 
of 
any 
unexpected 
fatal 
or 
life-threatening 
experience associated with the use of the drug as soon as 
possible but in no event later than 7 calendar days after the 
sponsor's initial receipt of the information. Each telephone call or 
facsimile transmission to FDA shall be transmitted to the FDA new 
drug review division in the Center for Drug Evaluation and 
Research or the product review division in the Center for Biologics 
Evaluation and Research that has responsibility for review of the 
IND. 
(3)Reporting format or frequency. FDA may request a sponsor to 
submit IND safety reports in a format or at a frequency different 
than that required under this paragraph. The sponsor may also 
propose and adopt a different reporting format or frequency if the 
change is agreed to in advance by the director of the new drug 
review division in the Center for Drug Evaluation and Research or 
the director of the products review division in the Center for 
Biologics Evaluation and Research which is responsible for review 
of the IND. 
(4) A sponsor of a clinical study of a marketed drug is not required 
to make a safety report for any adverse experience associated 
with use of the drug that is not from the clinical study itself. 
(d) Followup. 
(1) The sponsor shall promptly investigate all safety information received by it. 
(2) Followup information to a safety report shall be submitted as soon as the 
relevant information is available. 
(3) If the results of a sponsor's investigation show that an adverse drug 
experience not initially determined to be reportable under paragraph (c) of this 
section is so reportable, the sponsor shall report such experience in a written 
safety report as soon as possible, but in no event later than 15 calendar days 
after the determination is made. 
(4) Results of a sponsor's investigation of other safety information shall be 
submitted, as appropriate, in an information amendment or annual report. 
(e) Disclaimer. A safety report or other information submitted by a sponsor under this 
part (and any release by FDA of that report or information) does not necessarily reflect a 
conclusion by the sponsor or FDA that the report or information constitutes an admission 
that the drug caused or contributed to an adverse experience. A sponsor need not admit, 
and may deny, that the report or information submitted by the sponsor constitutes an 
admission that the drug caused or contributed to an adverse experience. 

 
26
We are also required by the FDA to provide annual reports, in accordance with  
21CFR312.33, as follows; 
A sponsor shall within 60 days of the anniversary date that the IND went into effect, 
submit a brief report of the progress of the investigation that includes: 
(a)Individual study information. A brief summary of the status of each study in progress 
and each study completed during the previous year. The summary is required to include 
the following information for each study: 
(1) The title of the study (with any appropriate study identifiers such as protocol number), 
its purpose, a brief statement identifying the patient population, and a statement as to 
whether the study is completed. 
(2) The total number of subjects initially planned for inclusion in the study; the number 
entered into the study to date, tabulated by age group, gender, and race; the number 
whose participation in the study was completed as planned; and the number who 
dropped out of the study for any reason. 
(3) If the study has been completed, or if interim results are known, a brief description of 
any available study results. 
(b)Summary information. Information obtained during the previous year's clinical and 
nonclinical investigations, including: 
(1) A narrative or tabular summary showing the most frequent and most serious adverse 
experiences by body system. 
(2) A summary of all IND safety reports submitted during the past year. 
(3) A list of subjects who died during participation in the investigation, with the cause of 
death for each subject. 
(4) A list of subjects who dropped out during the course of the investigation in 
association with any adverse experience, whether or not thought to be drug related. 
(5) A brief description of what, if anything, was obtained that is pertinent to an 
understanding of the drug's actions, including, for example, information about dose 
response, information from controlled trials, and information about bioavailability. 
(6) A list of the preclinical studies (including animal studies) completed or in progress 
during the past year and a summary of the major preclinical findings. 
(7) A summary of any significant manufacturing or microbiological changes made during 
the past year. 
(c) A description of the general investigational plan for the coming year to replace that 
submitted 1 year earlier. The general investigational plan shall contain the information 
required under 312.23(a)(3)(iv). 

 
27
(d) If the investigator brochure has been revised, a description of the revision and a copy 
of the new brochure. 
(e) A description of any significant Phase 1 protocol modifications made during the 
previous year and not previously reported to the IND in a protocol amendment. 
(f) A brief summary of significant foreign marketing developments with the drug during 
the past year, such as approval of marketing in any country or withdrawal or suspension 
from marketing in any country. 
(g) If desired by the sponsor, a log of any outstanding business with respect to the IND 
for which the sponsor requests or expects a reply, comment, or meeting 
 
Control of Investigational Drugs: The VA Pharmacy will order, store, and dispense 
Phenylephrine Hydrochloride USP and Sodium Nitroprusside USP. The investigative 
team will administer and dispose of the drugs, which will be documented according to 
FDA guidelines. 
  

 
28
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