The Role of Aldosterone on Augmented Exercise Pressor Reflex in Hypertension
Phase: PHASE2
Conditions: Hypertension
Sponsor: University of Texas Southwestern Medical Center

1 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
Protocol 
 
Title: The Role of Aldosterone on Exercise Pressor Reflex in Hypertension , NCT01996449 
December 12, 2018 
Investigators 
 
Principal Investigator: Wanpen Vongpatanasin, M.D., Cardiology Division, Department of 
Medicine, University of Texas Southwestern Medical Center 
 
Participating Institutions: University of Texas Southwestern Medical Center 
 
 
 
 
 
  
Funding: 
 NIH 
 
 
Table of Contents: 
 
Protocol Section 
 
 
 
 
                          Pages 
 
1) Introduction  
 
 
 
 
                              2  
2) Background 
 
 
 
 
                              3 
3) Concise Summary of Project                                                    4 -5 
 
 
  
4) Study Procedures                                                                        6-9 
5) Criteria for Inclusion of Subjects               
                              9 
6) Criteria for Exclusion of Subjects 
 
                              9-10 
7) Sources of Research Material  
                                         10 
8) Recruitment Methods and Consenting Process                          10 
9) Potential Risks  
 
 
 
                              10 
10) Subject Safety and Data Monitoring  
                              10-13 
11) Procedures to Maintain Confidentiality                                       13 
12) Potential Benefits 
                                                               13-14 
13) Biostatistics                                                                                 14-15 
14) References 
 
 
 
 
                               16 

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1. Introduction and Purpose: 
 
Hypertensive patients often show an exaggerated rise in blood pressure (BP) during exercise—
sometimes to the levels that raise safety concern. The mechanisms responsible for exercise 
pressor reflex (EPR) overactivity in hypertension remain unknown but an increasing body of 
evidence suggests a role for aldosterone in regulating resting central sympathetic outflow in both 
hypertensive rats and humans. Furthermore, local aldosterone synthesis in the brain has been 
shown to contribute to sympathetic activation and hypertension in SHR and Dahl-salt sensitive 
rats despite normal circulating levels of aldosterone. Whether aldosterone contributes to 
excessive sympathoexcitation and BP elevation during exercise remains to be investigated. 
Therefore, we now propose parallel translational studies in hypertensive patients with primary 
hypertension and a secondary form of hypertension caused by adrenal overproduction of 
aldosterone (primary aldosteronism) to systematically investigate the contribution of aldosterone 
to the generation of EPR dysfunction.   
 
Major New Hypothesis: Aldosterone augments the EPR by selectively potentiating muscle 
mechanoreflex activity in hypertensive humans. We further hypothesize that this effect of 
aldosterone is mediated by its direct action on the central nervous system and that 
mineralocorticoid receptor (MR) antagonists constitute an effective treatment for EPR 
overactivity in hypertension, independent of reductions in resting BP. 
 
 
In patients with uncomplicated essential hypertension (EH), hypertensive patients with primary 
aldosteronism (PA), and normotensive (NT) controls matched for age, gender, ethnicity, and 
BMI, we will perform multiple exercise interventions to determine if: 
a. Sympathetic nerve activity (SNA) and BP responses to EPR activation during rhythmic 
handgrip exercise and mechanoreflex activation during passive arm cycling are 
augmented in patients with EH and PA. 
b. SNA and BP responses to metaboreflex activation during post handgrip arm occlusion 
are unaltered in the EH and PA groups. 
c. Indices of central command (increases in skin SNA, heart rate, and rating of perceived 
effort) input during exercise are similarly increased in EH and PA groups compared to the 
NT group. In addition, the sensitivity of the arterial baroreflex to modulate SNA and BP 
responses to exercise is unaltered during rhythmic and static handgrip in EH and PA 
groups. 
d. Augmented EPR and mechanoreflex sensitivity in a subset of PA patients with 
aldosterone-producing adenoma (APA) is reversible upon surgical removal of the tumor. 
e. MR antagonists constitute an effective treatment for EPR dysfunction by attenuating 
mechanoreflex overactivity in patients with EH and PA. Such effects have not been 
observed with treatment with amlodipine despite similar reductions in resting BP. 

3 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
2. Background:  
 
Hypertensive patients are known to display exaggerated rise in blood pressure (BP) during exercise but 
the underlying mechanisms are poorly understood. Normally, exercise is accompanied by decreased 
parasympathetic activity and increased sympathetic activity caused by central command and activation 
of thin fiber muscle afferents that reflexively increase sympathetic outflow and BP. Traditionally, muscle 
afferents were dichotomized as metaboreceptors, which are activated slowly and only during intense or 
ischemic muscle contraction, or mechanoreceptors, which respond quickly to even mild deformation of 
their receptive fields. The increase in SNA and BP caused by activation of these receptors, known as 
exercise pressor reflex, is normally buffered by activation arterial baroreceptors, which are reset to 
operate at higher BP range but at the same level of sensitivity. Our recent work in spontaneously 
hypertensive rats (SHR) and patients with essential hypertension indicated an exaggerated rise in 
sympathetic nerve activity (SNA) and BP during muscle contraction even in the absence of congestive 
heart failure. Furthermore, the latency of the sympathetic response during exercise is much shorter than 
normal and the threshold level of exercise intensity required to increase muscle SNA is much lower than 
normal, implicating mechanoreceptor mediation. Mechanisms responsible for overactive exercise 
pressor reflex (EPR) in hypertension remain unknown but an increasing body of evidence suggested a 
role for aldosterone in regulating resting central sympathetic outflow in both hypertensive rats and 
humans. Furthermore, local aldosterone synthesis in the brain has been shown to contribute to 
sympathetic activation and hypertension in the SHRs and Dahl-salt sensitive rats despite normal 
circulating levels of aldosterone. Whether aldosterone contributes to excessive sympathoexcitation and 
BP elevation during exercise remains unknown. Therefore, we now propose parallel translational studies 
in hypertensive patients with primary hypertension and secondary form of hypertension caused by 
adrenal overproduction of aldosterone (primary aldosteronism), using multiple interventions to directly 
activate muscle mechanoreceptors, metaboreceptors, or arterial baroreceptors during exercise and 
microelectrode recordings of SNA to measure the reflex response. The distinctive features of this 
proposal include: (1) the use of state-of- the-art techniques (microelectrode recordings of SNA) to directly 
test mechanistic hypotheses in conscious humans; (2) assessment of mechanoreflex using a new 
paradigm which is developed in our laboratory which evokes sustained elevation in SNA and BP than 
other methods previously described in humans; and (3) the use of complimentary approach to asses 
central vs. peripheral effects of aldosterone on neural control of circulation.  
 
 
Significance and Cardiovascular Relevance: Hypertension is a major cause of cardiovascular 
morbidity and mortality in the United States and many countries worldwide. Excessive elevation in BP 
during exercise has been shown to contribute to impaired exercise tolerance in hypertensive patients 
even in the absence of coronary artery disease or left ventricular dysfunction 1-4. Furthermore, numerous 
epidemiological studies indicated that BP during exercise predicts development of left ventricular 
hypertrophy 5, 6, stroke, myocardial infarction, and death 7-9, independent of resting BP. Accordingly, 
current guidelines prohibit any exercise in hypertensive patients if BP during exercise is 220/105 or above 
for safety reasons 10, 11. Therefore, exercise prescription is often limited in hypertensive patients with an 
enhanced EPR 12. Because most current antihypertensive drugs approved are much less effective in 
reducing BP at rest than during exercise 9, 13-18, a more complete mechanistic understanding of the 
augmented pressor response to exercise in human hypertension could lead to effective antihypertensive 
treatment. This will in turn allow more hypertensive patients to engage in regular exercise and enjoy the 
cardiovascular benefits of exercise, which is also an important nonpharmacologic treatment of 
hypertension. The information derived from our proposed studies will be pertinent not only to patients 
with primary hypertension but also those with primary aldosteronism which has been identified in 5-13% 
of unselected patients with hypertension in the community and primary care practice 19, 20 and up to 20% 
of patients with resistant hypertension referred to the tertiary care hypertension clinic 21. 
 
 
3.Concise Summary of Project: 
 

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General Methods BP will be measured with an oscillometric device (Welch Allyn, N.C.).  
(1) Microneurography. With the subject supine, we will measure blood pressure, heart rate, and muscle 
SNA with microelectrodes in the arm (median nerve or radial nerve or posterior cutaneous nerve) or leg 
(peroneal nerve). The nerve signals are amplified, filtered, rectified and integrated to obtain a mean 
voltage display of SNA. The criteria for an acceptable recording of muscle SNA has been previously 
described 15. Sympathetic bursts will be detected by inspection of the filtered and mean voltage 
neurograms. A deflection on the mean voltage display is counted as a "burst" if it has a minimal signal 
to noise ratio of 2:1. SNA will recorded at rest in all protocols.  
 
(2) Reflex Interventions: (a) Handgrip exercise . Rhythmic HG (RHG) will be performed in the 
nondominant arm at 40 beats per minute (50% cycle duty) at 30% and 45% maximal voluntary contraction 
(MVC), each for 3 minutes. Static HG (SHG) will be performed at 30% MVC for 2 minutes. Ratings of 
perceived exertion (RPE) will be obtained at the end of each exercise by using a 6- to 20-unit Borg scale. 
For safety, the study will be terminated if BP during exercise at any time point is 220/105 mmHg or above 
in accordance with current guidelines 10, 11. (b) Post-Handgrip Exercise Circulatory Arrest (PECA). During 
the last 5 sec of static HG exercise, a pneumatic cuff on the upper exercising arm will be inflated to 50 
mmHg above systolic BP for 2 minutes to activate the metaboreflex. (c) Passive arm cycling. To activate 
mechanoreflex, the subject’s nondominant hand will be strapped to one of the arm pedal of the arm 
ergometer while the investigator put their hand on the other pedal to move the arm ergometer at 80 r.p.m. 
Subjects will be asked to relax their arms during passive arm cycling for 5 minutes. Surface 
electromyography (EMG) on the bicep muscles (Noraxon, Scottsdale, AZ) will be used to monitor the 
muscular activity and a biofeedback device to assist the subjects with relaxation of their arm muscles. (d) 
Arterial baroreflex sensitivity. Arterial baroreflex control of muscle SNA and HR will be assessed during 
bolus injection of nitroprusside (NTP) and phenylephrine (PE) using the modified Oxford technique 22. 
For safety reasons, NTP will not be given if BP is ≤90/60 mmHg and PE will not be given if BP is ≥ 
160/100 mmHg immediately prior to administration. 
 
(3) Cardiopulmonary testing: A subset of subjects who opt for this optional testing, will be asked to cycle 
on a rate-independent ergometer to measure peak VO2 using a metabolic cart (Medgraphics) during a 
separate visit. An established cycle ergometer ramp protocol will be performed. Standard indications for 
termination of exercise testing will be employed. Respiratory gases inclusive of VE, VO2, VCO2, and RER 
will be measured continuously throughout the exercise test. We will also measure continuous ECG and 
maximal heart rate, maximal oxygen uptake (peak VO2), pulmonary ventilation (VE), ventilatory 
equivalents for oxygen (VE/VO2), carbon dioxide (VE/CO2), end-tidal partial pressure of oxygen (PETO2), 
and carbon dioxide (PETCO2). Simultaneous cardiac output (CO) measurement will be performed at rest 
and during steady state exercise testing by inert gas rebreathing method using Inncor system (Innovision 
A/S, Odense, Denmark). Cardiac output will be measured separately from maximal exercise testing in 
three conditions: (1) at rest, (2) at 20 Watts, and (3) at 50% peak VO2/work load. After the participant 
has completed the peak exercise testing protocol and rested 5 minutes, exercise stress 
echocardiography will be performed at 50% of maximal workload. 
 
 
Premenopausal women will be studied during the early follicular phase (2-4 days after the onset of 
menstruation) to minimize confounding influence of sex hormones on SNA 23. Women taking oral 
contraceptives will be studied during low hormone (LH) phase (i.e., placebo pills). 
 
 
 
 
 
 
 
 
 
 
 
 
 
Agent/Device 
A. Amlodipine  
Trade name: Norvasc 
Chemical Formula: 3-Ethyl-5-methyl (±)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-l,4-dihydro-6-
methyl-3,5-pyridinedicarboxylate, monobenzenesulphonate 

5 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
Source: Pfizer Inc. 
            Pharmacology: Inhibits calcium ion from entering the “slow channels” or select voltage-sensitive areas of 
vascular smooth muscle and myocardium during depolarization, producing a relaxation of coronary 
vascular smooth muscle and coronary vasodilation; increases myocardial oxygen delivery in patients with 
vasospastic angina. 
Toxicity: Peripheral edema, fatigue, dizziness, hypotension, and gingival hyperplasia. 
 
B. Eplerenone  
Trade name: Inspra 
Chemical Formula: Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3oxo-, γ-lactone, methyl 
ester, (7α,11α,17α) 
Source: Pfizer Inc. 
            Pharmacology: Eplerenone binds to the mineralocorticoid receptor and blocks the binding of aldosterone, 
a component of the renin-angiotensin-aldosterone-system (RAAS). Aldosterone synthesis, which occurs 
primarily in the adrenal gland, is modulated by multiple factors, including angiotensin II and non-RAAS 
mediators such as adrenocorticotropic hormone (ACTH) and potassium. Aldosterone binds to 
mineralocorticoid receptors in both epithelial (e.g., kidney) and nonepithelial (e.g., heart, blood vessels, 
and brain) tissues and increases blood pressure through induction of sodium reabsorption and possibly 
other mechanisms. 
Toxicity: hyperkalemia, orthostatic hypotension, and acute renal failure 
 
 
 
4.Study Procedures: 
 
Experiments will be performed on 3 groups of nondiabetic subjects 1) stage I (140-159/90-99 mmHg) 
subjects with EH, 2) stage I hypertensive subjects with PA, and 3) NT controls. The diagnosis of PA will 
be established based on the current Endocrine Society guidelines 24. As part as standard medical 
care, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, renin inhibitors, central 
sympatholytic drugs and diuretics will be discontinued for at least 2 weeks and MR antagonists will be 
discontinued for at least 6 weeks to avoid interference with biochemical testing 24. During the period of 
biochemical evaluation, subjects will be treated with calcium channel blockers, hydralazine, and/or 
alpha-adrenergic receptor blockers, which do not interfere with measurement of plasma renin activity 
(PRA) or aldosterone 24. The screening evaluation is considered to be positive if plasma aldosterone 
levels are > 15 ng/dL and PRA are < 1 ng/ml/hr. Diagnosis of PA is established by presence of 
nonsuppressible aldosterone production after a salt loading test according to the guidelines 24. Subjects 
with biochemical confirmation of PA will undergo adrenal vein sampling (AVS) as part of standard 
care to distinguish between unilateral vs. bilateral adrenal overproduction of aldosterone. We need to 
determine if association between aldosterone and SNA as well as BP is independent of dietary pattern, 
including caloric intake. Dietary pattern will be assessed by food recall using the Automated Self-
Administered 24-Hour (ASA24®) Dietary Assessment Tool (https://epi.grants.cancer.gov/asa24/).   
 
 
The following procedures are experimental: 
 
We plan to assess muscle SNA and MAP at rest, during passive one-arm cycling exercise (see aim 2a), 
RHG, and SHG followed by PECA in 15 EH and 15 PA subjects (not included in previous aims). Subjects 
will be randomized to receive eplerenone (50-200 mg/day) or amlodipine (2.5-10 mg/day) each for 8 
weeks, using randomized, double-blinded, crossover design. The rationale for using amlodipine is that 
previous studies have shown that this drug reduces BP without affecting resting SNA 26. BP and serum 
K will be monitored every 2 weeks. Each subject will be started with 50 mg eplerenone/day or 2.5 mg 
amlodipine/day. If BP remains above 140/90 mmHg at week 2, the dose of eplerenone will be increased 
to 100 mg/day, whereas amlodipine will be increased to 5 mg/day. If BP remains elevated at week 6, 

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eplerenone will be increased to 200 mg/day and amlodipine to 10 mg daily. This dose titration of 
amlodipine and eplerenone is chosen because it was shown to cause equivalent reductions in BP in 
hypertensive patients in a large clinical trial 27. If hyperkalemia develops with eplerenone, the dose will 
be reduced by 50% and serum K will be rechecked in 2 weeks (see detail in section 10d). We predict that 
eplerenone will attenuate the increase in SNA and MAP during HG and passive arm cycling in subjects 
with EH and PA without affecting SNA and BP responses to PECA. Amlodipine will have no effect on 
SNA or BP in the same subjects during all exercise interventions, despite causing similar reduction in 
resting BP.  
 
Visit#1 (week 0): Obtain blood samples for basic metabolic panel, resting levels of plasma direct renin, 
aldosterone (aldo), norepinephrine (NE), and fasting plasma glucose and insulin. Then, assess muscle 
SNA and MAP at rest, during passive one-arm cycling exercise for 5 minutes (see general method 
#2c). During passive arm cycling, surface electromyography (EMG) on the bicep muscles (Noraxon, 
Scottsdale, AZ) will be used to monitor the muscular activity and a biofeedback device to assist the 
subjects with relaxation of their arm muscles.  Then, subjects will be asked to perform, RHG at 30% 
and 45% MVC each for 3 minutes, and SHG at 30% MVC followed by PECA for 2 minutes. Each 
exercise intervention will be separated by at least 30 minutes. After completion of exercise, each 
subject will be randomized to receive Eplerenone at the dose of 50 mg once daily or Amlodipine 2.5 mg 
once daily. 
 
Visit#2 (2 weeks later): Subjects will return for BP check and blood draw for basic metabolic panel. If 
BP is < 90/60 mmHg, subjects will be excluded from the study. If subjects BP > 140/90, the dose of 
Amlodipine will be increased to 5 mg once daily or Eplerenone will be increased to 100 mg once daily. 
3cc of blood will be drawn for basic metabolic profile. If If hyperkalemia develops with eplerenone, the 
dose will be reduced by 50% and serum K will be rechecked in 2 weeks. 
 
Visit#3 (2 weeks later): Subjects will return for BP check and blood draw. If BP is < 90/60 mmHg, the 
dose of study drugs will be reduced by 50%. If subjects BP > 140/90, the dose of Amlodipine will be 
increased to 7.5 mg once daily or Eplerenone will be increased to 150 mg once daily. 3cc of blood will 
be drawn for basic metabolic profile. If hyperkalemia develops with eplerenone, the dose will be 
reduced by 50% and serum K will be rechecked in 2 weeks.  
 
Visit#4 (2 weeks later): Subjects will return for BP check and blood draw. If BP is < 90/60 mmHg, the 
dose of study drugs will be reduced by 50%. If subjects BP > 140/90, the dose of Amlodipine will be 
increased to 10 mg once daily or Eplerenone will be increased to 200 mg once daily. 3cc of blood will 
be drawn for basic metabolic profile. If hyperkalemia develops with eplerenone, the dose will be 
reduced by 50% and serum K will be rechecked in 2 weeks. 
 
Visit#5 (2 weeks later): Subjects will return for blood draw for basic metabolic panel, resting levels of 
plasma direct renin, aldosterone (aldo), norepinephrine (NE), and fasting plasma glucose and insulin. 
Then, assess muscle SNA and MAP at rest, during passive one-arm cycling exercise for 5 minutes (see 
general method #2c). During passive arm cycling, surface electromyography (EMG) on the bicep 
muscles (Noraxon, Scottsdale, AZ) will be used to monitor the muscular activity and a biofeedback 
device to assist the subjects with relaxation of their arm muscles.  Then, subjects will be asked to 
perform RHG at 30% and 45% MVC each for 3 minutes, and SHG at 30% MVC followed by PECA for 2 
minutes. Then, subjects will be given BP medications in the remaining arm (either Amlodipine 2.5 mg 
once daily or Eplerenone at the dose of 50 mg once daily). 
 
Visit#6 (2 weeks later): Subjects will return for BP check and blood draw. If BP is < 90/60 mmHg, 
subjects will be excluded from the study. If subjects BP > 140/90, the dose of Amlodipine will be 
increased to 5 mg once daily or Eplerenone will be increased to 100 mg once daily. 3cc of blood will be 
drawn for basic metabolic profile. If hyperkalemia develops with eplerenone, the dose will be reduced 
by 50% and serum K will be rechecked in 2 weeks. 
 

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Visit#7 (2 weeks later): Subjects will return for BP check and blood draw. If BP is < 90/60 mmHg, the 
dose of study drugs will be reduced by 50%. If subjects BP > 140/90, the dose of Amlodipine will be 
increased to 7.5 mg once daily or Eplerenone will be increased to 150 mg once daily. 3cc of blood will 
be drawn for basic metabolic profile. If hyperkalemia develops with eplerenone, the dose will be 
reduced by 50% and serum K will be rechecked in 2 weeks. 
 
Visit#8 (2 weeks later): Subjects will return for BP check and blood draw. If BP is < 90/60 mmHg, the 
dose of study drugs will be reduced by 50%. If subjects BP > 140/90, the dose of Amlodipine will be 
increased to 10 mg once daily or Eplerenone will be increased to 200 mg once daily. 3cc of blood will 
be drawn for basic metabolic profile. If hyperkalemia develops with eplerenone, the dose will be 
reduced by 50% and serum K will be rechecked in 2 weeks. 
 
Visit#9 (8 weeks later): Subjects will return for blood draw for basic metabolic panel, resting levels of 
plasma direct renin, aldosterone (aldo), norepinephrine (NE), and fasting plasma glucose and insulin. 
Then, assess muscle SNA and MAP at rest, during passive one-arm cycling exercise for 5 minutes (see 
general method #2c). During passive arm cycling, surface electromyography (EMG) on the bicep muscles 
(Noraxon, Scottsdale, AZ) will be used to monitor the muscular activity and a biofeedback device to assist 
the subjects with relaxation of their arm muscles.  Then, subjects will be asked to perform RHG at 30% 
and 45% MVC each for 3 minutes, and SHG at 30% MVC followed by PECA for 2 minutes. End of study. 
 
5. Inclusion Criteria 
Experiments will be performed on 3 groups of nondiabetic human subjects 1) stage I (140-159/90-99 
mmHg) subjects with essential hypertension, 2) stage I hypertensive subjects with primary aldosteronism, 
and 3) normotensive controls. Only subjects without CHF, any heart diseases, or target organ disease 
with eGFR ≥ 90 ml/min/1.73 m2, can participate in the study.  
 
 
6. Exclusion Criteria 
 
 
 
 (1) Any evidence of cardiopulmonary disease, left ventricular hypertrophy or systolic dysfunction 
by echocardiography.  
 
 
(2) Blood pressure averaging ≥160/100 mmHg 
 
 
(3) Estimated glomerular filtration rate (eGFR) < 90 mL/min/1.73m2 
 
 
(4) Diabetes mellitus or other systemic illness   
 
 
 
   
 
(5) Pregnancy 
  
(6) Hypersensitivity to nitroprusside, phenylephrine, amlodipine or eplerenone 
 
 
(7) Any history of substance abuse or current cigarette use  
 
 
(8) Any history of psychiatric illness  
 
(9) History of malignancy 
 
 
 
7. Sources of Materials: 
 
  
 
The physician-investigators will have access to the patients' medical records. We also will have 
access to the Dallas Heart Study database, as all participants gave their written consent to be 
contracted for future research studies. The Dallas Heart Study health survey questionnaire data 
will be used to identify potentially eligible subjects to be recruited for our studies. However, all the 
new hemodynamic and hormonal data at rest and during handgrip exercise (SNA, BP, HR, plasma 
NE, insulin, glucose, serum K, renin, serum aldosterone, baroreflex sensitivity) will be obtained 
solely for research purposes.      
 
 
8. Recruitment Methods and Consenting Process: Informed written consent will be obtained from all 
subjects using consent forms approved by the UT Southwestern IRB. The physician-investigators will 
interview all potential subjects and will explain the nature of all experimental procedures in detail. They 

8 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
also will perform a pertinent medical history and physical examination. Normal controls and subjects with 
essential hypertension will be recruited via advertisement in the campus and letter of invitation (only for 
DHS subjects who had given consent for contact for participation in future studies). PA subjects will be 
recruited from the hypertension clinic at Aston ambulatory center by the P.I. and study investigators.  
.  
 
9. Potential Risks: 
The potential risks are related to: a) microneurography, b) intravenous infusion of nitroprusside 
(NTP), c) intravenous infusion of phenylephrine (PE), d) administration of eplerenone, e) 
venipuncture, and f) administration of amlodipine, and g) Potential risk of handgrip exercise. 
 
a) The potential risks of microneurography: 10% of subjects may experience transient arm or 
legtiredness, or increase sensitivity to touch (hyperesthesia), motor weakness  lasting less than 
1 week. Since 1979, microneurography has been performed on more than several thousand 
subjects without permanent complications related to procedure.  
 
 
b) The potential risks of administration of nitroprusside is flushing, hypotension and tachycardia. 
For safety reasons, NTP will not be given if resting BP is ≤90/60 mmHg. 
 
c) The potential risk of administration of phenylephrine is excessive increase in BP. PE will not be 
given if resting BP is ≥ 160/100 mmHg. 
 
d) Potential risk of Eplerenone is hypotension, hyperkalemia, and renal dysfunction. 
 
e) Potential risks of venipuncture are infection, bleeding, and vasovagal reaction.  
 
f) Potential risk of amlodipine is hypotension, gingival hyperplasia, and lower extremity edema. 
 
g) Potential risk of handgrip exercise is hand discomfort and excessive increase in BP. However, 
study will be terminated if BP during exercise at any time point is 220/105 mmHg or above for 
safety reasons according to the current guidelines 10, 11. 
 
h) Potential Risks for exercise testing: There is a finite probability of an adverse cardiovascular 
event (myocardial infarction or sustained ventricular tachycardia: 1.2/10,000) and death 
(<~1/10,000) in individuals undergoing exercise testing.  Exercise testing will be terminated based 
on standard indications, including ST-segment elevation, decreased systolic blood pressure > 10 
mmHg with exercise, sustained ventricular tachycardia. Resuscitative equipment and study 
personnel certified in Advanced Cardiovascular Life Support (ACLS) will be nearby in the rare 
case of a medical emergency. If there is cardiovascular or respiratory compromise, the “Code 
Blue” system will be triggered in the UT Southwestern campus while ACLS is administered by 
trained personnel within the room. A defibrillator is located in the exercise lab and can be 
immediately available should the situation arise. An attending cardiologist will be immediately 
available by phone call as needed. 
 
 
 
10. Subject Safety and Data Monitoring:  
 
a) Microneurography will be performed under the supervision of Dr. Vongpatanasin who has 
extensive experience.  A stimulus isolation unit (special electronic circuit) will be used to prevent 
electrocution from the recording apparatus. Subjects will be given a microneurographic 
questionnaire to report any adverse reactions occurring up to one week after the study.  
 

9 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
b) All subjects will have a routine history and physical examination performed by the physician-
investigators. In addition, hospital medical records will be reviewed prior to the initiation of these 
studies. Venipuncture will be performed by research nurse at the CTRC, using sterile technique. 
 
c) ACLS-certified personnel, including a board certified cardiologist, will be present during all 
experiments. Resuscitative equipment and drugs will be available in the laboratory. Blood 
pressure and the subject’s heart rate will be monitored continuously during all experiments.  
During baroreflex testing, NTP will not be given if resting BP is ≤90/60 mmHg and PE will not be 
given if BP is ≥ 160/100 mmHg.  
 
During handgrip exercise intervention, study will be terminated if BP at any time point is 220/105 
mmHg or above for safety reasons according to the current guidelines 10, 11.  
 
d) During treatment with eplerenone in aim 1e, BP and serum K will be monitored every 2 weeks. 
Each subject will be started with 50 mg eplerenone daily or 2.5 mg amlodipine daily. If BP remains 
above 140/90 mmHg at week 2, the dose of eplerenone will be increased to 100 mg daily, whereas 
amlodipine will be increased to 5 mg daily. If BP remained elevated at week 6, eplerenone will be 
increased to 200 mg daily and amlodipine to 10 mg daily If subjects develop hypotension (BP < 
90/60 mmHg persistently), the eplerenone and amlodipine will be reduced by 50%. BP will be 
rechecked within 2 weeks in these subjects. 
 
This dose range and pattern of titration of eplerenone and amlodipine is chosen because it was 
shown to produce equivalent reduction in BP in a large clinical trial 27 and there were no serious 
adverse events related to either drug with this dose range 27, 28. The incidence of hyperkalemia (K 
> 5.5 mmol/L) was between 1.1-3.0% in the eplerenone group 27, 28 and 1.5% in the amlodipine 
group 27 
 
If subjects develop serum K between 5.5-6.0 mmol/L with eplerenone, the dose will be reduced 
by half and serum K will be rechecked in 2 weeks.  
 
Those who develop hypokalemia during the follow up visit (K 3.0-3.5 mmol/L) will be started on K 
supplement 40 mmol daily and serum K will be rechecked in 2 weeks. 
 
Subjects who develop severe hypokalemia (< 3.0 mmol/L) or hyperkalemia ( > 6.0 mmol/L), EKG 
will be obtained immediately and the study procedures will be discontinued in these subjects. 
Subjects will be transferred to the emergency department for further evaluation and management. 
Known adverse events and the likelihood of occurrence are described in the IRB-approved informed 
consent document. 
The classification of adverse events will follow the Common Toxicity Criteria used by the National Cancer 
Institute at http://ctep.cancer.gov/forms/CTCv20_4-30-992.pdf.  Attribution of an adverse event to study 
procedures will be in accordance with IRB terminology ("definite," "probable," "possible," "unknown," or 
"unrelated" relationship). 
Ms. Beverley Adams-Huet, our collaborating investigator, will perform the aggregate analysis of adverse 
event reports. 
In the absence of adverse events, data analysis will occur at weekly intervals and reviewed at weekly lab 
meetings. 
Monitoring will be performed in conjunction with each subject's experience with study procedures, and 
conclusions of the monitoring reported to the IRB and CTRC no less than every 12 months in accordance 

10 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
with IRB and CTRC requirements. Such progress reports will include a discussion of data quality and 
timeliness, participant recruitment, accrual and retention; participant risk versus benefit, and other factors 
than can affect study outcome. 
Monitoring reported to the IRB and CTRC will also include consideration of factors external to the study 
when interpreting the data, such as scientific or therapeutic developments that may have an impact on 
the safety of the participants or the ethics of the study. 
Stop Points:  Any of the following criteria will constitute stop points.   
• 
Refractory hyperkalemia defined as more than one episode of serum potassium level > 5.5 
mEq/L.  The second episode must be confirmed by repeat measurement in the core laboratory. 
• 
Withholding of study medication for > 4 weeks for whatever reason. 
• 
Any reason including serious adverse events that results in participant’s inability to continue with 
study protocol and procedures.  
• 
Severe hypertension defined as persistent elevation of systolic BP > 180 mmHg or diastolic BP > 
110 mmHg on 3 consecutive measurements after randomization 
• 
Refractory hypotension defined as persistent systolic BP < 100 mmHg   
• 
Pregnancy 
• 
Development of diabetes mellitus or a new medical condition necessitating administration of ACE 
inhibitor, angiotensin receptor blocker, or spironolactone 
Conditions for temporarily halting study medications: These include 1) sudden increase in serum 
creatinine of > 50% of the baseline value obtained on the CTRC visit prior to randomization; 2) Severe 
uncontrolled hypertension in the outpatient setting defined as elevation of systolic BP > 180 mmHg or 
diastolic BP > 110 mmHg on two consecutive occasions measured at least 48 hours apart despite effort 
to lower BP by diet or nonpharmacologic intervention. 
 
  
Conditions for permanently halting study medication in an individual:  A stop point will be 
defined as any serious adverse event that in the opinion of the Principal investigator could be reasonably 
attributed to the study intervention. At this time the participants study medication will be permanently 
discontinued and a close out visit will be scheduled.   
  
 
 
Conditions for stopping the protocol entirely: Conditions for stopping the entire protocol are 1) 
development of severe hyperkalemia (serum K > 6.0 mEq/L) or acute renal insufficiency (eGFR reduces 
by > 30%) in 1 subject or more after they have been randomized to study drug, (2) any evidence of 
myocardial ischemia or infarction (chest pain, EKG changes), (2) any new onset of sustained cardiac 
arrhythmia including atrial fibrillation, supraventricular tachycardia, ventricular tachycardia, or ventricular 
fibrillation; (3) any death. Under such circumstances, the study will not resume without IRB and CTRC 
approval.   
Reports to Federal, University, and hospital offices 
The PI will immediately report all unexpected and any serious adverse events and the recommendations 
derived from data and safety monitoring (such as continuation or conclusion of the trial) to the IRB, CTRC, 
hospital, FDA, and NIH.   
The PI will immediately notify NIH of actions, if any, taken by the IRB as a result of its continuing review.  
The PI will submit copies of FDA reports about adverse events to the CTRC as well as IRB. 

11 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
The PI will anonymize all reports of local adverse events submitted to the IRB and CTRC to protect a 
subject’s privacy. 
The PI will educate all key personnel about the requirements to report unexpected and serious adverse 
events to the IRB, CTRC, and other agencies and offices. 
 
 
11. Procedures to Maintain Confidentiality   
 
 
1.  Information will be given only to the physician-investigators.   
2.  The nature of the information concerns pertinent medical and social history. 
3. The purpose of the disclosure is solely for research purposes, in particular to determine whether 
potential subjects meet criteria for inclusion or exclusion and to identify specific subgroups of 
subjects.     
4.  The subject’s right of confidentiality will be given strict priority. No mention of the subjects’ 
identities will be made either directly or indirectly in oral or written presentation of this work. The 
investigators, who are physicians, will be responsible for providing medical care in the event of 
any adverse effects to the subjects.  
5.  Copies of executed consent forms and all experimental records will be in the locked cabinet in 
our cardiovascular physiology laboratory in H4.136. All medical and biographical information will 
be held strictly confidential and no disclosures of personal identity will be allowed unless 
specifically requested by the subject. Copies of executed consent forms, as well as the 
experimental log book, are kept in a locked file cabinet in this laboratory. 
 
6.  Data management: The data repository will be developed using the Microsoft SQL Server 
 
relational database management system with Microsoft Access as the user interface.  
 
Data will be centralized and maintained upon a secure centralized network server and is backed 
up on a rotating schedule.  This system allows access to the database throughout UTSW campus 
university network but database privileges are only allowed on an as-needed basis and must be 
approved by the Principal Investigator.  Data that is electronically captured will be reviewed before 
being merged into the database.  Audit logs of all data access will be maintained for security 
purposes.  De-identified data will be shared with collaborating investigators according to 
University rules and regulations. 
 
12. 
Potential Benefits of the proposed Research to the Subjects and Others 
Hypertensive patients are known to have exaggerated rise in BP during exercise, which poses 
substantial health risks independent of resting BP 5-9. However, the underlying mechanisms of 
augmented exercise-induced BP elevation in human hypertension remain unknown. Our current 
research proposal is designed to assess the role of sympathetic nervous system and sympathetic 
mediated vasoconstriction during exercise in hypertensive patients, which may lead to 
identification of new drug target or treatment for augmented exercise pressor response.  
 
1.  Although these experiments are not designed to benefit the subjects directly, all subjects will 
receive monetary reimbursement for participation in the study.  
 
2.  However, the results of these studies may lead to understanding of mechanism of augmented 
exercise-induced BP elevation. If we can confirm that circulating aldosterone or local aldosterone 
synthesis in the central nervous system is the major cause of this abnormality by sensitization of 
mechanoreflex, our work could lead to identification of new action of MR antagonists which can 
prevent excessive rise in BP during exercise in hypertensive patients. This in turn will allow more 
hypertensive patients to participate in regular exercise.   
 

12 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
The anticipated large amount of new knowledge about the role of sympathetic nervous system and 
modulation of sympathetic vasoconstriction in pathogenesis of augmented exercise pressor response 
should justify the performance of this research. We anticipate that the results of our human integrative 
cardiovascular research will provide a new conceptual framework for implicating mechanoreceptor 
sensitization causing reflex sympathetic activation and exaggerated exercise BP response in human 
hypertension. Our study may also lead to identification of the origin of accentuated mechanoreflex and 
exercise intolerance in heart failure.  
 
 
13. Statistical Considerations SAS (SAS Institute, Cary, NC) software will be used for all analyses and 
PROC MIXED for repeated measures 29. Statistical Considerations Sample size and power estimates 
were computed using PASS 11 (NCSS, LLC, Kaysville, UT).  
 
The primary endpoint is muscle SNA (bursts/min, aim 2a, b, d, and e) and skin SNA (%total activity, aim 
2c). Secondary endpoints include SNA % total activity (only for muscle SNA since skin SNA units are 
already in % total activity), MAP, and HR. In all aims, the association between SNA responses and NE, 
insulin resistance variables will be analyzed with Pearson or Spearman correlation coefficients. 
 
To control for resting levels for SNA, BP, or other endpoints which could be affected by drug 
treatment (aim 2e) or surgical removal of tumor (aim2d), the resting or time 0 measurement prior to each 
activity (see fig 12-13) will be analyzed as a control covariate (analysis of covariance approach) in 
repeated measures models. This approach will also increase the precision of the response estimates.  
We will also evaluate changes and % changes from baseline levels to facilitate interpretation of the 
results. Repeated measures (RM) analysis will be conducted with mixed linear models with subject as a 
random effect.  All available data will be included in the analyses, including subjects with missing data. 
Model parameter estimates in the presence of missing data will be made with restricted maximum 
likelihood. Most analyses will include a repeated factor for comparing the activity gradations; covariance 
structure is selected on the basis of information criteria as well as model parsimony. For multiple 
comparisons, adjustments will be made as appropriate with SAS’s simulation adjustment method with a 
family-wise error rate of 0.05 and fixed seed 29. 
 
Due to our prior success with frequency matching enrollment 25, 30-32, study groups should be 
balanced with respect to gender, menopausal status, ethnicity, age, and BMI and we do not anticipate 
needing additional covariate adjustments. However, we will assess effects of these and other potential 
confounding variables from e.g., 24h urine Na, descriptive statistics, scatter plots, box plots, and by 
modeling the covariate.  
 
 
A two-period crossover study of amlodipine vs eplerenone medication will be conducted in 15 PA and 15 
EH subjects. This sample size accounts for ~20% attrition and is powered for a separate crossover 
analysis for each group to detect a mean difference of 6 bursts/min (std=8, =.7) when comparing the 
primary endpoint of SNA between the two medication phases. With 12 subjects completing both 
medication crossover phases, this study has power of 0.88 at alpha=0.05. Between group response 
differences are not a primary hypothesis; hence are not evaluated for power.  Randomization: Stratified 
by EH and PA, the order of amlodipine and eplerenone treatment will be determined by blocked 
randomization. Statistical analysis:  For each study group, descriptive statistics and 95% CI will be used 
to summarize responses of the two medication phases of this crossover study.  A mixed linear model 
with repeated factors to assess the activity level and medication effect plus their interaction will be 
constructed. Treatment sequence effect will be evaluated in the models. To account for reduction in BP 
after each drug treatment, resting BP will be analyzed as baseline covariates. Other analyses include 
controlling for final medication dose as potential confounding factor and evaluating differences between 
the PA and EH groups with regard medication response using models that include PA and EH groups to 
directly and efficiently assess the group by MR antagonist interaction component. 

13 STU072012-066, Vongpatanasin, Form A3, Mod_7, 12-12-18 
 
         
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