THE INTERPLAY BETWEEN ESTROGENS AND THE NITRIC OXIDE

SYSTEM IN CARDIAC FUNCTION AND STRUCTURE

By

JEWELL ANNETTE JESSUP

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND

SCIENCES

In Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

In Physiology and Pharmacology

August 2011

Winston-Salem, North Carolina

Approved by:

Leanne Groban, M.D., Advisor

Examining Committee:

Michael E. C. Robbins, Ph.D., Chair

Mark C. Chappell, Ph.D.

David C. Sane, M.D.

Carol A. Shively, Ph.D.

ii

ACKNOWLEDGMENTS

I would like to sincerely thank Dr. Leanne Groban for her continuing guidance

and for introducing me to new concepts, providing the opportunity for me to grow

professionally, all while encouraging my independence. I am grateful for the time,

efforts, and contributions of my dissertation committee, Dr. Mike Robbins, Dr. Mark

Chappell, Dr. David Sane, and Dr. Carol Shively. I would like to thank Dr. David Averill

for his extensive lectures on cardiac physiology. It is without any hesitation I state that

the work presented here would not be possible without the diligent efforts of my

colleagues and friends: Mrs. Linda Moore, Miss Jessica VonCannon, Mrs. Marina Lin,

Dr. Jasmina Varagic, Dr. Hao Wang, Mr. Brian Bernish, Mrs. Ellen Tommasi, and Ms.

Deanna Brown. These individuals have taught me techniques, assisted in troubleshooting

problems, bred rats needed for these studies, allowed me to use their workspace, edited

manuscripts, and have become great friends throughout the process who have never

failed to make me smile at the end of the day.

My sincerest appreciation goes to my family for their constant support and

understanding, for I would not be here today were it not for all they have done for me.

My parents, James and Bonnie, have always strived to provide for me, the opportunities

they themselves never had. They made immense sacrifices in order to provide for me a

sound education, while never failing to nurture my hopes and dreams. To Lane, Suzette,

Valicia, and Jon, I am grateful for your encouragement and patience along the way, and

for giving me two of the greatest joys in my life. My nephew, Alston, and niece, Kaylie,

thank you both for being such delightful, precious kids, for bringing many smiles to my

face, and for your understanding when I had to miss your games or performances.

iii

Finally, there simply are no words to adequately express my appreciation for Dr.

Carlos M. Ferrario. His ceaseless efforts, phenomenal intuition, remarkable fundamental

understanding, and unyielding drive make him the greatest scientist I know. His passion

for life, his motivation by injustice, and his compassion for humanity make him one of

the greatest people I know. His constant encouragement, inspiration, and unwavering

support have carried me through the past few years and for that, I am eternally in debt

and truly grateful.

iv

TABLE OF CONTENTS

LIST OF FIGURES AND TABLES……………………………………………………v

LIST OF ABBREVIATIONS………………………………………………………….vii

ABSTRACT……………………………………………………………………………..x

CHAPTER ONE:

I Introduction……………………………………………………………………1

II Statement of the Problem…………………………………………………….4

III The Research Hypothesis…………………………………………………….6

IV The Background

Physiological Aspects of Cardiac Function ………………………………7

Diastolic Dysfunction …………………………………………………...12

Prevalence and Progression of Diastolic Dysfunction……………….….22

The Heart and Estrogens……..………………………………………..…25

The Nitric Oxide System and Heart Function…………………………...28

Estrogens and Nitric Oxide Synthase……………………………………31

The Experimental Model………………………………………………...33

V References……………………………………………………………………36

CHAPTER TWO:

DUAL ACE-INHIBITION AND AT1 RECEPTOR ANTAGONISM

IMPROVES VENTRICULAR LUSITROPY WITHOUT

AFFECTING CARDIAC FIBROSIS IN THE CONGENIC

MREN2.LEWIS RAT…………………………………………….100

Published in Ther Adv Cardiovasc Dis 3(4):245-57, 2009

CHAPTER THREE:

nNOS INHIBITION IMPROVES DIASTOLIC FUNCTION AND

v

REDUCES OXIDATIVE STRESS IN OVARIECTOMIZED-

MREN2.LEWIS RATS………….………………………………..137

Published in Menopause 18(6), 2011

CHAPTER FOUR:

TETRAHYDROBIOPTERIN REPLETION PREVENTS

DIASTOLIC DYSFUNCTION IN OVARIECTOMIZED-

MREN2.LEWIS RATS……...........................................................176

In press Endocrinology; 2011

CHAPTER FIVE: SUMMARY AND CONCLUSIONS

I Summary of Findings………………………………………………………212

II Concluding Statements……………………………………………………..227

III References………………………………………………………………….228

CURRICULUM VITAE……………………………………………………………….254

vi

LIST OF FIGURES AND TABLES

CHAPTER ONE

Figure 1.1 Mosaic of factors influencing cardiac function 11

Figure 1.2 Illustration of factors affecting left ventricular

compliance and diastolic dysfunction

13

Figure 1.3 Schematic outline of the collagen synthesis and

degradation pathways leading to increased diastolic

stiffness

19

Figure 1.4 Representative pressure-volume loops diagraming

curve shifts in scenarios of diastolic dysfunction

21

Figure 1.5 Relationship of preserved ejection fraction to course of

heart failure

22

Figure 1.6 Estrogens and BH4 32

CHAPTER TWO

Table 2.1 M-mode-derived measures of Left Ventricular

Dimension, Wall Thickness, and Systolic Function.

130

Table 2.2. Echocardiographic Indices of Diastolic Function. 131

Figure 2.1 M-mode echocardiogram and relative wall thickness of

a mRen2.Lewis receiving either vehicle or combined

renin angiotensin system blockers.

132

Figure 2.2 Tissue Doppler changes in mRen2.Lewis rats under

treatment.

133

Figure 2.3 Histochemical evidence of perivascular fibrosis in

mRen2.Lewis rats with and without treatment.

134

Figure 2.4 Perivascular collagen in vehicle- and combination-

treated mRen2.Lewis rats.

135

Figure 2.5 Representative immunoblot of SERCA and

PLB…………..

136

vii

CHAPTER THREE

Table 3.1. Body and Heart Weight Values. 168

Table 3.2. Echocardiographic Indices of Systolic and Diastolic

Function.

169

Figure 3.1. Effects of L-VNIO on arterial pressure 170

Figure 3.2 Tissue Doppler image of intact or OVX mRen2.Lewis

females receiving either vehicle or combination

171

Figure 3.3. Quantification of picrosirius red staining of

perivascular collagen in the hearts of sham-operated,

ovariectomized, and L-VNIO treated rats.

172

Figure 3.4 Effect of L-VNIO on cardiac levels of fluorescent 2-

hydroxyethidium from dihydroethidium (DHE)

staining.

173

Figure 3.5 Cardiac concentrations of biopterin, BH2, BH4, and

nitrite.

174

Figure 3.6 Cardiac nNOS, total eNOS, and phosphorylated eNOS

protein expression in sham-operated or OVX rats

treated with VEH or L-VNIO treated rats

175

CHAPTER FOUR

Table 4.1. Body and Heart Weight Values. 202

Table 4.2. Echocardiographic Indices of Diastolic Function. 203

Table 4.3. M-mode-derived measures of Left Ventricular Dimension,

Wall Thickness, and Systolic Function. 204

Figure 4.1. Cardiac concentrations of total biopterins and BH4 in

Sham, OVX, and OVX+BH4 treated rats.

205

Figure 4.2. Tail-cuff systolic blood pressures in Sham, OVX, and

OVX+BH4 conscious mRen2.Lewis rats.

206

Figure 4.3. Indices of myocardial relaxation. 207

Figure 4.4. Changes in perivascular collagen in the hearts of Sham,

OVX, and OVX+BH4 treated rats.

208

viii

Figure 4.5. Composite of OVX and BH4 supplementation on

cardiac levels of fluorescent 2-hydroxyethidium and

cardiac concentrations of nitrate.

209

ix

LIST OF ABBREVIATIONS

a late mitral annular velocity

AAALAC Association for Assessment and Accreditation of

Laboratory Animal Care

Ang angiotensin

ACE angiotensin converting enzyme

ACE2 angiotensin converting enzyme 2

ACEi angiotensin converting enzyme inhibitor

AT1 angiotensin II type 1 receptor

AT2 angiotensin II type 2 receptor

ARB angiotensin II type 1 receptor blocker

Amax maximum late transmitral filling velocity

ATP adenosine triphosphate

AWT anterior wall thickness

AWTed anterior wall thickness at end diastole

BPM beats per minute

B biopterin

BH2 dihydrobiopterin

BH4 tetrahydrobiopterin

cAMP cyclic adenosine monophosphate

DTI Doppler tissue imaging

Edectime early-filling deceleration time

e' early mitral annular velocity

x

E/A early-to-late transmitral filling

E/e' early transmitral filling velocity-to-mitral annular

velocity

EF ejection fraction

ECM extracellular matrix

EndMT endothelial-mesenchymal transition

eNOS endothelial nitric oxide synthase

FS fractional shortening

GAPDH glyceraldehyde-3-phosphate dehydrogenase

g gram

h hour

HPLC high performance liquid chromatography

HSP heat shock protein

iNOS inducible nitric oxide synthase

IVRT isovolumic relaxation time

kg kilogram

LV left ventricle

LVH left ventricular hypertrophy

LVEDD left ventricular end-diastolic dimension

LVESD left ventricular end-systolic dimension

L-VNIO N5-(1-imino-3-butenyl)-L-ornithine

MAP kinase mitogen-activated protein kinase

MMP matrix metalloproteinase

Mg milligram

xi

µg microgram

µL microliter

µM micromolar

mL milliliter

mM millimolar

mRen2.Lewis mRen2.Lewis congenic rat

nNOS neuronal nitric oxide synthase

NO nitric oxide

OVX ovariectomized

PBS phosphate buffered saline

PLB phospholamban

PSR picrosirius red

PWTed posterior wall thickness at end diastole

RAS renin angiotensin system

SERCA2 sarcoplasmic endoplasmic reticulum ATPase

SR sarcoplasmic reticulum

SHR spontaneously hypertensive rat

SV stroke volume

VVG Verhoeff-van Gieson

xii

ABSTRACT

JEWELL A. JESSUP

THE INTERPLAY BETWEEN ESTROGEN AND NITRIC OXIDE SYSTEM IN

CARDIAC FUNCTION AND STRUCTURE

Dissertation under the direction of

Leanne Groban, M.D., Associate Professor

It is increasingly recognized that biological differences in the mechanisms

regulating cardiovascular function in women and men critically impact the expression,

clinical presentation, and outcomes of chronic diseases such as hypertension, diabetes,

and heart failure. This is borne out from epidemiological and clinical studies that have

explored the prevalence and presentation of heart disease in women, as well as a robust

experimental literature demonstrating a role of estrogens in cardioprotection. Sex

differences in mortality after myocardial infarction tell the story since data show a worse

prognosis of death post-myocardial infarction for women (38% within the first year)

compared to men (25% within the first year).

The studies described in this dissertation investigated estrogens‟ mechanisms

through the nitric oxide pathway in the expression of diastolic dysfunction. The research

was performed in an unique estrogen- and salt-sensitive rat model of chronic

hypertension and cardiac dysfunction that is associated with the presence of left

ventricular hypertrophy and eventual development of renal disease. Our research has

documented that female mRen2.Lewis rats display diastolic dysfunction and cardiac

fibrosis which is further aggravated by removal of the ovaries. To explore mechanisms

xiii

accounting for the interaction between estrogens, diastolic dysfunction, and the cardiac

actions of the nitric oxide system, we performed a series of experiments that documented

increased radical oxygen species associated with depleted levels of cardiac BH4 and

decreased nitric oxide release in mRen2.Lewis female rats. The changes in lusitropic

activity in female mRen2.Lewis rats following nNOS inhibition were associated with

diminished left ventricular remodeling, increased nitric oxide release, and limited

generation of radical oxygen species. In the same model, BH4 repletion exhibited

protection against ovarian hormonal loss on diastolic function and cardiac structure in

mRen2.Lewis rats through restoration of NO release and mitigation of superoxide

generation.

The data collected in this series of experiments has increased the understanding of

an important interaction between estrogens and the nitric oxide pathway in the regulation

of cardiac function, the composition of the cardiac extracellular matrix, and their role in

the development and progression of diastolic dysfunction. This knowledge will allow the

design of a more rational approach to the treatment of heart disease in women.

1

CHAPTER ONE

I. Introduction

As narrated by Roelandt and Pozzoli (2), the American-borne clinical physiologist

Yandell Henderson was the first to associate the relaxation properties of the myocardium

to alterations in cardiac function from observations of exercise intolerance in elderly

patients. In his 1923 comment Henderson stated, “…in the heart, the velocity and extent

of relaxation…is as important a factor in the heart‟s behavior as the force and rapidity of

the systolic contraction.” From this, Roelandt and Pozzoli (2) summarized the comment

by saying that “if an old man‟s heart relaxes slowly, his capacity for physical exertion is

thus limited.” This concept remained dormant for several decades, but was revived in the

1960‟s from studies associating myocardial ischemia with left ventricular diastolic

dysfunction. At that time, the concept of diastolic dysfunction was recognized as an

impaired capacity of the left ventricle to fill and maintain its stroke volume without a

compensatory increase in left atrial filling pressure (2).

By virtue of its primary function for distributing oxygen to the tissues, the

mechanical properties of the heart are intrinsically coupled to maintenance of normal

homeostasis. Changes in the contractility of the cardiac muscle, venous return, and the

load that the heart faces during the ejection phase of the cardiac cycle determine the

quantity of blood distributed to the tissues. In turn, neurohormonal mechanisms and the

content of nutrients and electrolytes in the blood can further markedly impact the ability

of the heart to act as a pump. Hypertension, obesity, atherosclerotic vascular disease,

renal insufficiency, and diabetes all lead to maladaptive alterations of heart mass,

2

composition, and structure. These pathologies result in various combinations of

myocardial ischemia, hypertrophy, and increased interstitial and perivascular cardiac

collagen content, the consequences of which result in impaired capacity of the heart to

maintain cardiac output at a level consonant with the metabolic demands of the tissues.

The research described in this dissertation documents our efforts in assessing the

mechanisms accounting for cardiac diastolic dysfunction and the influence that sex has in

accounting for this process. It has been clearly documented that diastolic dysfunction in

women is highly prevalent particularly after the cessation of menses and that the higher

prevalence of this type of cardiac dysfunction may be in part related to the loss of

estrogenic cardioprotection (6). In addition, the progression of diastolic dysfunction to

heart failure in women is higher than the corresponding progression in men and that, in

the presence of chronic hypertension, the heart muscle becomes thicker in women than in

men, which exhibit left ventricular (LV) dilation (7-10). In a subgroup of the

Framingham Heart Study, women with isolated systolic hypertension had increased wall

thickness and mass without LV chamber enlargement, while men had LV dilation and

increased LV mass without increased wall thickness (8). Sex differences impact

cardiovascular mortality. Recent data of lengthened survival in male heart failure

patients have not shown comparable benefits in women (5;11), in part because research

conducted todate, as well as current treatment approaches, target systolic dysfunction, the

more common type of heart failure in men as opposed to diastolic dysfunction in women.

As indicated above, the prognosis for women with diastolic heart failure is poor,

accounting for a significant increase in hospitalizations (12;13) and resulting in deaths

rates greater than 20% (14;15).

3

In the pursuit of the described research, my previous work in understanding the

altered characteristic of the neural and hormonal mechanisms affecting the long term

blood pressure control in experimental hypertension, with a specific focus on exploring

the antihypertensive actions of angiotensin-(1-7) [Ang-(1-7)] and angiotensin converting

enzyme 2 (ACE2), provided the necessary background and the learned expertise in the

utilization of critical methods to address the hypothesis proposed by the current research.

In chronological order this research included: a) assessing the modulatory effect of

angiotensin II (Ang II) in the control of baroreceptor function within the dorsomedial

medulla oblongata of normotensive and the [mRen2]27 transgenic hypertensive rat (16);

b) determining the effects of blockade of Ang II synthesis or activity on the cardiac

expression and function of angiotensin converting enzyme 2 (ACE2) (17;18); c)

characterization of the mechanisms accounting for the intrarenal synthesis of the

vasodilator and anti-proliferative peptide –angiotensin-(1-7) – [Ang-(1-7)] (19); d)

demonstration that experimental hypertension is associated with reduced expression and

activity of Ang-(1-7) and ACE2 (20); e) uncovering the antiarrhythmic properties of

Ang-(1-7) by combination of immunohistochemical and electrophysiological measures of

cardiac trans-membrane electrical activity in the ventricle of cardiomyopathic hamsters

(21); f) determining the existence and function of a the novel angiotensin I (Ang I)

precursor – angiotensin-(1-12) [Ang-(1-12)] – in the heart and kidney of Wistar Kyoto

(WKY) and spontaneous hypertensive (SHR) rats (22-24); g) documenting for the effect

of low-dose thiazide diuretics on the activity of the ACE2/Ang-(1-7)/mas-receptor axis

(25); and h) uncovering the importance of cardiac lusitropic activity in accounting for the

4

beneficial remodeling of cardiac function following dual blockade of ACE and type 1

Ang II receptors (AT1) (26).

II. Statement of the Problem

The mechanisms underlying diastolic dysfunction, whereby systolic function as

defined by a normal ejection fraction is preserved, are poorly understood. The use of the

congenic mRen2.Lewis hypertensive rat as the experimental model of renin-dependent

hypertension in our laboratory represented a new undertaking since no data exist as to the

characteristics of heart function in this model. To bridge this gap, we first evaluated the

cardiac function in male mRen2.Lewis adult hypertensive rats as these data could be most

easily compared with previous published work on the [mRen2]27 parent strain.

Furthermore, the investigation on the impact of dual ACE-inhibition and AT1 receptor

blockade on the lusitropic response of mRen2.Lewis rats represents a critical element of

the thematic significance of our studies as it underscores that mechanisms of cardiac

regulation, on a common genetic background of increased tissue expression of renin, are

markedly influenced by sex hormones. The comparison of the results obtained in male

and female mRen2.Lewis congenic hypertensive rats affirms the critical importance of

sex hormones in determining the characteristics of diastolic function in the face of

elevated blood pressure and increased renin angiotensin system expression. This

interpretation is supported by previous studies by Pendergrass et al. (27) who

documented marked sex differences in the circulating and kidney contents of

angiotensins in congenic mRen2.Lewis rats. Their observations of lower plasma renin

activity and circulating Ang II levels in female compared to male mRen2.Lewis rats was

associated with lower levels of renal cortical content of Ang II in females. On the other

5

hand, cardiac content of Ang II, Ang-(1-7), and ACE activity were comparable in male

and female mRen2.Lewis rats while cardiac ACE2 activity was increased in male, but not

female rats (27). The differential changes in expression of renin angiotensin system

components in male and female mRen2.Lewis buttressed the need to determine the

diastolic characteristics of the male rats in the presence of their robust hypertension based

upon the acceptance that elevated blood pressure is a primary risk factor for developing

diastolic dysfunction. Our suspicion that mechanisms driving diastolic dysfunction are

not purely dependent on hypertension, led us to characterize both cardiac structure and

functional differences in the male mRen2.Lewis whose extreme hypertension was

prevented from developing, yielding their blood pressure status more similar to levels of

their female counterparts of equivalent age. Moreover, the goal of the chosen therapy, a

combination that blocked the Ang II type-1 receptor and ACE, served to evaluate the

effect of complete renin angiotensin system blockade on the blood pressure and cardiac

function of mRen2.Lewis model.

The loss of estrogens together with activation of the renin angiotensin system has

been linked to the development of diastolic dysfunction (28-31). Indeed, our previous

data in the female mRen2.Lewis rat showed that oophorectomy increased serum

aldosterone and interstitial fibrosis, that results in the accelerated development of

diastolic dysfunction (28). Additionally, ovarian hormone loss increases renal neuronal

nitric oxide synthase (nNOS) expression, decreases eNOS expression in the kidney, and

exacerbates the effects of salt on renal damage and proteinuria, which were reversed by

treatment with a specific nNOS inhibitor. These findings lead Yamaleyeva and

6

colleagues to suggest that nNOS may be uncoupled in mRen2.Lewis depleted of

estrogens or those fed high salt diets (32;33).

Our initial data in male mRen2.Lewis shows that the presence of diastolic

dysfunction was associated with significant alterations in Ca2+

handling suggesting that

the pathology may originate from the inability of the myocytes to sufficiently relax. In

contrast, in female mRen2.Lewis rats of similar age, increased cardiac collagen content

appears to be the primary mechanisms responsible for the altered cardiac function, thus

implicating altered extracellular matrix dynamics as the source of diminished compliance

(28). Based on these data as well as the renal data previously published by Yamaleyeva

et al. (33), we hypothesized that the loss of estrogens in female mRen2.Lewis rats will be

accompanied by suppression of the “protective” cardiac endothelial-derived NOS isoform

(eNOS). Furthermore, we proposed that the loss of eNOS may be associated with a

concomitant reduction in nitric oxide production and/or an increase in the presumed

“maladaptive” nNOS isoform (non-NO producing) that contributes to LV remodeling.

This shift in the cardiac nNOS pathway from an adaptive (NO-producing NOS) to a

maladaptive NOS (reactive oxygen species or ROS-producing) after the loss of estrogens

can be blocked following treatment with N5-(1-imino-3-butenyl)-L-ornithine (L-VNIO) a

specific inhibitor of nNOS (34).

III. The Research Hypothesis

The overall hypothesis of this research is that the anti-fibrotic effects of estrogens

in the heart are mediated through nitric oxide synthases such that the loss of estrogens

leads to maladaptive processing of the cardiac NOS system, or NOS “uncoupling,”

7

forming reactive oxygen species (ROS), that contribute to left ventricular remodeling and

diastolic dysfunction.

To examine this hypothesis, research was conducted to: 1) assess the diastolic

characteristics and cardiac structural composition of male mRen2.Lewis in their normal,

hypertensive state and following dual blockade of the renin angiotensin system in an

attempt to achieve blood pressures similar to their female counterparts; 2) characterize

the functional and structural changes occurring in the heart of intact and ovariectomized

(OVX) female congenic mRen2.Lewis rats in the absence and in the presence of chronic

administration of selective nNOS inhibition; and 3) quantitate the extent of which

tetrahydrobiopterin (BH4) repletion restores nNOS uncoupling and/or attenuates the

detrimental effects of depleted estrogens on LV remodeling and diastolic function in the

OVX-mRen2.Lewis compared to intact and OVX-mRen2.Lewis not treated with BH4.

IV. The Background

Physiological Aspects of Cardiac Function

World-renowned physiologist, Carl Ludwig, once stated that “a strong heart is

filled with blood empties itself more or less completely, in other words, [filling of the

heart with blood] changes the extent of contractile power” (35). In fact, Folkow and Neil

(36) state that “no pump yet devised has the long-term performance of a mammalian

heart.” In each ventricle of the resting human heart the output of the two ventricles is

about 400 million liters over an average life time, of which at a resting rate of 65 to 75

beats per minute, 0.3 of a second is spent in contraction of the ventricles (systole) and 0.5

seconds in relaxation (diastole). The metabolic requirements of the body are met by the

8

amount of the cardiac output that is distributed to the living organs of the body through a

fine regulation of both intrinsic and extrinsic mechanisms precisely adjusted to deliver

adequate amounts of oxygen to all cells of the body. The landmark studies of Ernest

Henry Starling and colleagues at the University College Hospital, London, UK (37-41)

and those of Linden at the former National Heart Institute of the National Institutes of

Health (42-51) established the physiological and neuro-humoral basis of cardiac

contractility control and function. Their respective contributions in terms of the critical

role of end-diastolic volume regulation (a manifestation of the length-tension relationship

in skeletal muscle) and that of the role of the inotropic influence of the sympathetic

postganglionic release of catecholamines represented, as commented by Arnold M. Katz

(52), the basis for our current understanding of the critical mechanisms regulating cardiac

function.

The Cardiac Cycle:

The cardiac cycle, or the composite activity of both myocardial contraction and

relaxation, is physiologically divided into the two components known as systole and

diastole. While systole and diastole occur as continuous rapid, sequential events, the

phases through which heart function occurs are conceptually divided into individual

events in order to understand the activity at each point in the cycle (36). During systole

or the contraction stage, three key events occur. The first, known as isovolumic

contraction, results from the depolarization of both the left and right ventricles and the

entrance of calcium ions into the myocytes through sarcolemma L-type calcium channels.

The increase in calcium within the cell stimulates the release of additional calcium from

the sarcoplasmic reticulum (SR) via ryanodine receptors or calcium-release channels. As

9

the intracellular calcium concentration increases, the calcium ions bind to the troponin-C

protein bound to thin filaments, inducing a conformational change that allows actin to

bind to the ATPase on the myosin head (53). The sarcomere can then shorten as the

hydrolyzed ATP provides energy for the movement of the actin-myosin complex, thereby

providing contraction of the myocytes. While these events are occurring within the

cardiomyocytes during this first phase of systole, it is important to note that the two sets

of atrioventricular valves, serving as gateways between the atria and ventricles, are closed

as the pressure within the ventricles increases in the absence of any change in volume.

These increases in intraventricular pressures lead to the second key event of

systole, which is the opening of the aortic and pulmonic valves allowing for rapid

ejection of the blood into the systemic circulation and lungs because of the pressure

gradient from the ventricles to the aorta and pulmonary arteries. The third segment of

systole begins a rapid 200 msec later and consists of repolarization of the ventricles and

diminished ejection (54). As the left ventricle contracts to expel the blood, pressure

briefly decreases in the left atrium, but then begins to increase as blood returns to the

atria.

As systole concludes, diastole must follow in order for the ventricle to relax and

reset the system so that the pump can refill and begin again (36). Diastole occurs as a

four-phase sequence of events beginning when the aortic and pulmonary valves close

following the decrease in intraventricular pressure at the end of systole. Known as

isovolumic relaxation, this first phase of diastole is characterized by low, constant

volume in the ventricle in the presence of the declining pressure. Mechanistically, this

phase is energy-dependent as relaxation of the myocytes requires ATP in order to drive

10

the sarco-endoplasmic reticulum calcium ATP-ase-2 (SERCA2) pump (55). SERCA2 is

responsible for restoring calcium homeostasis within myocardial cells by pumping the

ions, located in the cytosol and on the troponin-C complex, back into the SR. The efforts

of SERCA2 are facilitated by sodium-calcium exchangers that further remove Ca2+

from

within the cells. The end result is that the length of the sarcomeres composing the

myocytes, return to their pre-systolic conformation. The rate at which the sarcomeres

elongate, known as lusitropy, determines the rate at which ventricular pressure decreases

(56).

The second phase of diastole begins when the left atrial pressure exceeds the

pressure within the left ventricle resulting in the opening of the mitral valve. This early

filling phase is characterized by an immediate surge in blood flow into the ventricle.

Ventricular pressure decreases slightly due to the overlap in relaxation and filling

activity. This phase creates the overlap between the active relaxation of the ventricle

providing the suction mechanism of filling and the introduction of the passive stretching

or distensibility of the ventricular wall (57).

During the third phase, or diastasis, the ventricle continues to fill, but at much

slower rate than the prior filling phase. The expansion of the ventricle due to the volume

of blood that has filled the cavity leads to stiffer or less compliant ventricular walls.

Compliance is the ratio of volumetric changes over changes in pressure (58).

Finally, the last segment of diastole is the contraction of the atrium, which leads

to increased pressure with the atrial chamber that forces open the atrioventricular values

to expel the remaining blood into the ventricle before contraction occurs. Closing of the

11

AV valves after the final contribution of the atrium to obtain maximum ventricular

volume [end-diastolic volume (EDV)] the heart is ready to initiate the contraction phase.

Regulation of Cardiac Function

Figure 1 provides a basic overview of the mechanisms regulating cardiac

function. The contractile force

of the heart is directly

influenced by the intrinsic

mechanical properties of the

cardiac myocytes in terms of

both automaticity of

pacemaker cells and Ca2+

-

mediated inotropism. These

two properties have an

influence on venous return, the

critical factor influencing

diastolic volume and cardiac

work as first described by Starling as the “law of the heart” (40). Changes in venous

return, end-diastolic and stroke volumes may be modified by extrinsic factors affecting

both cardiac pre-load and the after-load. Release of catecholamines from post-synaptic

nerve endings lying within the heart tissue milieu have a direct effect on increasing the

inotropic performance of the heart while vagal nerve cholinergic nerve endings, primarily

innervating the pacemaker cells of the sinus and atrioventricular nodes, have the opposite

effect. The energetic requirements of the heart tissue are met by the coronary circulation

Figure 1.1. Schematic representation of the interplay of

intrinsic and extrinsic influences regulating cardiac

function. The interconnecting lines of the mosaic

illustrates the multiplicity of factors influencing the

regulation of cardiac function based on the concept of

equilibrium first postulated by Page (3) in his now famous

mosaic theory of hypertension.

Chemical Factors

Coronary Blood Flow

Catecholamines

Renin Angiotensin Aldosterone

System

CytokinesContractility

Extracellular Matrix

Composition

Venous ReturnCARDIAC FUNCTION

12

so that adequate perfusion and blood flow within the heart can be appropriately adjusted

to meet myocardial metabolic demands.

Extrinsic circulating and locally borne hormones as well as autocoid factors can

superimpose upon the contractile myocardium, an influence that can affect both diastolic

and systolic function. Beside the recognized importance of the sympathetic nervous

control of cardiac contractility and coronary blood flow, the renin angiotensin aldosterone

system can have a modest impact on cardiac contractility and a more profound impact in

terms of regulation of venous return through a direct action of Ang II on peripheral

venoconstriction (59-63), inhibition of presynaptic uptake of norepinephrine from cardiac

and peripheral sympathetic nerve endings (61-63), the effects of Ang II acting as a

growth factor (64-92), and a stimulus for cardiac and vascular collagen deposition (93-

104). The modulatory effect of Ang II on cardiac myocyte growth may be related, in

part, to stimulation of the potent profibrotic factors such as aldosterone (105-112) and

transforming growth factor (TGF-β1) (113-122). These growth and profibrotic actions of

Ang II have been demonstrated to be primarily mediated by the synthesis of the peptide

specifically in cardiac tissue, a finding that expands upon the concept of tissue renin

angiotensin systems and their independent regulation and function from the circulatory

one (71).

Diastolic Dysfunction

As commented at the beginning of this chapter, Henderson‟s work (123) and that

of Wiggers & Katz (124) first called attention to the impact that the diastolic properties of

the heart had on its pumping action. Almost a century past their illuminating

observations, the importance of alterations in the heart‟s diastolic properties as a

13

Figure 1.2. The illustration of factors affecting left

ventricular compliance and diastolic dysfunction using

the Page‟s mosaic (2) best documents the interplay

among the various mechanisms

Asynchrony

Collagen Deposition

AbnormalLoading

Renin Angiotensin Aldosterone

System

EstrogensCellular Disarray

AbnormalCa++ flux

OxidativeStress

DIASTOLIC DYSFUNCTION

mechanism contributing to abnormal cardiac function reached of age through advances in

molecular and cellular biology, genetics, and imaging techniques. Accelerated interest in

this field was prompted by the observation of a normal left ventricular ejection fraction

(EF) in approximately 30% of subjects with a diagnosis of heart failure (125-127). As

reviewed by Gaasch and Zile (128), similar patterns were then found in elderly subjects

with ischemic heart disease and hypertensive hypertrophic cardiomyopathy. Advances in

echocardiography and the introduction of Doppler techniques for visualization of cardiac

motion properties led to a prodigious explosion of new information, including non-

invasive measurements of LV filling pressure (129), and the acceptance of the concept of

the clinical importance of diastolic dysfunction as a predictor of heart disease (130).

It is generally accepted that alterations in the mechanical properties of the

myocardium defined as reduced

LV diastolic distensibility,

impaired filling, and slow or

delayed relaxation constitute the

syndrome of “diastolic

dysfunction” (128;131-134). The

fundamental basis of the altered

cardiac performance thus relates

to an increase in the passive

elastic stiffness of the

myocardium with a consequent

augmentation of the diastolic-pressure volume relationship. These changes result in

14

decreased left ventricular compliance or distensibility, adaptive changes in dynamic

filling of the ventricles, and augmentation in the end-diastolic pressure (128;131;135-

138). Figure 2 is a composite of the principal factors that affect the diastolic properties of

the heart and the mechanisms leading to diastolic dysfunction. Both intrinsic and

extrinsic factors may lead to abnormalities in diastolic function. Within the myocardium

itself, altered relaxation properties of the cardiomyocytes or changes in the composition

of the collagen content and type constituting the extracellular matrix reduce ventricular

distensibility and compliance (134;139). Neuro-hormones acting via endocrine,

paracrine, or even intracrine mechanisms (140-142) can alter the overall contractile

properties of the cardiomyocytes or markedly affect the composition of the extracellular

matrix (139). Lastly, cytokines stimulating an increase in radical oxygen species can also

have a direct effect on the relaxation properties of the myocytes or stimulate the

production of type III collagen (143-152).

Mechanical cardiac asynchrony, defined as the presence of a delayed contraction

of specific myocardial segments, is usually associated with altered relaxation properties

as well as systolic dysfunction (153-160). Coronary artery disease and hypertension-

induced cardiac hypertrophy are other situations rendering segments of the myocardium

with altered contractile properties, as foci of cardiac fibrosis and sub-endocardial

ischemia affect the normal propagation of electrical impulses spreading through the

cardiac ventricles (154;155;158;161-165). In this context, the report of Nii et al. (158),

using radionuclide ventriculography and echocardiography, showed that left ventricular

asynchrony plays a role in early diastolic filling abnormalities in hypertensive patients.

15

Alterations in both pre- and afterload have an influence on myocardial relaxation

properties. Abnormal loading may result from systemic hypertension (pressure overload)

and valvular heart disease (volume overload) (74;137;157;166;167). In addition, an

increase in pericardial pressure resulting in an upward shift of the pressure-volume curve

is another mechanism that can affect myocardial relaxation, in part through a restriction

of the late filling phase in the cardiac cycle (168-172).

The third factor influencing cardiac diastolic properties are intrinsic changes in

the contractile properties of the myocytes, particularly the entrance and release of Ca2+

and the intracellular myocardial ATP content (173-183). A decrease in the activity of

SERCA2a, the ATP-driven pump that translocates Ca2+

from the cytoplasm to the lumen

of the sarcoplasmic reticulum, has been found in senescent (>60 years of age) human

myocardium (184) as well as in children and adolescents with type 1 diabetes and

diastolic dysfunction (180). Using an experimental model of diabetes produced by

administration of streptozotocin to mice, Shao et al. (180) reported a reduced ability of

SERCA2a to hydrolyze ATP and transport Ca2+

in the cardiac myocytes. Mutations of

carbonyl adducts on select basic residues of SERCA2a demonstrated an important role of

carbonylation in accounting for the prolongation of cardiac and myocyte relaxation times

in this model of experimental diabetes. In keeping with these findings, high-energy

phosphate (HEP) metabolism assessed by magnetic resonance (MR) and phosphorus-31-

nuclear MR spectroscopy in type 2 diabetic patients, revealed that early (E) acceleration

peak, deceleration peak, peak filling rate, and transmitral early-to-late diastolic peak flow

(E/A) ratio, all indexes of diastolic function, were significantly decreased in diabetic

patients compared with controls (174). Similar conclusions were reported in

16

hypertensive patients with cardiac hypertrophy in whom LV filling was impaired as

reflected by a decreased peak rate of wall thinning (PRWThn), the ratio of the E/A

waves, early peak filling rate, and early deceleration peak (176). In these subjects, the

myocardial phosphocreatine (PCr)/ATP ratio was significantly reduced compared with

controls both at rest and during dose atropine-dobutamine stress (176). Furthermore,

using a porcine model of volume-overload, the insertion of recombinant adeno-associated

virus type 1 carrying SERCA2a, which ultimately led to SERCA2a overexpression,

preserved systolic function, attenuated diastolic dysfunction, and improved ventricular

remodeling (185). In another study in patients with diastolic dysfunction due to ischemic

heart disease, a 10-day administration of ATP was associated with improvements in

echocardiographic parameters of left ventricular relaxation (173). Changes in energy

metabolism and Ca2+

disposition may be also modulated by extrinsic factors since

stimulation of cardiac matrix metalloproteinases (MMPs) by Ang II regulates high-

energy phosphate (HEP) stores by mechanisms that were independent of changes in

myocardial collagen content, collagen subtype, and collagen cross-linking (178). These

studies suggest a critical role of ATP depletion in contributing to a state of energy

depletion (178). The biological effects of altered metabolic energy requirements on

diastolic function are further intertwined with the unbalance induced by concomitant

changes in cardiac structure and function such as inadequate growth of the capillary

network in hypertrophied myocardium, impaired subendocardial perfusion due to

increased diastolic wall stress, and the modulatory effect of ATP on calcium efflux by

allosteric effects (179).

17

An abundance of literature exists as to the contribution of cellular disarray and

collagen deposition in altering the diastolic properties of the functional myocardium. The

myocardial extracellular matrix (ECM) is composed of three important structural

constituents: a) fibrillar proteins such as collagen I, collagen III and elastin, b)

proteoglycans, and c) basement membrane proteins such as collagen IV, laminin, and

fibronectin. While it is likely that each of these ECM constituent proteins plays a role in

remodeling, there is good evidence that suggests that ECM fibrillar collagen plays a

pivotal role in the development of diastolic dysfunction. This evidence follows four lines

of experimentation: a) disease processes that alter diastolic function also alter ECM

fibrillar collagen structure and composition, b) treatment which is successful in correcting

diastolic function is associated with normalization of fibrillar collagen, c) experiments in

which an acute, isolated alteration in collagen structure is performed result in an

alteration of diastolic function, and d) experiments in which a chronic rather than acute,

isolated alteration in collagen metabolism is performed also alters diastolic function (186-

198).

The heart‟s three-dimensional extracellular fibrillar collagen scaffolding plays a

critical role in maintaining the efficiency of the systolic and diastolic pump performance

and the appropriate spreading of the electrical impulses across the cardiac contractile

components of the heart tissue (106). An imbalance in heart tissue organization due to

excessive synthesis or inhibition of collagen protein deposition by fibroblasts impairs

myocyte contractility. Great strides are being made in the field examining the

biosynthesis, modification, and maturation of collagen and elastin. Unraveling the

processes leading to the formation, secretion, deposition and hydroxylation of collagen

18

within the cells, and the cross-linking, and degradation of collagen along with its various

subtypes outside the cardiac fibroblasts have now assumed importance in understanding

the changes in both systolic and diastolic function of normal and disease states. An

extensive field of endeavor involves the investigation of the molecular and temporal

events in the genesis of collagen synthesis, intramolecular cross-linking, and procollagen

to tropocollagen cleavage. Cardiac fibrosis is characterized by excessive synthesis and

deposition of ECM proteins especially Type I collagen and altered level of matrix

metalloproteinases (MMPs), key regulators of tissue remodeling. Tissue homeostasis is

maintained by coordinating the synthesis of extracellular matrix by activated fibroblasts

and degradation of it by proteolytic activities of urokinase Plasminogen Activator/tissue

Plasminogen Activator (uPA/tPA) and MMPs (106;199-204). Additionally, novel data

suggest an involvement of TGF-β1 in inducing vascular endothelial cells to transform

into fibroblast-like cells via endothelial-mesenchymal transition (EndMT); EndMT-

derived fibroblast-like cells synthesize extracellular matrix proteins and contribute to

cardiac fibrosis (204). The concept that fibroblasts contributing in the pathogenesis of

cardiac fibrosis may derive from vascular endothelial cells provides an additional

mechanism by which the vascular endothelial cells may interplay with cardiac resident

fibroblasts in the remodeling of the extracellular matrix. In this context our observations

regarding the effects of L-VNIO in the reversal of left ventricular perivascular but not

cardiac interstitial fibrosis (147) might support the concept of EndMT transition proposed

by Ghosh (204).

Figure 3 provides a summary of the complex processes that regulate collagen

homeostasis. Fibrillar collagen biosynthesis begins within the fibroblast endoplasmic

19

reticulum with the

synthesis of

procollagen α chain

monomeric proteins,

which through

glycosylation and

disulfide bond

formation constitute the

triple helical structure

of a procollagen

molecule. The procollagen molecule is then secreted into the extracellular space where a

mature cross‐linked insoluble structural collagen fibril is formed through a series of post-

synthetic processing events. Newly synthesized procollagen can associate with the cell

surface of fibroblasts or cardiomyocytes through interactions with receptors such as,

α1β1 integrin, or may be subject to degradation by collagenases, such as membrane

bound MT1‐MMPs. Changes in the balance between procollagen processing and

procollagen degradation due to ischemia or altered neuro-hormonal imbalances will

increase fibrillar collagen content and thus lead to impaired relaxation and increased

diastolic stiffness (93;98;109;118;178;191;195;197;201;204-207). The intrinsic

regulatory processes involved in collagen synthesis and degradation are also modulated

by neuro-hormonal mechanisms including neuronal catecholamines, growth hormone,

Ang II, Ang-(1-7), aldosterone, estrogen, and nitric oxide (68;93;94;97-99;105-

108;110;112;115;116;119;178;186;187;208-217).

Figure 1.3 Schematic outline of the collagen synthesis and

degradation pathways leading to increased diastolic stiffness.

20

Lastly, but certainly no less important is the chemical electrolyte composition of

the blood and extracellular fluid bathing the contractile and skeletal system of the heart,

as well as, the direct influence of cytokines such as interleukin 6 and 10, tumor-necrosis

factor alpha, nuclear factor kappa-light-chain-enhancer of activated B cells, and

peroxisome proliferator-activated receptor-alpha on regulation of cell metabolism and

cell-to-cell communication (212;214;218-236). As reported by Burchfield (219), these

paracrine factors have the potential to directly modify the healing process in the heart,

including neovascularization, cardiac myocyte apoptosis, inflammation, fibrosis,

contractility, bioenergetics, and endogenous repair.

Assessing Diastolic Function

Prior to the 1990s, assessment of diastolic function was restricted to invasive

cardiac catheterization whereby a micromanometer was inserted into the left ventricle in

order to measure changes in pressure and volume through the principle of changes in

conductance (123). Experimentally, this remains an accepted means by which the

relationship between LV pressure and volume is established. The acquisition of pressure-

volume curves allows for the calculation of stroke volume, cardiac output, ejection

fraction, end-systolic and end-diastolic pressure-volume relationships as indicators of

hemodynamic function (123). As illustrated in Figure 1.4, the normal depiction of

changes in pressure and volume over a cardiac cycle are altered in situations of impaired

diastolic function whether it be a result of impaired relaxation or a stiff myocardium.

Clinically, obtaining pressure-volume curves was impractical due to the potential risks

associated with cardiac catheterization and the lack of a precise technique for determining

left ventricular volume throughout the cardiac cycle. The advent of echocardiography

21

and tissue Doppler imaging had a major impact in the field of non-invasive diagnostic

cardiology including the capability of accurate measures of LV relaxation and filling

dynamics (128). The technology is not only used clinically, but in experimental settings

on rodent and non-human primate models (28;237;238). This non-invasive approach

allows for direct measures of the velocity of myocardial displacement upon expansion of

the LV during diastole. In our laboratory, M-mode echocardiography or time-motion

mode echocardiography is used to obtain an “ice-pick” glimpse through the heart with the

vertical axis providing the distance from the transducer while the horizontal axis tracks

time. M-mode images allow for the determination of LV wall dimensions and

thicknesses (211). Pulsed Doppler ultrasonography provides blood flow measurements

through the heart, which derives data on mitral inflow velocities, components of flow

dynamics during diastole, and LV wall motion. A key feature of Doppler tissue imaging

Adapted from Carroll et al. (4)

22

from a diagnostic perspective is its ability to assess early mitral valve annular velocity

(e'), which is an essential determinant of diastolic function (28).

Prevalence and Progression of Diastolic Dysfunction:

Diastolic dysfunction is estimated to occur in 23.5% of older people between 60

and 86 years of age (239). In older women, diastolic dysfunction is more prevalanent

than in older men and the female sex, not male, accounts for a greater portion of

moderate to severe diastolic dysfunction cases (6). Patients with asymptomatic diastolic

dysfunction with comorbid conditions including hypertension, hyperlipidemia, coronary

artery disease, and renal dysfunction begin developing symptoms within two years,

which is associated with an increase in

hospitalizations (240). If left

untreated, diastolic dysfunction can

ultimately lead to overt heart failure in

some patients.

Heart failure associated with

reduced ejection fraction, (termed

systolic HF), leads to profound

contractile dysfunction as it is caused

by either primary cardiac myopathy or

cardiac injury. It must be emphasized

that EF is not a precise index of

contractility, which by definition is

independent of loading conditions.

Figure 1.5. Panel A shows the increase during

the study in the percentage of patients with heart

failure who had preserved ejection fraction.

Panel B shows that the number of admissions for

heart failure with preserved ejection fraction

increased during the study period, whereas the

number of admissions for heart failure with

reduced ejection fraction did not change. The

solid lines represent the regression lines for the

relation between the year of admission and the

percentage of patients with heart failure who had

preserved ejection fraction (Panel A) and the

number of admissions for heart failure with

preserved or reduced ejection fraction (Panel B).

The dashed lines indicate 95 percent confidence

intervals. Reprinted from (5)

23

Indeed, Larsen and coworkers demonstrated a significant decrease in LV time varying

elastance (LV Emax) in 16 month old mice despite a seemingly normal LV EF that was

above 50%. Humans are also burdened with other chronic disease processes,

hypertension, obesity, atherosclerotic vascular disease, renal insufficiency, and diabetes.

All of these factors contribute to maladaptive alterations of heart mass and function. In

this regard, stiffening of the vasculature due to atherosclerotic heart disease leads to early

wave reflection and an increased systolic load which, over time, can lead to both systolic

and diastolic dysfunction.

As documented in Figure 5, diastolic HF or HF with preserved ejection fraction

(EF) accounts for more than 50% of total heart failure patients (5). Heart failure due to

diastolic dysfunction is rapidly increasing in prevalence and owing to the lack of

effective therapies, survival has not improved (241). As compared to healthy or

hypertensive controls without HF, age and sex adjusted analysis showed that human HF

with preserved EF is characterized by cardiac chamber defects including impaired

relaxation, increased diastolic stiffness, subtle resting contractile dysfunction, and

impaired chronotropic, contractile, diastolic and arterial-ventricular coupling reserve

(242-274).

The secular trends in the prevalence of HF with preserved EF were studied by

Owan et al. (5) at Mayo Clinic Hospitals in Olmsted County, Minnesota, from 1987

through 2001. Of 4,595 patients, for which data on EF (mean ± SD) were available, 53

percent had a reduced EF (29 ± 10%, n = 2,429) and 47 percent had a preserved EF (61 ±

7%, n = 2,167, p < 0.001) (5). Importantly, Owan et al. (5) also showed that

hypertension was present in 62.7% of patients with the diagnosis of HF with preserved

24

EF compared with 48% of patients with the diagnosis of HF and reduced EF. As

compared to age and gender matched healthy or hypertensive controls, patients with a

diagnosis of HF with preserved EF have more severe vascular dysfunction including

endothelial dysfunction (275-279).

As noted above, the epidemiology of HF with preserved EF strongly suggests that

age, hypertension, and being female interact to produce the unique forms of myocardial

and vascular dysfunction (5;11;243;250;251;256;261;266;280-283). While myocardial

and vascular alterations characteristic of hypertensive remodeling in young (primarily

male) adult animals and of non-failing, aged animals have been studied, it remains

unclear as to whether HF with preserved EF occurs as the mere summation of age and

hypertension related remodeling, a unique, synergistic interaction between these

processes or whether additional factors such as renal impairment or neurohumoral

dysfunction are pivotal in mediating progression to HF. Further, whether age and

hypertensive remodeling or their interaction are fundamentally different in females and

promote the increased prevalence of HF with preserved EF in females is unclear.

Despite traditional teaching, HF with preserved EF is not inevitably associated

with marked LV hypertrophy (LVH) in the elderly; most studies suggests that only ≈

40% of HF with preserved EF patients have a LV mass above normal range at

echocardiography. Indeed, in a large, community based cohort of HF with preserved EF

patients, only 42% of patients had LVH despite the presence of impaired relaxation,

increased diastolic stiffness and subtle contractile dysfunction (284). Further, HF with

preserved EF is not simply due to diastolic dysfunction, but is a complex process with

both cardiac and vascular abnormalities (248;276;285-291). As compared to healthy or

25

hypertensive non-HF controls, in age and sex adjusted analysis, it has been shown that

human HF with preserved EF is characterized by cardiac chamber defects including

impaired relaxation, increased diastolic stiffness, subtle resting contractile dysfunction,

and impaired chronotropic, contractile, diastolic and arterial-ventricular coupling reserve

(284;288;289;292). While these studies have nicely defined the integrative phenotype of

HF with preserved EF, an understanding of the tissue, cellular and molecular

perturbations which contribute to organ dysfunction remains to be fully investigated.

The Heart and Estrogen

Estrogens and the activation of estrogen receptors have been implicated as key

modulators of the cardiovascular protection exhibited by females (293-300). This idea is

further supported by data demonstrating an increase in cardiac pathology following

menopause (150;282;301-303). In order to understand potential mechanisms of

estrogens‟ regulation of cardiac structure and function in abnormalities such as diastolic

dysfunction, it is important to recognize the plethora of actions estrogens have on

baseline cardiac physiology and pathology.

Estrogens bind to three types of receptors: the two classical estrogen receptors,

ER and ER (304-307), and the more recently identified G protein-coupled estrogen

receptor, GPER (293;308-310). These receptors have been localized to both atrial and

ventricular cardiomyocytes as well as cardiac fibroblasts (311-313). The activation of

estrogen receptors is dependent upon various stimuli or in response to injury, which in

turn leads to either estrogens‟ genomic effect of altering gene expression or the more

rapid, non-genomic effects through alternative signal transduction pathways.

26

Within the heart, ER participates in calcium homeostasis, a genomic effect

demonstrated in ER knockout mice, which exhibit increased expression of L-type

calcium channels within myocytes and prolonged action potential duration with

consequent conduction disturbances (314). An additional genomic influence of estrogens

is augmented expression of atrial natriuretic peptide (ANP), which is released by

myocytes in response to stretch and limits hypertrophy primarily through its actions on

blood volume (306). Furthermore, estrogens offer cardioprotection through their

participation in the transcriptional regulation of connexin 43 (315). Acting as a gap

junction protein, connexin 43, facilitates coordination of ventricular depolarization by

activating potassium ATP channels within cardiomyocyte mitochondria. Estrogenic

influences on the renin angiotensin system include suppression of ACE expression,

diminished AT1 receptor density, decreased renin, while circulating angiotensinogen is

increased (316-318). Finally, estrogen has a modulatory effect on the expression of nitric

oxide synthases within cardiomyocytes (319).

As previously mentioned, estrogens mediate non-genomic effects within the heart

that occur rapidly upon initiation of a signal cascade from receptors located on the cell

surface. In addition to altering the expression the L-type calcium channels, estrogens

regulate Ca2+

mobilization and influx within cardiac myocytes (320). Moreover,

estrogens, acting through cGMP, can activate potassium channels located on coronary

vessels leading to vasodilation (321). Activation of GPER through the

phosphatidylinositol 3-kinase (PI3K)/AKT pathway was recently shown to reduce infarct

size (310). Bopassa et al. (322) demonstrated that acutely, ischemia/reperfusion injury

was decreased due to inhibition of mitochondria permeability transition pore opening, an

27

effect mediated by ERK activation (309). Data from our own laboratory revealed that

activation of the GPER improved diastolic impairment, decreased cardiac hypertrophy,

and ventricular wall thickness in mRen2.Lewis rats fed a high salt diet (323).

Physiologically, sex-differentiated data illustrate greater distensibility of the left

ventricles in females, which are smaller and fill at lower pressures compared to men,

documenting that estrogens modulate hemodynamics and myocardial function (324).

The EF and rate of systolic contractility is also higher in females than in males (324).

Prior to menopause, women have higher afterload-corrected EF compared to men (7),

observations which are further supported by data showing higher ejection fractions,

pressure-volume ratios, and improved aortic in pre- versus post-menopausal women

(325;326). Additionally, young women exhibit better diastolic function than age-

matched males (327;328). Similar data exist in animals studies which show sex

differences in hemodynamics and heart function (329-332).

The role of estrogens on pathological changes within the heart was documented in

early reports by Scheuer et al. (333) that illustrate estrogenic modulation of cardiac

hypertrophy. Since then, estrogen replacement therapy in women was shown to diminish

cardiac fibrosis and hypertrophy (334;335). Recent attempts to understand the

contribution of estrogens toward anti-remodeling of the heart have shown that activation

of ER prevents the development of cardiac fibrosis (336), which may occur in light of

increased Akt phosphorylation within the myocardium (337). In a model of chronic

volume overload, 17β-estradiol therapy blockade MMP-9 activation and attenuated

perivascular fibrosis, was associated with improvements in the TIMP/MMP balance and

the expression of collagen type I/III ratio (338). Additionally, ER modulation of PI3

28

kinase activity related to the increased myocyte-enriched calcineurin-interacting protein

(MCIP) 1 expression (336) is yet another aspect of the involvement of estrogens in

hypertrophy since MCIP1 is responsible for blocking calcineurin signaling, a key

component to hypertrophic signaling (339). Corroborating these observations is the

report by Donaldson et al. (340) that shows estrogens participate in calcineurin

ubiquitination and degradation. Furthermore, the actions of estrogen on Akt activation to

increase NOS activity, phosphorylation of MAP kinases impacting growth, and

alterations in the renin-angiotensin system are viable mechanisms that can participate in

estrogenic alterations within the heart (311).

The Nitric Oxide System and Heart Function

The identification of the elusive “endothelial-derived relaxant factor” (EDRF) in

1987 as the gaseous nitric oxide (NO) molecule led to a new era in understanding the

complex nature of the molecular mechanisms mediating biological processes in the heart

and the blood vessels (341-343). NO is a simple diatomic gas and free radical that is

endogenously synthesized by a family of enzymes called NO synthases (NOS). NO is

involved not only in vascular relaxation, but in neurotransmission both centrally and

peripherally; immune responses by macrophages; and in various pathophysiological

diseases through its actions ranging from impairing DNA and protein synthesis, eliciting

protein nitrosylation, inducing posttranslational protein modifications, inhibiting

mitochondrial respiration, to the generation of oxygen radicals. As commented by Otani

(344) the crucial role of NO in the pathophysiology of HF is a doubled edged sword since

at physiological concentrations it inhibits ischemia-reperfusion, represses inflammation,

29

and prevents left ventricular (LV) remodeling, whereas excess NO and co-existence of

reactive oxygen species (ROS) are injurious.

Within the body, NO is endogenously generated by NOS, which converts L-

arginine to L-citrulline (341;345) through a nicotinamide adenine dinucleotide

phosphate-oxidase (NADPH) and oxygen-dependent oxidation of a guanidino nitrogen

on the L-isomer of the amino acid molecule. Just as diverse as the physiologic actions of

NO, is the variety of NOSs (346). Type I NOS or neuronal NOS (nNOS) is located in

cardiac, smooth, and skeletal muscle as well as neurons, renal macula densa, adrenal

medulla, sympathetic ganglia, peripheral nerves, epithelial cells of lung, uterus and

stomach, pancreatic islet cells (347). The second NOS enzyme, NOS II or inducible

NOS (iNOS) is predominantly found in macrophages, but its presence has been

documented in polymorphonuclear leukocytes, hepatocytes, hepatic endothelial cells,

Kupffer cells, and under certain conditions in cardiac myocytes, vascular smooth muscle,

human myometrium, and endothelial cells in colon. Finally, NOS III or endothelial NOS

(eNOS) is most notably found within endothelial cells, but in hippocampal CA1 neurons,

cardiac myocytes, renal tubular epithelium, mitochondria of skeletal muscle, and in rat

myometrium.

The mechanism by which NOSs catalyze the release of NO from the conversion

of arginine to citrulline has requisite cofactors including NADPH, O2, flavin adenine

dinucleotide (FAD), flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) (348).

While the roles of the majority of the cofactor panel are understood, the actual

requirement of BH4 was less understood until recently. BH4 appears to lack a

stoichiometric role in the complex, acting rather as an allosteric modulator by

30

contributing to the dimerization of the NOS monomers (349;350). Maintaining the

cohesion of the dimerized complex, or “coupling” (351-354), ultimately leads to the

generation of NO, without which results in ROS generation thus implicating BH4 as a

critical constituent of the NOS system.

BH4 is synthesized through either its de novo pathway using guanosine

triphosphate (GTP) as a precursor or through “salvage” mechanisms that use pre-existing

dihydropterins in order to produce BH4 (355). The de novo pathway requires NADPH,

Mg2+

, and Zn2+

, while forming two intermediates from GTP, 7,8-dihydroneopterin

triphosphate and 6-pyruvoyl-5,6,7,8-tetrahydropterin before concluding the reaction with

BH4 as the final product. GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin

synthase, and sepiapterin reductase are the key enzymes involved in the process (356).

The most important of which is GTPCH as it is the first and rate-limiting step in the

cascade. BH4 synthesis, via the de novo pathway, is a requisite component of NO

release. Importantly, upregulation of GTPCH gene transcription precedes BH4

accumulation in numerous laboratory studies, further illustrating the importance of the

enzyme to BH4 generation (357-362).

Numerous studies have documented the role of NO on heart function. Vascular

(363;364) as well as cardiomyocyte-derived (365;366) NO affect contractility through

signalling events that lead to the opening of sarcolemmal voltage-gated and sarcoplasmic

ryanodine receptor calcium channels. Inhibition of NOS elicits bradycardia, which

underscores the positive chronotropic effects of NO (367). NO has been shown to limit

cardiac remodeling (368) following myocardial infarction while providing protection

31

both pre- and post-conditioning by opening mitochondrial ATP-dependent K+ channels

in ischemic-reperfusion injury (369).

Estrogen and Nitric Oxide Synthase

Estrogens were first recognized as key regulators of NOS expression within the

myocardium following the studies by Weiner et al. who showed that 17-estradiol

elicited increases in calcium-dependent NOS activity via augmentation of NOS

expression (370). In subsequent studies, it was observed that within the myocardium

specifically, 17-estradiol stimulates the expression of not only ERα and ERβ, but NOS

protein as well (319). While activation of ERα and ERβ upregulates eNOS expression,

the signalling events through GPER and subsequent activation of Akt leads to enhanced

eNOS activity (309). Because activation of NOS and production of NO have been shown

to be cardioprotective (297;300;322;344;371-374), the relationship between estrogens

and NOS is possibly a key mechanistic component of sex-differentiated cardiovascular

disease. Indeed, females maintain higher resting calcium concentrations compared to

males, which leads to a slower decline in intracellular calcium concentrations and

therefore, an increase in NO release (375). Within the heart, NOSs are differentially

expressed among males and females. nNOS expression was found to be higher in female

isolated rat cardiac myocytes compared to their male counterparts (376). Similarly, the

concentration of circulating estrogens parallels nNOS expression in both humans (377)

and rodents (374;378). Interestingly, men treated with low doses of 17-estradiol yielded

had increased nNOS protein expression in circulating neutrophils, an observation that

suggests participation in inflammatory processes (377).

32

As previously mentioned, one of

the key components of NO release is the

rate-limiting action of GTPCH on BH4

synthesis and therefore, NOS activity

(356). Estrogens have been shown to

regulate GTPCH mRNA as well as

activity (379), and modulate

bioavailability of BH4 (380). These

observations combined with data

showing normalization of BH4 levels

and suppression of ROS generation

following 17β estradiol therapy (380),

intriguingly suggests that these specific

effects of estrogens on the stability of the

NOS complex could participate in cardiac dysfunction and remodeling. Indeed, as

detailed in the introductory remarks, Yamaleyeva et al. (33) first reported that

oophorectomy evoked increases renal nNOS expression in mRen2.Lewis rats, which

exhibited exacerbated hypertension in the absence of ovarian hormones (381). The

authors went on to show that inhibition of nNOS, significantly reduced blood pressure in

the estrogen-depleted rats, while having no sustained effect on blood pressure in intact

littermates. Data from our laboratory shows that in the same rat model, oophorectomy

evokes impairments in diastolic function, increased collagen deposition, and increased

serum aldosterone (21). Taken together, these observations serve as the foundation for

Figure 1.6. In the presence of estrogen,

GTPCH catalyzes the production of BH4,

when coupled with nNOS drives the “coupled”

arm of the pathway. In the absence of

estrogen, BH4 is oxidized to BH2 shifting the

pathway to generate ROS. SGC, soluble

guanylate cyclase; Trx, thioredoxin; PKG,

protein kinase G; GTPCH; guanosine

triphosphate cyclohydroxylase. Adapted from

Takimoto et al. (1).

nNOS nNOS

BH4

NONO

Soluble Guanylate CyclaseSoluble Guanylate Cyclase

cGMPcGMP

Protein Kinase GProtein Kinase G

LV Remodeling LV Remodeling Cardiac DysfunctionCardiac Dysfunction

Coupled nNOS

GTPCH

Estrogen-intact

EstrogenEstrogen

Zn2+nNOS

BH2

ROS ROS

Uncoupled nNOS

GTPCH

Estrogen-deplete

Estrogen

Zn2+

nNOS nNOS

BH4

NONO

Soluble Guanylate CyclaseSoluble Guanylate Cyclase

cGMPcGMP

Protein Kinase GProtein Kinase G

LV Remodeling LV Remodeling Cardiac DysfunctionCardiac Dysfunction

Coupled nNOS

GTPCH

Estrogen-intact

EstrogenEstrogen

Zn2+nNOS

BH2

ROS ROS

Uncoupled nNOS

GTPCH

Estrogen-deplete

Estrogen

Zn2+

33

the studies described in this dissertation that elaborated on the interplay among estrogens,

the NO system and diastolic dysfunction in a genetic model of increased tissue renin

expression.

The Experimental Model

In 1990, Mullins, Peter, and Ganten {Mullins, 1990 502 /id} describe in Nature

the first successful introduction of the murine Ren-2 gene into the genome of the

Hannover substrain of the Sprague Dawley rat. In this landmark paper they report that

the insertion of the Ren-2 gene results in a model of hypertension in “…which the genetic

basis of the disease is known..,” and are characterized by reduced levels of active renin in

both the kidney and the circulation {Mullins, 1990 502 /id}. The pertinence of this

conclusion reflects the fact that at the time the paper from the German investigators was

published many researchers were not supportive of the idea that the renin angiotensin

system was a primary culprit in the causation of chronic hypertension. To date, this

transgenic model of hypertension, [mRen2]27 transgenic hypertensive rat, has become a

critical experimental tool in assessing the mechanisms by which a state of high tissue-

specific renin expression contributes to the evolution of high blood pressure, the

remodeling of cardiovascular tissue, the interplay between the renin angiotensin system

and other hormones to the disease state, and the mechanisms by which increased

expression of tissue renin alters organ function in the brain, heart, kidney, endocrine, and

reproductive organs. The importance of this model to biomedical research is underscored

by the fact that more than 7,000 publications using this model are currently in circulation.

Because the hypertension in homozygous [mRen2]27 transgenic rats is associated with

the rapid development of fulminant “malignant” hypertension (382), the increased

34

lethality can only be ameliorated through continuous treatment with ACE inhibitors.

Heterozygous [mRen2]27 transgenic hypertensive rats have a lower incidence of

malignant hypertension and thus are more commonly used as the experimental model. A

robust literature has documented that the hypertension in [mRen2]27 transgenic

hypertensive rats is associated with cardiac and vascular hypertrophy, increased oxidative

stress, and progressive decline in renal function (382-405). In addition, early studies by

Brosnihan et al. (406) first demonstrated a vascular protective effect of estrogen in

[mRen2]27 transgenic hypertensive rats, an important finding given the prior observation

of a strong sexual dimorphism of the blood pressure phenotype in this model (382-

384;391;392). Later studies further demonstrated that [mRen2]27 transgenic

hypertensive rats mimic some aspects of the cardiometabolic syndrome in humans (407-

411) as their hypertension was associated with dyslipidemia and insulin resistance (412-

414).

A contractual agreement between Professor D. Ganten (formerly of Heidelberg,

Germany) and Professor C.M. Ferrario (formerly at the Cleveland Clinic Foundation) in

1990 allowed for the development of a second colony of [mRen2]27 transgenic

hypertensive rats at the Cleveland Clinic. The colony was moved to the Wake Forest

University School of Medicine in 1992 with the transfer of the program to this site. Here

the extensive use of the [mRen2]27 transgenic hypertensive rat by investigators at the

Medical Center prompted interest in the development of a congenic strain from the parent

transgenic one. The rationale for this approach was a desire to insert the mouse Ren-2

transgene into a genetically homogenous inbred rat strain to eliminate effects associated

with the heterogenous outbred background of the parent strain (Hannover SD) of the

35

original [mRen2]27. Through instrumental efforts of Dr. D. Averill, the mRen2.Lewis

congenic rat strain was obtained by mating a homozygous male [mRen2]27 rat with a

female Lewis rat. From a series of backcrosses of mRen2 transgene positive offspring

with a Lewis rat, the founder line of congenic mRen2.Lewis congenic rats was identified

for the establishment of this colony.

The congenic mRen2.Lewis strain retained many of the critical characteristics of

the parent [mRen2]27 transgenic hypertensive animals, but with a reduced frequency of

the malignant phase of hypertension found in [mRen2]27 animals. Given the relative

short history of this model, there is limited information on the overall characteristic of

mRen2.Lewis congenic hypertensive rats. Nevertheless, early investigations document

the effects of blockade of Ang II synthesis or activity on the course of hypertension as

well as the response of mRen2.Lewis to treatment with either ACE inhibitors or AT1

receptor blockers (20;381).

There are profound sex-differences in the degree of hypertension in that males

m[Ren2]27 and mRen2.Lewis rats exhibit higher systolic blood pressures compared to

females of similar age (20;27;381). As discussed above, the characterization of this

genetic model of hypertension has further illuminated the importance of sex differences

in the expression of renin angiotensin system components and the possible contributions

the differences have on pathology and disease progression. Male mRen2.Lewis rats have

higher circulating levels of Ang II compared to higher concentrations of Ang-(1-7) in

their female counterparts (27). The differential expression of renin angiotensin system

peptides is associated with the males having higher plasma angiotensinogen, plasma renin

concentration, and serum ACE activity, compared to females (27). Pendergrass et al.

36

revealed tissue-specific alterations in the RAS components of the mRen2.Lewis model,

which in males include higher tissue levels of Ang II as well as ACE2 activity (27).

Conversely, neprilysin expression and activity is substantially higher in females

compared to males (27). Further studies have characterized the salt-sensitivity of male

and female mRen2.Lewis rats, both dependent and independent of estrogenic contribution

(28;32;323;415;416).

Beyond the sex differences and more pertinent to the theme of this dissertation is

the estrogen sensitivity of the female mRen2.Lewis model. It was first shown that 17-

estradiol treatment, initiated at OVX, prevented the development of exacerbated

hypertension present in untreated OVX rats (381). Recent data on vasodilatory effects of

the novel estrogen receptor, GPER, provide further support for estrogen‟s participation in

the mRen2.Lewis model (417). These observations, combined with the two previously

discussed reports that document the presence of diastolic dysfunction (28) following

estrogen loss and show alterations in NOS regulation (33), underscores the uniqueness

the mRen2.Lewis model provides to examine the role of the cardiac of nitric oxide

system and estrogen play in the development and progression of diastolic dysfunction.

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CHAPTER TWO

DUAL ACE-INHIBITION AND AT1 RECEPTOR ANTAGONISM IMPROVES

VENTRICULAR LUSITROPY WITHOUT AFFECTING CARDIAC FIBROSIS

IN THE CONGENIC MREN2.LEWIS RAT

Jewell A. Jessup,1 Brian M. Westwood,

2 Mark C. Chappell,

2 Leanne Groban

1,3

From the Department of Physiology and Pharmacology,1 the Hypertension and Vascular

Research Center,2 and the Department of Anesthesiology,

3 Wake Forest University

School of Medicine, Winston Salem, North Carolina, USA

The following manuscript is published in Therapeutic Advances in Cardiovascular

Disease 3(4):245-57, 2009. Differences in formatting and organization reflect

requirements of the journal. Jewell A. Jessup completed the histological analyses and

prepared the manuscript. Dr. Leanne Groban performed the echocardiographic studies,

edited the manuscript, and served in an advisory capacity to Ms. Jessup. Mr. Brian

Westwood was responsible for the care and treatment of the animals. Dr. Chappell

provided the animals and assisted with editing the manuscript.

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Abstract

Purpose: Hypertension and left ventricular (LV) hypertrophy often precede diastolic

dysfunction and are risk factors for diastolic heart failure. Although pharmacologic

inhibition of the renin angiotensin system (RAS) improves diastolic function and

functional capacity in hypertensive patients with LV hypertrophy, the effects of

combination therapy with an angiotensin converting enzyme inhibitor (ACEi) and an

angiotensin receptor blocker (ARB) are unclear.

Methods: We assessed the effects of the combined 10-week administration of lisinopril

(10 mg/kg/day, p.o.) and losartan (10 mg/kg/day, p.o.) (LIS/LOS) on diastolic function

and LV structure in seven young (5 wks), prehypertensive congenic mRen2.Lewis male

rat, a model of tissue renin overexpression and angiotensin II (Ang II)-dependent

hypertension compared to vehicle (VEH) treated (n=7), age-matched rats.

Results: Systolic blood pressures were 64% lower with the combination therapy

(P<0.001), but there were no differences in heart rate or systolic function between groups.

RAS inhibition increased myocardial relaxation, defined by tissue Doppler mitral annular

descent (e׳), by 2.2 fold (P< 0.001). The preserved lusitropy in the LIS/LOS-treated rats

was accompanied by a reduction in phospholamban-to-SERCA2 ratio (P<0.001). Despite

lower relative wall thicknesses (VEH: 1.56 0.17 vs. LIS/LOS: 0.78 0.05) and filling

pressures, defined by the transmitral Doppler-to-mitral annular descent ratio (E/e, VEH:

28.7 ± 1.9 vs. LIS/LOS: 17.96 ± 1.5), no differences in cardiac collagen were observed.

Conclusion: We conclude that the lusitropic benefit of early dual RAS blockade may be

due to improved vascular hemodynamics and/or cardiac calcium handling rather than

effects on extracellular matrix reduction.

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Key words: diastolic function; fibrosis; hypertension; myocardial relaxation; renin-

angiotensin system (RAS)

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Introduction

Recent treatment guidelines emphasize the use of combination therapy to control

blood pressure thereby decreasing the risk of hypertension-related events such as

diastolic dysfunction (Chobanian et al., 2003; Mancia et al., 2007; Williams et al., 2004).

Among the potential therapeutic combinations, dual renin angiotensin system (RAS)

blockade using angiotensin converting enzyme (ACE) inhibitors (ACEi) and angiotensin

II (Ang II) type 1 receptor (AT1) blockers (ARBs), has been proposed to achieve more

complete RAS suppression, better blood pressure control, and incremental reno- and

cardioprotective effects when compared to either drug alone (Kolesnyk et al., 2007;

Werner et al., 2008). The additional rationale for this type of combination therapy is

based on mechanistic evidence regarding incomplete suppression of Ang II formation by

ACE inhibitors due in part to increased renin activity and higher Ang I. This “escape

phenomenon” ultimately results in the plasma levels of Ang II returning to their

pretreatment values (Roig et al., 2000; van den Meiracker et al., 1992). However, clinical

experience with the combination of ACE-inhibitors and ARBs has produced conflicting

results. Most studies show a modest reduction in systolic and diastolic pressure when an

ARB is combined with an ACE inhibitor and visa versa (Doulton et al., 2005). In

addition, a meta-analysis involving more than 6,000 hypertensive patients found that dual

RAS blockade reduced proteinuria to a greater extent (~ 25%) than either drug alone that

cannot be attributed to differences in blood pressure (Kunz et al., 2008). In spite of this,

findings from the ONTARGET (Renal Outcomes With Telmisartan, Ramipril, or Both, in

People at High Vascular Risk) study of more doubling of creatinine and need for dialysis

in the combination arm of telmisartan and ramipril when compared to monotherapy have

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cast doubts on the potential synergistic advantage of dual RAS blockade (Mann et al.,

2008). Likewise, in systolic heart failure patients, addition of candesartan to an ACE

inhibitor in the CHARM-Added trial (Effects of Candesartan in Patients With Chronic

Heart Failure and Reduced Left-Ventricular Systolic Function Taking Angiotensin-

Converting-Enzyme Inhibitors), reduced heart failure hospitalizations, but more patients

discontinued study medications in the combination arm due to adverse side effects,

including increases in plasma creatinine and potassium, than in the placebo/ACEi arm

(McMurray et al., 2003). Hypotension, worsening of renal function, and hyperkalemia

were also more common with combination therapy than with ACE inhibitors alone in

over 18,000 patients with LV dysfunction (Lakhdar et al., 2008). While these outcome

and safety data have evoked skepticism with regard to dual RAS blockade for

hypertension, renoprotection and heart failure, the therapeutic potential of combination

RAS blockade for preclinical diastolic dysfunction remains unclear.

Several experimental and clinical studies have suggested the beneficial effects of

RAS blockade, using either ACE inhibitors or ARBs in both hypertensive and

hypertensive-prone animals and patients to improve diastolic function, via reductions in

LV hypertrophy, myocardial fibrosis, and vascular stiffness. Our laboratory and others

have demonstrated that ACEi and ARBs normalize blood pressure, improve left

ventricular (LV) function, reverse cardiac hypertrophy and proteinuria in experimental

rodent models of hypertension; changes that are often associated with a compensatory

activation of the angiotensin converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang-(1-

7)]/mas receptor axis (Chappell, 2007; Ferrario et al., 2005a; Ferrario et al., 2005b;

Ferrario et al., 2005c; Jessup et al., 2006; Varo et al., 1999; Varo et al., 2000). In

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addition, sub depressor doses of ARBs attenuated diastolic impairment and LV

remodeling in the Dahl, salt-sensitive rat, in part, through their anti-inflammatory effects

(Nishio et al., 2007). Interestingly, the cardio-, vascular-, and renoprotective effects of

ACEi and ARBs persisted through adulthood when treatment was initiated in young,

prehypertensive rats (Baumann et al., 2007a; Baumann et al., 2007b; Baumann et al.,

2007c; Berecek et al., 2005). Long-lasting effects of the AT1 antagonist, olmesartan,

were observed following cessation of treatment in the female hypertensive mRen2.Lewis

strain (Chappell et al., 2003). Preemptive therapeutic RAS blockade with either an ACEi

or an ARB in prehypertensive humans, characterized by “high normal” blood pressures,

also delayed the development of hypertension (Julius et al., 2006; Luders et al., 2008).

Likewise, the Vascular Improvement with Olmesartan medoxomil Study (VIOS) showed

that AT1 blockade using olmesartan normalized blood pressure and reversed vascular

hypertrophy in prehypertensive subjects who otherwise would not have been treated by

current clinical guidelines (Smith et al., 2008). This attenuation in vascular remodeling

was a pressure-independent effect of olmesartan as similar pressure reductions using

atenolol had no effect on arterial morphology (Smith et al., 2008).

Therefore, we investigated the effect of combined early administration of ACEi

and ARB in a model of experimental hypertension that over-expresses the mouse Ren2

gene. The mRen2.Lewis, which we have extensively characterized elsewhere (Groban et

al., 2008; Jessup et al., 2006; Pendergrass et al., 2006; Pendergrass et al., 2008;

Yamaleyeva et al., 2007a; Yamaleyeva et al., 2007b) expresses higher circulating Ang II,

but similar cardiac levels of the peptide (Pendergrass et al., 2006; Pendergrass et al.

2008) The congenic strain is derived from the original transgenic [mRen2]27 rat

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developed by Mullins et al (Mullins et al., 1990; Mullins and Ganten, 1990) and

backcrossed in the inbred Lewis. The transgenic model exhibits accelerated hypertension

accompanied by hypertension-induced cardiac and vascular hypertrophy (Bachmann et

al., 1992; Bohm et al., 1996; Ohta et al., 1996) as well as disturbances in body fluid

regulation and renal function (Gross et al., 1996; Springate et al., 1994); key factors

leading to diastolic dysfunction. Accordingly, we hypothesized that early administration

of chronic antagonism of Ang II synthesis and AT1 signal transduction prevents the

development of hypertension and preserves diastolic function in the mRen2.Lewis strain.

Methods

Animals:

Male mRen2.Lewis rats were obtained from the Hypertension and Vascular

Research Center Congenic Colony of Wake Forest University School of Medicine. Rats

were housed in an Association for Assessment and Accreditation of Laboratory Animal-

approved facility individual cages (12-hr light/dark cycle) with ad libitum access to rat

chow and tap water.

Experimental protocol:

Rats (5 wks of age) were randomly assigned to drink either tap water (vehicle, n =

4) or tap-water to which lisinopril and losartan (combination, 10 mg/kg/day of each, n =

7) were added for 10 consecutive weeks. Doses were chosen based on efficacy in other

rodent models using blood pressure, neurohormonal, and renal endpoints (Ferrario et al.

2005a, Ferrario et al. 2005b, Jessup et al. 2006). Systolic blood pressure was determined

by tail-cuff plethysmography at the conclusion of treatment. Immediately prior to

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decapitation, an experienced echocardiographer (L. Groban) who was masked to the

treatment protocol performed transthoracic echocardiography on the anesthetized rats

[ketamine HCl (60 mg/kg) and xylazine HCl (5 mg/kg)]. Once the echocardiograms

were completed, the rats were decapitated. The hearts were removed and weighed with

one half being submerged in 10% formalin and the other frozen on dry ice for

biochemical analyses.

Echocardiographic studies:

The echocardiograms were obtained in sedated animals as previously described

using a Philips Envisor echocardiograph (Philips Medical Systems, Andover, MA) and a

12 MHz phased array probe (Groban et al., 2008). Left ventricular end-diastolic and end-

systolic diameters (LVEDD and LVESD, respectively), LV posterior wall thickness

(PWT), and anterior wall thickness (AWT) were measured from midpapillary, short-axis

images obtained by M-mode echocardiography. The percentage of LV fractional

shortening (%FS), an index of contractile function, was calculated as FS (%) = [(LVEDD

– LVESD)/LVEDD] × 100. LV mass was calculated using a standard cube formula,

which assumes a spherical LV geometry according to the formula: LV mass (LVmass) =

1.04 × [[LVEDD + PWT + AWT]3 – LVEDD], where 1.04 is the specific gravity of

muscle. Relative wall thickness (RWT) was calculated as: 2 x PWT/ LVEDD. Mitral

inflow measurements of early and late filling velocities (Emax and Amax, respectively),

deceleration slope (Edec slope), isovolumic relaxation time (IVRT), and early deceleration

time (Edec time) were obtained using pulsed Doppler, with the sample volume placed at

the tips of mitral leaflets from an apical four-chamber orientation. Doppler tissue

imaging (DTI) to assess early mitral valve annular velocity (e') was measured from the

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apical four-chamber view with the pulse-wave Doppler sample volume placed at the base

of the left ventricle, in the region of the septal mitral annulus. All measurements were

performed with an off-line analysis system (Cardiac Workstation, Freeland Systems) by

one observer who was blinded to previous results. An average of at least five consecutive

cardiac cycles to minimize beat-to-beat variability was used for all represented measured

and calculated systolic and diastolic indices.

Histopathology:

Vertical, long-axis sections of the formalin-fixed heart were taken through the left

ventricle. Specimens were dehydrated with ethanol and embedded into paraffin blocks.

Following microtome sectioning, the 4 µm tissue sections were stained with Verhoeff-

van Gieson (VVG) and picrosirius red (PSR) for assessment of collagen and elastin

fibers. The sections were examined under both bright field and polarized light using a

Leica DM4000B microscope system (Bannockburn, IL) and an Olympus polarizing

microscope system (Center Valley, PA), respectively. Bright field photomicrographs

were captured with a Leica DFC digital camera and processed using Leica Application

Suite software, while polarized images were taken using Diagnostic Instruments Inc.

Digital SPOT RT, 3-pass capture, thermoelectrically cooled charge-coupled camera

(Sterling Heights, MI) and processed using the SPOT® Advanced software. A blinded

observer took 2 images from each of 4 randomized quadrant fields totalling 8 images per

section under bright field and with polarization magnified 200-times. The digitized

images of equal pixel composition were analyzed using Adobe Photoshop Creative Suite

3. The quantified collagen content was determined as a ratio of VVG-stained or PSR-

stained pixels divided by total pixels.

109

Western blot hybridization:

Sarcoplasmic reticulum (SR) membranes were prepared as previously reported

(Groban et al., 2006). Immunoblots were ran and probed for sarcoplasmic endoplasmic

reticulum ATPase (SERCA2) antibody (1:1000 dilution; Abcam, Cambridge, MA) and

phospholamban (PLB) (1:5000 dilution; Abcam, Cambridge, MA) as previously detailed

(Groban et al., 2006). The PLB-to-SERCA2 ratio was used as a measure of SERCA2

inhibition. To normalize the variability of protein loading, the antibody to

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000 dilution; Cell Signaling,

Danvers, MA) was probed onto the SERCA2 and PLB-stripped membranes (Western

Blot Recycling Kit; Alpha Diagnostic International, Inc., San Antonio, TX). The bands

corresponding to SERCA2, PLB, and GAPDH were scanned and digitized (MCDI image

analysis software; Imaging Research Inc., Ontario, Canada). Each SERCA2 and PLB

bands were normalized to its own GAPDH band.

Statistical analysis:

All values are expressed as mean ± SEM. Two-tailed Student‟s t-tests were used

for comparing differences in the vehicle vs. combination groups at a p value < 0.05.

Results

Ten weeks of dual RAS blockade in the mRen2.Lewis rat, significantly reduced body

weights compared to vehicle treatment (LIS/LOS: 357 ± 6 g vs. VEH: 426 ± 8 g,

respectively), but did not affect urine output (LIS/LOS: 22.7 1.1 mL/24 h vs 25.0 1.7

mL/24 h). The tail-cuff systolic arterial pressure in congenic rats medicated with the

combination therapy was 64% less than the rats that were maintained on vehicle

110

treatment (210 ± 2 mmHg vs. 76 ± 4 mmHg, respectively). The treatment had no effect

on cardiac rate (Table 1). Table 1 summarizes the M-mode measurements of LV

dimensions, wall thicknesses, LV mass and systolic function. LV end-diastolic and end-

systolic dimensions were higher in treated rats, which was accompanied by a lower

relative wall thickness (Figure 1). Since body weights were significantly lower in treated

rats, LV mass was normalized to body weight. LV mass normalized to body weight, also

known as LV mass index, was significantly lower in LIS/LOS-treated rats compared to

saline-treated control rats (.0023 .0002 vs .0036 .0004 mg/gram body weight). There

was no effect of the treatment on percent fractional shortening. Using Doppler flow and

Doppler tissue imaging, assessment of diastolic function revealed a significantly lower

isovolumic relaxation time and a higher mitral annular descent (e') in treated rats (Table 2

and Figure 2). RAS blockade resulted in a greater E wave to A wave ratio, a function

most likely due to the 1.4-fold higher maximum early filling velocity of the left ventricle

through the mitral valve. The preserved myocardial relaxation elicited by inhibiting Ang

II synthesis as well as the activity at its receptor resulted in a 37% lower filling pressure,

as determined by the ratio of early transmitral filling velocity to early mitral annular

velocity (E/e') (P < 0.001) (Figure 2).

Systolic blood pressure and relative wall thickness in the mRen2.Lewis rats were

linked to TDI measures of septal mitral annular descent. Specifically, tissue Doppler

imaging of myocardial motion, or e', showed significant inverse correlations with blood

pressure and relative wall thickness (r = -0.83, P = 0.002 and r = -0. -0.72, P < 0.01,

respectively). As expected, systolic blood pressure was significantly correlated with

RWT (r = 0.95, P < 0.0001).

111

Interstitial (Figures 3 and 4) and perivascular (0.4 ± 0.1 % vs. 0.5 ± 0.2 % total

area, n=6 each group, P = 0.86) collagen content following RAS blockade was not

different compared to vehicle treatment (Figures 3 and 4). Displayed in Figure 5, the

resulting immunoblots probed for SERCA2 and Phospholamban (PLB) and their

respective GAPDH blots revealed single bands of molecular weight 110 kDa, 25 kDa,

and 40 kDa, respectively. The ratio of PLB- to -SERCA2 levels normalized to their

respective GAPDH decreased 74% in the mRen2.Lewis medicated with the ACEi and

ARB compared to VEH treatment (Figure 5).

Discussion

In the current study we examined the effect of early dual RAS blockade on

cardiac structure and diastolic function in young, prehypertensive male mRen2.Lewis

rats. Chronic inhibition of Ang II synthesis combined with blockade of Ang II‟s actions

at the AT1 receptor effectively prevented the development of LV hypertrophy and the

functional alterations of the LV relaxation phase. While these effects were associated

with reduced LV filling pressures, indicated by a relatively lower E/e' ratio compared to

vehicle-treated mRen2.Lewis rats, markers of collagen deposition were comparable in

both treated and vehicle rats. The preserved LV lusitropy with dual RAS blockade was

coupled by decreases in phospholamban-to-SERCA2 ratios and low, normal systemic

blood pressures compared to untreated rats. Taken together, our data show that early

commencement of dual RAS blockade attenuated the development of hypertension and

early diastolic dysfunction in the 15 week old mRen2.Lewis male rat.

112

Arterial hypertension is a common etiologic factor in the development of diastolic

dysfunction, even with the presence of normal systolic function. The transmitral Doppler

filling and time interval abnormalities specific to our 15 week old untreated hypertensive

mRen2.Lewis rats include decreases in E velocity and E/A ratio, and prolongation in

IVRT compared to the RAS blockade-treated cohort. Although the echocardiographic

changes are similar to that which has been reported in patients with hypertension and

delayed relaxation (Gardin et al., 1987; Pearson et al., 1987) the lack of an increase in

late filling (A velocity) or a prolongation in deceleration time or reversal in E/A suggests

a transitioning to a pseudonormal pattern. It is well known that diastolic filling is

determined by the atrial-ventricular pressure gradient and is, therefore, regulated not only

by relaxation but the driving pressure and compliance of the LV (Appleton et al., 1988;

Thomas and Weyman, 1991). Accordingly, we used pulsed-wave tissue Doppler imaging

of mitral annular descent, or e', as a preload independent index of LV relaxation to

differentiate pseudonormal from normal Doppler inflow patterns (Nagueh et al., 1997).

Indeed, the untreated mRen2.Lewis rats exhibited a reduced e' and increased E/e' ratio,

compared to treated mRen2.Lewis, suggesting that the increased afterload in the

hypertensive mRen2.Lewis rats may have partially contributed to the impaired relaxation

and increased filling pressure.

In the presence of LVH, lower mitral annular velocities in diastole have been

reported and reduced early filling velocities have been correlated with the time constant

of relaxation or tau (Kasner et al., 2007). Moreover, reductions in e' have been noted in

patients with LVH in whom mitral inflow velocities have been “normalized” due to

elevations in left atrial pressure (Di, V et al., 2004; Wachtell et al., 2000). Although we

113

cannot discount changes in chamber stiffness or compliance, increases in wall thickness

were observed in our untreated, mRen2.Lewis cohort in the absence of myocardial

fibrosis. These finding are in keeping with the observation that in 13 week old Dahl salt-

sensitive hypertensive rats, increases in tau and LV mass were observed without a change

in myocardial fibrosis (Masuyama et al., 2000). Indeed, the mechanisms by which LVH

alters diastolic function are associated with changes in the extracellular matrix such as

collagen infiltration (Weber et al., 1987), but also with increases in LV mass, myocyte

diameter, altered calcium currents, subendocardial ischemia, and LV dyssynchrony

(Gradman and Alfayoumi, 2006; Periasamy and Janssen, 2008; Tan et al., 2008).

Given that LVH is independently an important risk factor in hypertension, preventing its

development and enforcing its regression are indisputable necessities. LVH regression

leads to beneficial alterations in diastolic filling, limits the propensity for ventricular

arrhythmias, improves systolic midwall performance, autonomic function, and coronary

reserve (Agabiti-Rosei et al., 2006; Cuspidi et al., 2008; Muiesan et al., 2000; Shimizu et

al., 2000; Teniente-Valente et al., 2008; Tsuyuki et al., 1997). Numerous studies have

demonstrated that the use of ARBs facilitates the regression of LVH. Previous

documentation has shown that an ARB (olmesartan 0.6 mg/kg/day) given to 17 week old

Dahl salt-sensitive rats for three weeks reversed diastolic dysfunction, which has been, in

part, attributed to the anti-inflammatory effects of the drug as assessed by measurements

of NADPH oxidase activity, transforming growth factor-ß1 (TGF-ß1), and monocyte

chemoattractant protein-1 (MCP-1) (Nishio et al., 2007). Rothermund et al.(Rothermund

et al., 2001) reported that administration of eprosartan prevented LV diastolic functional

impairment in [mRen2]27, independent of its anti-hypertensive action. Submaximal

114

doses of eprosartan (6 mg/kg given for 20 wks from age 10 wk - 30 wk) elicited profound

decreases in LV end-diastolic pressure and increases in LV relaxation (Rothermund et al.,

2001), suggestive of improvements in LV compliance and lusitropy, respectively. It has

been shown in canine experiments that acute exposure to the AT1 antagonist, L-158,809,

significantly decreases LV end-diastolic pressure and chamber- as well as myocardial

stiffness constants after saline loading in dogs with LVH (Hayashida et al., 1997).

Clinically, ARB therapy improves diastolic function in diabetic patients with LV diastolic

dysfunction (Kawasaki et al., 2007). The authors attributed the improvement in cardiac

diastolic function to increased collagen turnover since the ratio of synthesis versus

degradation decreased (Kawasaki et al., 2007). On an outcomes level, the CHARM-

Preserved clinical trial found that candesartan reduced hospitalizations in the sub-group

of heart failure patients with preserved ejection fraction (Yusuf et al., 2003).

Blocking ACE activity, similar to blocking AT1 receptors, results in beneficial

outcomes when it comes to cardiac diastolic function, however, the mechanisms appear

to be different. Satoh et al. (Satoh et al., 2003) demonstrated that ACEi restored

sarcoplasmic reticulum Ca2+

uptake in 17 week old Dahl rats that had been treated for

seven weeks with temocapril (10 mg/kg/day). Similarly, restoration of Ca2+

handling and

correction of the overall Ca2+

homeostasis through the use of ACEi or even an ARB, was

mechanistically deemed responsible for reducing LV systolic and end-diastolic pressures

in 14 week old [mRen2]27 rats, while simultaneously increasing the ventricle‟s relaxation

velocity (Flesch et al., 1997). Furthermore, in a publication detailing the impact of ARB

add-on therapy to ACEi there was even more attenuation of diastolic dysfunction in that

LV end-diastolic pressure and the percentage of fibrosis were significantly less in ACEi

115

and ARB (temocapril 0.2 mg/kg/day and olmesartan 0.3 mg/kg/day, respectively) treated

Dahl rats compared to animals receiving only ACEi (Yoshida et al., 2004).

The role of dual RAS blockade in collagen synthesis and degradation remains

unclear as in the mRen2.Lewis model treated for 10 consecutive weeks there were no

measurable differences in interstitial or perivascular collagen content. However, Ferrario

et al. (Ferrario et al., 2005a) showed that up-regulation of the cardiac ACE2/Ang-(1-7)

axis induced by a 12-day administration of either lisinopril or losartan in Lewis rats was

abrogated when both drugs were given in combination. Other factors may contribute as

well. The age at which treatment is initiated may play a role since the collagen content of

the mRen2.Lewis compared to a Lewis of the same age was not different (Figure 4). In

keeping with this interpretation, the reduction in collagen volume fraction reported by

Varo et al. (Varo et al., 1999; Varo et al., 2000) in SHR occurred in 30 week-old rats in

which losartan was given at doses of 20 mg/kg/day for 14 weeks. Comparative results

reported by Lambert's group were found in SHR in which ACEi treatment was initiated at

age 4 weeks and continued for 12 to 20 weeks (Gagnon et al., 2004). Thus far, no studies

have evaluated the effect of combining an ACE-inhibitor and ARB on cardiac collagen

deposition in hypertensive rats.

N-acetyl-Ser-Asp-Lys-Pro, a tetrapeptide specifically degraded by ACE, has

potent anti-fibrotic actions and its novel anti-inflammatory mechanisms in hypertension-

induced target organ damage may in part be related to the inhibition of the inflammatory

cytokine tumor necrosis factor alpha (TNF-α) (Liu et al., 2004; Sharma et al., 2008).

Likewise, the effects of AT1 blockade on cardiac fibrosis may be more complex than

previously anticipated as the net action may depend not only on the tissue concentration

116

of Ang II, but also on interactions among kinins, AT1, and AT2 receptors (Liu et al.,

2004).

A limitation of the current study is that that the analysis of cardiac function was

based on noninvasive evaluation of hemodynamics and myocardial performance.

Therefore, the direct filling pressure data as well as the decrease in LV isovolumetric

pressure or its time constant are not available. Regardless, we used transmitral and tissue

Doppler echocardiography, both of which are the methods of choice for routine

noninvasive evaluation of diastolic function in humans as previously discussed (Groban

et al., 2008). Additionally, whether the changes in diastolic function following early

initiation of combined RAS blockade are merely reflective of a reduced afterload is

unclear. Indeed, vascular load is an important determinant of ventricular function as it

comprises both resistive load (arising primarily at the arteriolar resistance vessels) and

pulsatile load (determined by aortic stiffness and early return to the heart of reflected

peripheral waves). Since other cellular factors have been commonly associated with

maladaptive cardiac hypertrophy (Izumo et al. 1987, Izumo et al. 1988), such as the ratio

of α- and β-myosin heavy chain mRNA and myocyte hypertrophy, future studies will

investigate whether combination RAS blockade in the mRen2 will limit these markers of

LV remodeling.

In summary, we show that the beneficial lusitropic effects of combined RAS

blockade by angiotensin converting enzyme inhibition and angiotensin II receptor

blockade in young, prehypertensive rats are more likely due to improvements in cardiac

calcium handling and/or vascular hemodynamics rather than its effects on myocardial

fibrosis.

117

Acknowledgments

The work described here was supported in part by grants from the National Institute of

Health grants KO8-AG026764-04 Paul Beeson award (LG), HL-56973 (MCC), and HL-

51952(PI-C.M.Ferrario).

118

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Table 2.1. M-mode-derived measures of Left Ventricular Dimension, Wall

Thickness, and Systolic Function

Variables Vehicle Combination

Heart Rate, bpm

P =

243 ± 11 260 ± 2

0.18

LVEDD, cm

P =

0.68 ± 0.02 0.79 ± 0.02

0.004

LVESD, cm

P =

0.37 ± 0.02 0.44 ± 0.02

0.04

RWTed, cm

P <

0.78 ± 0.05 0.37 ± 0.02

0.0001

PWTed, cm

P <

0.27 ± 0.02 0.15 ± 0.01

0.0001

AWTed, cm

P <

0.23 ± 0.01 0.14 ± 0.01

0.0001

LV Mass, g

P <

1.56 ± .17 0.81 ± .07

0.001

%FS

P =

46 ± 3 45 ± 2

0.77

Data are expressed as mean ± SEM. LVEDD = left ventricular end-diastolic dimension;

LVESD = left ventricular end-systolic dimension; RWTed = relative wall thickness;

PWTed = posterior wall thickness at end diastole; AWTed = anterior wall thickness at

end diastole; LV Mass = left ventricular mass; FS = fractional shortening.

131

Table 2.2. Echocardiographic Indices of Diastolic Function

Variables Vehicle Combination

Emax, cm/sec

P <

54 ± 2 74 ± 1

0.0001

Amax, cm/sec

P =

43 ± 5 39 ± 2

0.54

E/A

P =

1.4 ± 0.2 1.9 ± 0.1

0.02

Edectime, sec

P =

0.058 ± 0.003 0.058 ± 0.001

0.87

e׳, cm/sec

P <

2.0 ± 0.2 4.3 ± 0.4

0.0001

E/e׳

P <

28.7 ± 1.9 18.0 ± 1.5

0.0001

IVRT, sec

P <

0.037 ± 0.003 0.021 ± 0.0001

0.0001

Data are expressed as mean ± SEM. Emax = maximum early transmitral filling velocity;

Amax = maximum late transmitral filling velocity; E/A = early-to-late transmitral filling

ratio; Edectime = early-filling deceleration time; e' = early mitral annular velocity; E/e' =

early transmitral filling velocity-to-mitral annular velocity; late mitral annular velocity;

IVRT = isovolumic relaxation time.

132

Figure 2.1

A. Representative M-mode echocardiogram of a mRen2.Lewis receiving either vehicle

(left panel) or combination (right panel). B. Relative wall thickness (RWT). Data

represent mean ± SEM. *, P < 0.001 compared to vehicle.

A.

B.

Vehicle Combination0.0

0.2

0.4

0.6

0.8

1.0

*RW

Te

d

(cm

)

Male mRen2-vehicle Male mRen2 + Lis/Los

133

Figure 2.2

A. Representative tissue Doppler image of a mRen2.Lewis receiving either vehicle (left

panel) or combination (right panel). E = early transmitral filling velocity; A = late

transmitral filling velocity; e' = early mitral annular descent (septal) velocity. B. Early

mitral annular velocity (e´) and early transmitral filling velocity – to – mitral annular

velocity ratio (E/e´). Data represent mean ± SEM. *, P < 0.0001 compared to vehicle.

A.

B.

Vehicle Combination0

1

2

3

4

5 *

e'

(cm

/se

c)

Vehicle Combination0

5

10

15

20

25

30

35

*

E/e

'

134

Figure 2.3

Representative VVG staining showing perivascular and interstitial collagen content in

vehicle- and combination-treated rats (upper panel). Quantification of interstitial collagen

shown in lower panel. Data represent mean ± SEM. *, P < 0.05 compared to vehicle, n =

6. Magnification = 200x.

A.

B.

Vehicle Combination0.000

0.005

0.010

0.015

Inte

rsti

tial

Co

llag

en

(sta

ined

: t

ota

l p

ixels

)

135

Figure 2.4

Representative picrosirius red staining showing perivascular collagen in vehicle- and

combination-treated rats mRen2.Lewis rats compared to a normotensive Lewis rat.

Magnification = 200x.

136

Figure 2.5

A. Representative immunoblots of SERCA and PLB with their respective GAPDH

loading controls from vehicle and combination treated rats. B. Protein concentration of

phospholamban to sarco/endoplasmic reticulum ATPase (SERCA) 2. Data represent

mean ± SEM. *, P < 0.05 compared to vehicle.

A.

.

B.

Vehicle Combination0.0

0.5

1.0

1.5

2.0

2.5

*

PL

B/S

ER

CA

137

CHAPTER THREE

NNOS INHIBITION IMPROVES DIASTOLIC FUNCTION AND REDUCES

OXIDATIVE STRESS IN OVARIECTOMIZED-MREN2.LEWIS RATS

Jewell A. Jessup, BS,1 Lili Zhang, MD,

3 Alex F. Chen, MD, PhD,

3 Tennille D. Presley,

PhD,4,5

Daniel B. Kim-Shapiro, PhD,4,5

Mark Chappell, PhD,6,7

Hao Wang, MD, PhD2,

Leanne Groban, MD1,2

Department of Physiology and Pharmacology1 and the Department of Anesthesiology

2,

Wake Forest University School of Medicine, Winston Salem, North Carolina;

Department of Surgery3, University of Pittsburgh School of Medicine, and Vascular

Surgery Research, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pa;

Department of Physics4 and Translational Science Center

5 Reynolda Campus, Wake

Forest University, Winston-Salem, NC; Hypertension and Vascular Research Center6,

Women‟s Health Center of Excellence7

Wake Forest University School of Medicine, Winston-Salem, North Carolina

The following manuscript is published in Menopause 2011; 18(6):698-708. Differences

in formatting and organization reflect requirements of the journal. Jewell A. Jessup

designed and performed the experiments, analyzed the data, and prepared the manuscript.

Dr. Leanne Groban oversaw the design of the experimental protocol, performed the

echocardiographic studies, edited the manuscript, and served in an advisory capacity to

Ms. Jessup. Drs. Zhang and Chen provided biopterin analyses. Drs. Presley and Kim-

Shapiro performed the nitrite detection. Dr. Hao Wang edited the manuscript. Dr.

Chappell participated in study conceptualization and assisted with editing the manuscript.

138

Abstract

Objective: The loss of estrogen in mRen2.Lewis rats leads to an exacerbation of diastolic

dysfunction. Since specific neuronal nitric oxide synthase inhibition reverses renal

damage in the same model, we assessed the effects of inhibiting neuronal nitric oxide on

diastolic function, left ventricular remodeling, and the components of the cardiac nitric

oxide system in ovariectomized and sham-operated mRen2.Lewis rats treated with L-

VNIO (0.5 mg/kg/day for 28 days) or vehicle (saline).

Methods: Female mRen2.Lewis rats underwent either bilateral oophorectomy (OVX;

n=15) or sham-operation (Sham; n=19) at 4 weeks of age. Beginning at 11 weeks of age,

the rats were randomized to receive either L-VNIO or vehicle.

Results: The surgical loss of ovarian hormones, particularly estrogen, led to exacerbated

hypertension, impaired myocardial relaxation, diminished diastolic compliance, increased

perivascular fibrosis, and increased relative wall thickness. The cardiac

tetrahydrobiopterin (BH4) – to – dihydrobiopterin (BH2) levels were lower among OVX

rats compared to sham-operated rats and this altered cardiac biopterin profile was

associated with enhanced myocardial superoxide production and decreased nitric oxide

release. LVNIO decreased myocardial reactive oxygen species production, increased

nitrite concentrations, attenuated cardiac remodeling and improved diastolic function.

Conclusions: Impaired relaxation, diastolic stiffness and cardiac remodeling were found

among OVX mRen2.Lewis rats. A possible mechanism for this unfavorable cardiac

139

phenotype may have resulted from a deficiency in available BH4 and subsequent increase

in nNOS-derived superoxide and reduction in NO metabolites within the heart. Selective

nNOS inhibition with L-VNIO attenuated cardiac superoxide production and limited

remodeling, leading to improved diastolic function in OVX mRen2.Lewis rats.

Key words: diastolic function; nitric oxide; fibrosis; myocardial relaxation;

postmenopausal; left ventricular remodeling

140

Introduction

Research directed to explore the underlying mechanisms associated with the

development and progression of heart failure is an important area of investigation as the

incidence of this disease is reaching epidemic proportions.1 Recent data of lengthened

survival in male heart failure patients have not shown comparable benefits in women.2, 3

This is partly because research conducted to-date, as well as current treatment

approaches, target systolic dysfunction, the more common type of heart failure in men as

opposed to diastolic dysfunction in women. In fact, some estimates claim that 75% of

diastolic heart disease patients are women over the age of 65.4, 5

The prognosis for these

women is severe as there is a significant increase in hospitalizations compared to systolic

heart failure patients1, 6

, and women with diastolic heart disease yield deaths rates greater

than 20%.7, 8

The mechanisms underlying diastolic dysfunction, the precursor of heart failure

with normal ejection fraction or diastolic heart failure, are poorly understood, particularly

in older women. The loss of estrogen following menopause has been linked to the

development of hypertension and left ventricular hypertrophy9, 10

; two risk factors for

diastolic dysfunction.11

However, estrogen replacement therapy remains controversial

given that data from the Women‟s Health Initiative have not shown protective

cardiovascular benefits in postmenopausal women receiving estrogen therapy.12

Additionally, the precise mechanism by which estrogen acts in females to offer

cardioprotection is not completely clear though data demonstrates that estrogen

modulates the production of nitric oxide (NO).13-15

A role of the NO system in the

141

progression of heart failure in postmenopausal women may be related to estradiol

upregulation of endothelial nitric oxide synthase (eNOS) by stimulation of

serine/threonine protein kinase Akt (protein kinase B).16

The actions of eNOS as well as

neuronal NOS (nNOS) have profound impacts on diastolic function by affecting nitric

oxide bioavailability.17

Furthermore, dysregulation of the NOS system may contribute to

diastolic dysfunction based on several lines of evidence, including: a) regulated

production of nNOS by estrogen;18-21

and b)-the observation that estrogen receptor beta

(ERß) mediates increases in cardiomyocyte eNOS levels.22

Surgical depletion of endogenous estrogen in experimental models results in

significant changes in blood pressure and cardiovascular function. In our prior study, the

development of diastolic dysfunction, interstitial fibrosis, and increased serum

aldosterone was accelerated in female hypertensive mRen2.Lewis following

oophorectomy.23

The mRen2.Lewis strain, which expresses the mouse renin 2 (mRen2)

gene in various tissues, is a congenic model of angiotensin II-dependent hypertension

produced by the successive backcross of the (mRen-2)27 transgenic rat onto the Lewis

background.24

The female mRen2.Lewis is an estrogen-sensitive model since

ovariectomy significantly exacerbates the degree of hypertension and estrogen

replacement normalizes the blood pressure.25

This model emulates the syndrome

exhibited clinically in postmenopausal women.

Interestingly, in estrogen-depleted mRen2.Lewis rats, Yamaleyeva et al. reported

that the renal expression of endothelial nitric oxide synthase (eNOS) was attenuated

while neuronal NOS (nNOS) content was increased.26

Subsequent treatment with the

selective nNOS inhibitor L-VNIO partially reversed the effects of oophorectomy on high

142

blood pressure in the female mRen2.Lewis.26

These authors speculated that L-VNIO

may inhibit uncoupled nNOS to reduce the formation of superoxide resulting in a

reduction in blood pressure. With this in mind, the current study was designed to

investigate the hypothesis that suppression of the protective cardiac eNOS isoform with a

concomitant reduction in nitric oxide production and/or an increase in the presumed

deleterious nNOS isoform contributes to LV remodeling in the ovariectomized

mRen2.Lewis rats. In addition, we evaluated whether the shift in the cardiac nNOS

pathway from an adaptive (NO-producing NOS) to a maladaptive NOS [reactive oxygen

species (ROS)-producing] is attenuated by the nNOS inhibitor.

Methods

Experimental Model:

Experiments were conducted in congenic female mRen2.Lewis rats obtained from the

transgenic rat colony of the Hypertension and Vascular Research Center of Wake Forest

University School of Medicine. All experimental procedures were approved by the

Animal Care and Use Committee of Wake Forest University. Rats were weaned at 3

weeks of age and allowed to acclimate in a temperature-controlled, Association for

Assessment and Accreditation of Laboratory Animal-approved facility (12-hr light/dark

cycle) with free access to rat chow and tap water. At 4 weeks of age, rats were randomly

assigned to undergo either bilateral oophorectomy (OVX; n=15) or sham-operation

(Sham; n=19) performed under 2% isoflurane anesthesia, as previously described.23

At

age 11 weeks, the groups were further randomly divided to receive the specific nNOS

143

inhibitor, N5-(1-Imino-3-butenyl)-l-ornithine (L-VNIO, Enzo Life Sciences International,

Inc., Plymouth Meeting, PA) diluted in saline for a targeted dose of 0.5 mg/kg/day or

pure saline as a vehicle control (VEH) both delivered intraperitoneally via an osmotic

minipump (Model 2ML4, 2.5 µL/hr, 4 weeks) [Sham+VEH n=9; Sham+L-VNIO n=10;

OVX+VEH n=6; OVX+L-VNIO n=9].26

Systolic blood pressure was monitored once

weekly via tail-cuff plethysmography (NIBP-LE5001, Panlab, Barcelona, Spain)

beginning with a training phase at 5 wk of age and continued through the termination of

the study at 15 wk of age. Serum estradiol was measured at the completion of the study

to verify complete oophorectomy and confirm depletion of circulating estrogen with a

minimal detection limit of 5 pg/mL [Polymedco (Cortlandt Manor, NY)].

Echocardiographic evaluation:

At 15 weeks of age, cardiac dimensions and function were assessed in anesthetized

(ketamine/xylazine: 80/12 mg/kg) animals using a Philips 5500 echocardiograph (Philips

Medical Systems, Andover, MA) and a 12 MHz phased array probe as previously

described.23

LV mass was calculated using a standard cube formula, which assumes a

spherical LV geometry according to the formula: LV mass (LVmass) = 1.04 × [[LVEDD

+ PWT + AWT]3 – LVEDD], where 1.04 is the specific gravity of muscle. Relative wall

thickness (RWT) was calculated as: 2 × PWT/LVEDD.

Morphometry and Histopathology:

Cardiac cross-sections, 2 mm thick, were fixed for 24 h in 4% paraformaldehyde

followed by dehydration in graded ethanols before being embedded into paraffin blocks.

144

4 µm slices were mounted onto Superfrost Plus slides and stained with hematoxylin and

eosin in order to measure myocyte cross-sectional area. Approximately one hundred

cardiomyocytes from 2 sections per rat were analyzed at 400× magnification using a

Leica DM4000B microscope (Bannockburn, IL) with a tandem Leica DFC digital camera

and Simple PCI 6.0 software. Additional slides were stained with Picrosirius red (PSR)

to evaluate interstitial and perivascular collagen deposition within the tissue using

polarized dark-field microscopy [Olympus polarizing microscope system (Center Valley,

PA) equipped with a Digital SPOT RT, 3-pass capture, thermoelectrically cooled charge-

coupled camera (Sterling Heights, MI) and SPOT® Advanced software] as previously

described. Adobe Photoshop Creative Suite 3 (Adobe Inc., San Jose, CA) was used to

determine the ratios of collagen positive stained pixels to unstained pixels in each

photomicrograph (4 randomized quadrant field images per rat, magnified 200-times).

In situ ROS production:

In situ production of reactive oxygen species (ROS) was determined using

dihydroethidium (DHE), which reacts with O2- yielding the fluorescent product, 2-

hydroxyethidium. Slices (3 sections per rat) were cut from frozen cardiac cross-sections

and mounted in slides. The sections were then incubated with 10 μM DHE (Invitrogen

Molecular Probes, Carlsbad, CA) in a humidified, light-protected chamber for 30 min at

37°C and then the nuclei counterstained with 4',6-diamindino-2-phenylindole (DAPI, 30

µg/mL, Sigma-Aldrich, St. Louis, MO). Slides were the rinsed in PBS, cover slipped,

and analyzed at 400x using a fluorescence microscope (Leica DM4000B, Bannockburn,

IL; excitation = 510-550 nm, emission = 590 nm for DHE; excitation = 330-380 nm,

145

emission 420 nm for DAPI) connected to a Leica DFC digital camera. The fluorescent

signal intensities were analyzed within the heart using Simple PCI 6.0 software. All data

are presented as average gray scale intensities + SEM.

Western blot hybridization:

Cardiac microsomes were prepared and protein concentrations were determined as

previously reported.23

50 μg of homogenate proteins were separated by SDS–PAGE on a

7.5% acrylamide gel and transferred to a polyvinylidene fluoride membrane (PVDF, Bio-

Rad, Hercules, CA). Immunocomplexes were detected with an anti-nNOS (1:2000), anti-

eNOS (1:500), anti-phospho eNOS (1:1000) (BD Transduction Laboratories, Franklin

Lakes, NJ), anti-sarcoplasmic calcium (SERCA) 2 ATPase (1:1000 dilution; Abcam,

Cambridge, MA), anti-PLB (1:2000; Millipore, Temecula, CA), and anti-phospho-PLB

Ser16 (1:5000; Millipore, Temecula, CA). To normalize the variability of protein

loading, the antibody to elongation factor 1 (EF1, 1:10,000 dilution, Millipore,

Temecula, CA) or glyceraldehyde-3-phosphtate (GAPDH, 1:5000 dilution; Cell

Signaling, Danvers, MA) was used as a loading control. The bound antibodies were

resolved with a Super Signal West Pico chemiluminescent/enhancer kit (Pierce ECL

Western Blotting Substrate, Pierce, Rockford, IL, USA), exposed to Amersham

Hyperfilm (Amersham Biosciences, Piscataway, NJ), and analyzed using densitometry.

The resulting densities were corrected for background and expressed as arbitrary units

normalized to the EF1 intensities. Human endothelial cell lysate (eNOS) and rat

cerebrum cell lysate (nNOS) were used as positive controls (BD Transduction

Laboratories, Franklin Lakes, NJ).

146

Measurement of total biopterin and BH4 levels:

The biopterin profile of the cardiac tissue was measured by HPLC with

fluorescence detection as previously documented.28

The cardiac supernatant was

subjected to protein assay and BH4 detection. Protein was removed from supernatant by

adding 10 μL of a 1:1 mixture of 1.5 M HClO4 and 2 M H3PO4 to 90 μL of extracts,

followed by centrifugation. Total biopterin (BH4, dihydrobiopterin [BH2] and oxidized

biopterin [B]) was determined by acid oxidation whereby 10 μL of 1% iodine and 2% KI

in 1 M HCl solution were added to 90 μL protein-free supernatant. BH2 plus B was

determined by alkali oxidation by the addition of 10 μL of 1 M NaOH to 80 μL of the

supernatant, followed by 10 μL of 1% iodine and 2% KI in 1 M NaOH solution. The

acid-oxidation samples were protected from light and incubated for 1 hr at room

temperature. 5 μL of fresh ascorbic acid (20 mg/mL) was then added to samples to

reduce iodine, and 20 μL of 1 M H3PO4 was added to acidify alkaline-oxidation samples

prior to the ascorbic acid. The samples were centrifuged and 90 μL of the supernatant

was injected into a 250-mm long, 4.6-mm inner diameter Spherisorb ODS-1 column (5

μm particle size; Alltech Associates, Inc., Deerfield, IL) isocratically eluted with a

methanol-water (5:95, v/v) mobile phase running at a flow rate of 1.0 mL/min.

Fluorescence (350 nm excitation, 450 nm emission) was detected by a fluorescence

detector (RF10AXL, Shimadzu Co., Columbia, MD). BH4 concentration was calculated

by subtracting BH2 and B from total biopterins.

Nitrite Measurements

147

Nitrite levels were measured using a chemiluminescence-based Nitric Oxide Analyzer

(Sievers, Inc., GE Analytical Instruments, Boulder, CO). Nitrite is reduced in a sample

chamber to release nitric oxide, which then goes to a reaction chamber where it reacts

with ozone to form a quasi-stable chemiluminescent species. The sample chamber

contains sodium iodide in glacial acetic acid to reduce the nitrite. The amount of light

detected on a photodiode is proportional to the amount of released NO and hence nitrite

in the sample. The integral of the voltage recorded is proportional to the detected light.

The values observed in this study are typical of these measurements.29

In addition to

variation due to sample handling and instrumentation, nitrite is influenced mostly by

NOS function, but also by diet. 29

Overall, the variability is small. For all measurements,

standard curves were obtained and used for quantitative measurements.

Statistical analysis:

All values are expressed as mean ± SEM. For serial blood pressure measurements, a 2 x

2 x 2 x 5 multivariate analysis of variance (MANOVA) was conducted to determine the

relationship among the four groups and the changes in blood pressure over the duration of

the experiment. The differences in the blood pressure levels were modeled as

interactions between procedure (Sham vs. OVX); treatment (VEH vs. L-VNIO); before

and after treatment; and time. The one 4-way factorial analysis and the three 3-way

analyses indicated significant interactions among all the factors, which lead to further

characterization of the differences after treatment commenced using one-way analyses of

variance and Bonferroni post hoc tests for each time point. For all other endpoints, two-

way ANOVA evaluated significant effects of estrogen or L-VNIO. Significant

148

interactions between the groups were further characterized using Bonferroni post hoc

tests. Analyses were performed using GraphPad Prism 5 (GraphPad, San Diego, CA) or

SPSS (SPSS, Inc., Chicago, IL). Differences were considered significant at p < 0.05.

Results

Removal of the ovaries was associated with an increase in body weight,

independent of nNOS inhibition (Table 1). Body weight in sham rats were not affected

by treatment with L-VNIO. Estrogen depletion was confirmed by serum estradiol levels

of <5 pg/mL and 27 ± 1.6 pg/mL in OVX- and sham-treated mRen2.Lewis rats,

respectively. Heart weight was increased in estrogen-deficient rats compared to intact,

but this can be attributed to the increased body size of the OVX-rats given that HW/BW

were similar (Table 1), as reported previously.23

Progressive increases in systolic blood pressure (SBP) occurred in estrogen-intact

and OVX-mRen2.Lewis rats from 5 to 11 wks (Figure 1). Similar to earlier findings,25

substantial rises in blood pressure were observed by week 7 in OVX-rats as compared to

intact littermates (P < 0.0001). This exacerbated hypertension continued throughout the

duration of the 15-week study [λ (4,26) = 0.13, P <0.0001]. Commencement of chronic

L-VNIO treatment led to modest reductions in systolic blood pressure in both intact and

OVX rats g [λ (1,29) = 0.58, P <0.0001]; however, the blood pressure lowering effects of

L-VNIO- were not sustained in the estrogen-intact group (P > 0.05)

The effects of estrogen and nNOS inhibition on echocardiographic-derived

variables indicative of systolic and diastolic function are summarized in Table 2 and

149

Figure 2. Estrogen deficiency had no effect on M-mode measurements of LV chamber

dimensions or systolic function, as signified by the absence of changes in %FS.

Assessment of diastolic function using Doppler flow and Doppler tissue imaging showed

impairment in diastolic function in estrogen-deplete mRen2.Lewis as demonstrated by a

22% decrease in mitral annular descent (e') in OVX rats despite there being no effect on

systolic function (Figure 2). Furthermore, the E/A ratio tended to be blunted in estrogen-

deficient rats compared to sham-operated littermates as estrogen accounted for 9%

percent of the variance in E/A [Estrogen: F(1,33) = 3.18, P = 0.08]. Treatment with L-

VNIO offered preservation of myocardial function as reflected in a 21% reduction in

filling pressure, combined with a positive effect on myocardial relaxation represented as

the ratio of early to late mitral annular velocity (e'/a'; P =0.04; Table 2).

The reduction in myocardial relaxation in the OVX-mRen2.Lewis rats was not

associated with changes in protein levels of SERCA2 or phospholamban, key proteins in

cytosolic Ca2+

reuptake to the sarcoplasmic reticulum. Furthermore, the phosphorylation

level of phospholamban was not altered by the removal of the ovaries nor by L-VNIO

(data not shown).

Although relative heart weights were similar among groups, estrogen and nNOS

inhibition had notable effects on LV structure. There were increases in relative wall

thickness in OVX-rats relative to intact rats (Estrogen effect: P<0.05). Administration of

L-VNIO partially reversed the increased wall thickness [L-VNIO: F(1,33) = 9.51, P =

0.004] from significantly elevated values following OVX (Table 2). Moreover,

ovariectomy in the mRen2.Lewis rats increased LV mass compared to intact littermates,

and this remodeling effect was significantly attenuated by 4 weeks of nNOS inhibition

150

[L-VNIO: F(1,33) = 4.65, P = 0.039]. Importantly, these L-VNIO-mediated structural

benefits were also observed in the estrogen-intact mRen2.Lewis rats, despite nominal

changes in blood pressure.

Evaluation of LV hypertrophy following surgical estrogen depletion further

showed that an increase in myocardial fibrosis was the primary trigger for remodeling

rather than an increase in myocyte size. Perivascular collagen deposition was increased

in OVX-rats compared with intact littermates (P=0.005), and this effect was improved by

nNOS inhibition [L-VNIO effect: F(1,100) = 51.91, P < 0.0001] (Figure 3). While

myocyte size was not altered by estrogen loss or L-VNIO (data not shown),

dihydroethidium staining within myocytes revealed increased ROS generation in the

OVX-mRen2.Lewis rats compared to intact littermates [Estrogen effect: F(1,266) = 48.8,

P < 0.0001] (Figure 4). Importantly, the 4-week administration of L-VNIO attenuated the

DHE staining, particularly in OVX-rats [L-VNIO effect: F(1,266) = 4.62, P = 0.0326].

A potential explanation for enhanced ROS generation in the hearts from OVX-

mRen2.Lewis rats is nNOS uncoupling, as estrogen influences the levels of

tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase.30, 31

To examine

the potential role of BH4 degradation as the limiting factor in NO versus ROS

production, we measured the cardiac biopterin profile (B + BH2 +BH4). While there was

no overall change in biopterin homeostasis in estrogen deficient rats, the balance between

BH4 and BH2 was shifted such that the concentration of BH4 was decreased and BH2

increased (Figure 5). Specifically, estrogen accounted for 34% of the total variance in

BH4 (P = 0.001), while simultaneously increasing cardiac concentrations of BH2

151

[Estrogen: F(1,25) = 6.36, P = 0.019] (Figure 5). Specific nNOS inhibition with L-

VNIO did not alter the cardiac biopterin profile.

In addition to the biopterin data, nitrite concentration, as an indicator of cardiac

NO availability, showed that estrogen accounted for 21% of the total variance in tissue

nitrite levels [Estrogen: F(1,25) = 7.35, P = 0.012], while L-VNIO did not change the

cardiac concentrations of nitrite and hence, NO (Figure 5).

To determine whether estrogen or L-VNIO influences the cardiac NOS isoforms,

we examined cardiac nNOS and eNOS expression. nNOS expression was increased 1.3-

fold in the homogenates from OVX rats compared to estrogen-intact rats [Estrogen:

F(1,8) = 56.27, P < 0.005] (Figure 6). The protein levels of eNOS were similar between

OVX and intact littermates (Figure 6) as was the ratio of phosphorylated eNOS to total

eNOS between the groups.

Discussion

The primary finding of the current study is that in the ovariectomized

mRen2.Lewis rats, a model that emulates the cardiovascular phenotype in women

following ovarian hormone loss, selective nNOS inhibition with L-VNIO attenuated

cardiac superoxide production and limited cardiac remodeling, leading to improvements

in diastolic function. As in previous reports, 23

diastolic function was worsened by

removal of the ovaries. In the absence of estrogen, left ventricular relaxation was

impaired while filling pressures tended to increase. These effects were coupled with

myocardial structural alterations indicated by an increase in LV relative wall thickness

152

(RWT) as well as increased cardiac collagen deposition. Four weeks of targeted nNOS

inhibition attenuated the diastolic dysfunction phenotype of the ovariectomized

mRen2.Lewis rats as revealed by echocardiographic-derived measures of myocardial

relaxation and ventricular compliance. These functional changes were accompanied by

reverse LV remodeling, as L-VNIO limited cardiac hypertrophy and perivascular

collagen deposition in OVX-mRen2.Lewis rats. Importantly, these data are the first to

show in the heart that OVX-elicited potentiation in ROS are abrogated by the nNOS

inhibitor L-VNIO. These effects within the heart of the ovariectomized mRen2.Lewis

appear to be due to diminished or oxidized BH4, the necessary cofactor for NOS activity,

with concomitant increases in uncoupled nNOS and/or a reduction in NO production.

The adverse structural remodeling of the myocardium, indicated by increases in

wall thickness, LV mass, and perivascular collagen, that contributes to the lusitropic

dysfunction in the OVX-mRen2.Lewis rat appears to be rooted at the level of

extracellular matrix. Several studies showed that estrogen loss is accompanied by

increases in tissue collagen deposition which may in part be a consequence of increased

cardiac Ang II tissue content or activity in this model.23

Since serum levels of

aldosterone are increased in ovariectomized mRen2.Lewis hypertensive rats23

, this may

contribute to collagen deposition within the heart. Other studies have implicated

aldosterone as a potent profibrotic factor32-34

and in previous reports in mRen2.Lewis

rats25

, L-VNIO treatment was associated with a decrease in serum aldosterone to levels

found in non-ovariectomized rats.26

It is plausible that the loss of estrogen accounts for

the increased deposition, given that estrogen has been shown to regulate collagen

turnover and fibroblast proliferation.35, 36

Certainly, the exacerbated hypertension in the

153

estrogen-deplete mRen2.Lewis rats could contribute to extracellular matrix remodeling

and diastolic dysfunction.

Importantly, L-VNIO offered a degree of cardioprotection among OVX-

mRen2.Lewis rats that mitigated the diastolic dysfunction and attenuated LV remodeling.

Since there were no overt changes in blood pressure in sham-operated rats at the end of

the 4 weeks of treatment, the action of LVNIO appears to be directed to the myocardium

rather than the peripheral vasculature. Indeed, diastolic functional derangements have

been reported in isolated cardiomyocytes from hypertensive mice.37

Moreover, L-VNIO

treatment in our sham-operated (estrogen-intact) rats improved myocardial relaxation and

reduced wall thicknesses, perivascular fibrosis, and ROS generation in the absence of

changes in systolic blood pressure. In a recent report by Silberman at al.,37

hypertensive

mice given either hydralazine or BH4 experienced equivalent blood pressure reductions

while improvements in diastolic function were detectable only in the BH4-treated mice.

Taken together with our findings, these data implicate a direct role of cardiac NOS in the

preservation of cardiac structure and diastolic function. Our data corroborate clinical and

experimental studies which show cardiac functional improvements associated with

decreased oxidative stress following blockade of the renin angiotensin system that are

independent of blood pressure.38-40

The loss of the estrogenic stimulus on BH4 synthesis may uncouple nNOS

shifting it to a maladaptive ROS generating state. In the ovariectomized mRen2.Lewis

hypertensive rat, a discoordinate regulation of renal NOS isoforms was evident with

reduced renal cortical mRNA and protein levels of eNOS while renal cortical nNOS

mRNA and protein were increased.26

In this situation, just as in our current study, nNOS

154

is presumed to transition from catalyzing NO formation to generating superoxide.41, 42

While the precise mechanism for this conversion of nNOS is not clear, it may reflect

estrogen sensitive regulation of guanosine triphosphate cyclohydrolase I (GTPCH), the

rate limiting enzyme of BH4 synthesis.43

Thus, in the absence of estrogen, the nNOS

cofactor, BH4, may be oxidized or decreased overall leading to an increase in ROS at the

biological expense of reduced NO. White and colleagues44

emphasized the importance

of age-related decreases in requisite components of NOS activity while demonstrating

that nNOS specifically is an estrogen target in coronary vessels that can lead to

detrimental or beneficial effects depending upon the surrounding milieu that determines

the coupling state of the enzyme. In a coupled state, NOS is considered positive in that it

generates NO. Alternatively, in situations where NOS becomes uncoupled, NOS activity

can be detrimental by generating reactive oxygen species.

This idea of “uncoupling” is a rather novel phenomenon, which was first

demonstrated in the heart and vascular beds predominantly related to eNOS.45, 46

More

recently it has been shown that nNOS uncoupling in the heart may regulate cardiac

function.47-49

Specifically, nNOS stimulates cardiac O2- production, modulates cardiac

nitroso-redox balance, and stimulates development of diastolic dysfunction.37

L-VNIO

appears to have targeted this pathway as chronic administration of the inhibitor reduced

the increase in ROS production while improving diastolic function following

oophorectomy. According to Babu and Griffith,50

L-VNIO is a potent, mechanism-based

inhibitor blocking the heme cofactor of NOS which also shows a marked selectivity for

nNOS. Typically, in the presence of NADPH and O2, L-VNIO irreversibly inactivates

nNOS through a Ca2+

/calmodulin-dependent effect. In the absence of BH4, inhibition of

155

nNOS activity by L-VNIO is increased.50

It is plausible that estrogen depletion and the

subsequent BH4 reduction may have augmented the inhibitory capacity of L-VNIO on

nNOS.50

Taken together, these findings support the notion that estrogen has a regulatory

role in the maintenance of cardiac structure and diastolic function in part through its

modulation of myocardial NO production or by direct attenuation of the profibrotic

actions of reactive oxygen species.

The present study has a few limitations. First, the pharmacological specificity of

L-VNIO for nNOS as opposed to the other NOS isoforms is unconfirmed under the

existing physiologic setting. In order to adequately address the precise contribution of

nNOS blockade to alterations in NOS activity, and to improvements in cardiovascular

endpoints, parallel studies using nonspecific and specific-inhibitors of the other NOS‟s

would be needed. While this is beyond the scope of our current study, several ex vivo

cardiomyocyte studies performed by others do indeed show that L-VNIO is specific for

nNOS. The initial study by Babu et al.50

found the Ki of L-VNIO for nNOS to be 0.1 µM

compared to values at least 100 times higher in eNOS (12 µM) and iNOS (60 µM).

Another study utilizing isolated myocytes from wild-type and selective nNOS knockout

mice show that L-VNIO-treated cells of wild-type mice have the same relaxation

potential as that of cells rendered from knockout mice.51

Elaborating upon their previous

work, Casadei‟s group also showed that L-VNIO-treated cardiomyocytes from wild-type

mice increase the sarcoplasmic reticulum Ca2+

load to a similar degree as myocytes

isolated from nNOS knockout mice.52

Therefore, taken together with the increased

expression of nNOS in hearts from OVX-mRen2.Lewis rats and the abrogation of cardiac

156

ROS production after administration of L-VNIO, suggests that the in vivo administration

of L-VNIO conferred specificity for nNOS rather than eNOS.

Second, the physiologic perturbations from abrupt (via surgical manipulation)

ovarian hormone loss in adult rodents are not analogous to that which occurs in middle-

aged women following natural menopause. While artificial or surgical menopause

represents less than 13% of women (see the web site http://www.menopause.org), reports

have also linked premenopausal oophorectomy with unfavorable health consequences

including increased cardiovascular risk.53, 54

As in humans, the mRen2.Lewis female

rodent shows accelerated increases in blood pressure and left ventricular hypertrophy

following ovarian hormone depletion. This strain, therefore, provides a unique model to

study the role of ovarian hormones, independent of age-related cardiovascular changes, in

the pathogenesis of diastolic heart disease in a „high risk‟ population.

Conclusion

In summary, we show that estrogen depletion, due to the removal of the ovaries,

in mRen2.Lewis female rats exacerbates diastolic dysfunction while invoking

functionally diminished myocardial relaxation and altered cardiac pathology including

increased LV relative wall thickness and collagen deposition. These detrimental effects

were significantly attenuated following administration of a selective nNOS inhibitor,

which we propose is due to the ability of L-VNIO to mitigate ROS generation.

Acknowledgments

157

The authors wish to thank Marina Lin, MS for her technical contributions and Timothy T.

Houle, PhD for his statistical expertise.

158

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Table 3.1. Body and Heart Weight Values

Sham OVX P value

Vehicle L-VNIO Vehicle L-VNIO Estrogen L-VNIO Interaction

N = 9 N=10 N=6 N=9

Body weight, g 240 ± 9 238 ± 5 276 ± 13 280 ± 7 0.0001 0.781 0.533

Heart weight, mg 818 ± 0.02 822 ± 0.02 925 ± 0.01 932 ± 0.01 0.002 0.873 0.960

Heart weight/Body

weight, mg/g 3.42 ± 0.08 3.47 ± 0.09 3.35 ± 0.12 3.33 ± .09 0.461 0.918 0.773

Data are expressed as mean ± SEM.

168

Table 2. Echocardiographic Indices of Systolic and Diastolic Function

Data are expressed as mean ± SEM. LVEDD = left ventricular end-diastolic dimension;

LVESD = left ventricular end-systolic dimension; RWT = relative wall thickness; %FS =

percent fractional shortening; Emax = maximum early transmitral filling velocity;

Edecslope = early-filling deceleration slope; Amax = maximum late transmitral filling

velocity; E/A = early-to-late transmitral filling ratio; e' = early mitral annular velocity;

E/e' = early transmitral filling velocity-to-mitral annular velocity; a' = late mitral annular

velocity; e'/a' = early-to-late mitral annular ratio.

Sham OVX P value

Vehicle

L-VNIO

Vehicle

L-VNIO Estrogen L-

VNIO

Interaction

N = 9 N=10 N=6 N=9

Heart Rate, bpm 250 ± 9 244 ± 5 206 ± 13 228 ± 7 0.156 0.357 0.753

LVEDD, cm 0.68 ± 0.02 0.68 ± 0.02 0.68 ± 0.01 0.69 ± 0.01 0.757 0.752 0.763

LVESD, cm 0.37 ± 0.02 0.37 ± 0.02 0.37 ± 0.02 0.40 ± 0.01 0.518 0.271 0.350

RWT 1.07 ± 0.08 1.28 ± 0.09 1.02 ± 0.11 1.16 ± 0.09 0.002 0.004 0.211

%FS 46 ± 2 46 ± 1 46 ± 2 43 ± 2 0.562 0.365 0.499

Emax, cm/sec 64 ± 2 66 ± 3 61 ± 3 61 ± 3 0.147 0.909 0.693

Edecslope,

cm/sec2

11 ± 1 13 ± 1 10 ± 1 11 ± 1 0.033 0.128 0.875

Amax, cm/sec 42 ± 3 38 ± 2 42 ± 3 41 ± 2 0.637 0.357 0.652

E/A 1.57 ± 0.09 1.76 ± 0.13 1.47 ± 0.12 1.49 ± 0.08 0.085 0.348 0.461

e', cm/sec 3.40 ± 0.22 3.52 ± 0.19 2.66 ± 0.14 3.24 ± 0.15 0.011 0.076 0.235

E/e' 19.5 ± 1.23 19.2 ± 1.32 23.6 ± 1.7 18.7 ± 0.55 0.162 0.048 0.077

a', cm/sec 3.20 ± 0.09 2.82 ± 0.16 2.75 ± 0.31 2.95 ± 0.28 0.451 0.718 0.191

e'/a' 3.20 ± 0.09 2.82 ± 0.16 2.75 ± 0.31 2.95 ± 0.28 0.617 0.038 0.492

169

Figure 3.1.

Tail-cuff systolic blood pressure in sham-operated or OVX conscious female

mRen2.Lewis rats treated with L-VNIO or saline vehicle for 4 weeks. Values are means

± SEM.

6 7 8 9 10 11 12 13 14 15

100

125

150

175

200

OVX-VEH

Sham-L-VNIO

OVX-L-VNIO

Sham-VEH

Age (Weeks)

Systo

lic B

loo

d P

ressu

re

(mm

Hg

)

170

Figure 3.2.

A. Representative tissue Doppler image of intact or OVX mRen2.Lewis female receiving

either vehicle or combination. E = early transmitral filling velocity; A = late transmitral

filling velocity; e' = early mitral annular descent (septal) velocity. B. Early mitral annular

velocity (e´). Open bars are vehicle treated, while filled bars received L-VNIO. Data

represent mean ± SEM; * P < 0.01 (Estrogen effect). C. Early transmitral filling velocity

– to – mitral annular velocity ratio (E/e´). Data represent mean ± SEM; * P < 0.05 (L-

VNIO effect).

Sham OVX0

1

2

3

4 *

e'

(cm

/sec)

Sham OVX0

10

20

30#

E/e

' (L

V f

illin

g p

res

su

re)

B C

A

171

Figure 3.3.

Quantification and representative picrosirius red staining of perivascular collagen in the

hearts of sham-operated, ovariectomized (OVX), and rats given the nNOS inhibitor (L-

VNIO). Open bars are vehicle treated, while filled bars received L-VNIO. Data

represent mean ± SEM; * P = 0.0005 (Estrogen effect), # P < 0.0001 (L-VNIO effect).

Sham OVX0.00

0.02

0.04

0.06

0.08 *

#

#

Co

lla

ge

n C

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B Sham - Veh Sham –

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OVX - Veh OVX – L-VNIO

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Figure 3.4.

Quantification and representative images illustrating the effect of L-VNIO on cardiac

levels of fluorescent 2-hydroxyethidium from dihydroethidium (DHE) staining in

estrogen intact (sham) or OVX. Open bars represent vehicle treated rats, while filled bars

received L-VNIO. Fluorescent images are shown at 400x magnification. Data represent

mean ± SEM; * P < 0.0001 (Estrogen effect), # P = 0.0326 (L-VNIO effect).

Sham OVX0

20

40

60

80

100 *

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#

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rayscale

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B Sham - VEH

OVX - VEH

Sham – L-VNIO

OVX – L-VNIO

173

Figure 3.5.

Cardiac concentrations of biopterin, BH2, BH4, and nitrite. Open bars are vehicle

treated, while filled bars received L-VNIO. Data represent mean ± SEM; * P < 0.01

(Estrogen effect).

Cardiac Biopterin

Sham OVX0

1

2

3

4

pm

ol/m

g

Cardiac Dihydrobiopterin

Sham OVX0

1

2

3

4

*

pm

ol/m

g

Cardiac Tetrahydrobiopterin

Sham OVX0.0

0.5

1.0

1.5

*

pm

ol/m

g

Cardiac Nitrite

Sham OVX0

100

200

300

400

500*

nM

174

Figure 3.6.

Values are means ± SEM of cardiac nNOS, total eNOS, and phosphorylated eNOS

protein expression in sham-operated or OVX rats treated with VEH or L-VNIO treated

rats. Open bars are vehicle treated, while filled bars received L-VNIO. * P < 0.005

(Estrogen effect).

175

CHAPTER FOUR

TETRAHYDROBIOPTERIN RESTORES DIASTOLIC FUNCTION AND

ATTENUATES SUPEROXIDE PRODUCTION IN OVARIECTOMIZED

MREN2.LEWIS RATS

Jewell A. Jessup, BS,1 Lili Zhang, MD,

2 Alex F. Chen, MD, PhD,

2 Tennille D. Presley,

PhD,3,4

Daniel B. Kim-Shapiro, PhD,3,4

Hao Wang, MD, PhD5, Leanne Groban, MD

1,5

Department of Physiology and Pharmacology1 and the Department of Anesthesiology

5,

Wake Forest University School of Medicine, Winston Salem, North Carolina;

Department of Surgery2, University of Pittsburgh School of Medicine, and Vascular

Surgery Research, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pa;

Department of Physics3 and Translational Science Center

4 Reynolda Campus, Wake

Forest University, Winston-Salem, NC;

The following manuscript in published in Endocrinology 2011; (152(6):2428-2436.

Differences in formatting and organization reflect requirements of the journal. Jewell A.

Jessup designed and performed the experiments, analyzed the data, and prepared the

manuscript. Dr. Leanne Groban oversaw the design of the experimental protocol,

performed the echocardiographic studies, edited the manuscript, and served in an

advisory capacity to Ms. Jessup. Drs. Zhang and Chen provided biopterin analyses. Drs.

Presley and Kim-Shapiro performed the nitrite detection. Dr. Hao Wang assisted with

data interpretation and editing the manuscript.

176

Abstract

Following oophorectomy, mRen2.Lewis rats exhibit diastolic dysfunction,

associated with elevated superoxide, increased cardiac neuronal nitric oxide synthase

(nNOS) expression, and diminished myocardial tetrahydrobiopterin (BH4) content,

effects that are attenuated with selective nNOS inhibition. BH4 is an essential cofactor of

nNOS catalytic activity leading to nitric oxide production. Therefore, we assessed the

effect of 4-week BH4 supplementation on diastolic function and left ventricular (LV)

remodeling in oophorectomized mRen2.Lewis rats compared to Sham-operated controls.

Female mRen2.Lewis rats underwent either bilateral oophorectomized OVX (n=19) or

Sham-operation (Sham; n=13) at 4 weeks of age. Beginning at 11 weeks of age, OVX

rats were randomized to receive either BH4 (10 mg•kg–1

•day–1

) or saline, while the Sham

rats received saline via subcutaneous mini-pumps. Loss of ovarian hormones reduced

cardiac BH4 when compared to control hearts; this was associated with impaired

myocardial relaxation, augmented filling pressures, increased collagen deposition, and

thickened left ventricular walls. Additionally, superoxide production increased and nitric

oxide decreased in hearts from OVX compared to Sham rats. Chronic BH4

supplementation after ovariectomy improved diastolic function and attenuated left

ventricular remodeling while restoring myocardial nitric oxide release and preventing

reactive oxygen species generation. These data indicate that BH4 supplementation

protects against the adverse effects of ovarian hormonal loss on diastolic function and

cardiac structure in mRen2.Lewis rats by restoring myocardial NO release and mitigating

myocardial O2- generation. Whether BH4 supplementation is a therapeutic option for the

177

management of diastolic dysfunction in postmenopausal women will require direct

testing in humans.

Key words: diastolic function; nitric oxide; BH4; fibrosis; postmenopausal; left

ventricular remodeling

178

Introduction

Cardiovascular disease in women has gained attention in recent years in light of

the recognized sex differences between males and females (1-4). Prior to menopause, the

incidence of heart disease is significantly lower in women compared to men (5).

However, both the risk and the prevalence of heart failure, dramatically increases in

women following the onset of menopause (6). Unlike their male counterparts, women

predominantly exhibit diastolic impairment and eventual diastolic heart failure rather than

systolic dysfunction (7;8). The interplay between age and declining estrogens were

deemed the common denominators in women‟s heart disease and led to robust

examination of hormone replacement therapy in the cardioprotection of postmenopausal

women. However, conflicting results failed to show cardioprotective benefit with

replacement of various estrogen therapies (9), underscoring the need to gain a further

insight into the mechanisms which influence the development of heart disease in women.

Using the female mRen2.Lewis rat, an angiotensin-II dependent model of

hypertension, we show diastolic functional abnormalities and remodeling of the cardiac

interstitium following early bilateral oophorectomy, a cardiac phenotype that bears a

resemblance to the cardiac and vascular changes resulting from surgically-induced or

natural menopause (10). In this same rodent model, the loss of ovarian hormones

exacerbates the increases in systolic blood pressure, which can be corrected with 17β

estradiol replacement (11). In a recent study, we further show that the pathogenesis of

diastolic dysfunction in the female mRen2.Lewis rat involves a critical interaction

between neuronal nitric oxide synthase (nNOS) and ovarian hormones, particularly

estrogens (12). That is, selective nNOS inhibition with L-VNIO attenuates diastolic

179

dysfunction and cardiac remodeling in ovariectomized (OVX) mRen2.Lewis females,

presumably due to the ability of L-VNIO to mitigate myocardial reactive oxygen species

(ROS) generation. Taken together, these findings suggest that a dynamic shift occurs in

the nNOS pathway within the female heart such that the loss of estrogens leads to

uncoupling of nNOS and the subsequent generation of superoxide (O2-) rather than nitric

oxide (NO) (12).

The effects of nNOS on diastolic function based upon the bioavailability of NO

are also documented (13). Additional data show that estrogens can modulate the

production of nNOS (14-17) as well as regulate guanosine triphosphate cyclohydrolase I

(GTPCH) (18-20). GTPCH is the rate limiting enzyme for the synthesis of

tetrahydrobiopterin (BH4), the necessary cofactor responsible for maintaining nNOS in a

“coupled” state which allows the enzyme‟s catalytic activity to generate NO (21). In our

previous study, diminished BH4 levels within the heart tissue of oophorectomized rats

was associated with compromised ventricular relaxation and cardiac structural alterations

including increased collagen deposition and increased relative wall thickness (12).

Theorizing that the loss of estrogens and consequent depletion of BH4 may lead to nNOS

uncoupling and the release of reactive free radicals instead of NO, we hypothesized that

BH4 supplementation would exert beneficial effects on diastolic function in terms of

improving myocardial relaxation, attenuating LV remodeling, and decreasing the

generation of ROS in the heart of the OVX-mRen2.Lewis rat.

Methods

Experimental Model

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Congenic female mRen2.Lewis rats obtained from the transgenic rat colony of the

Hypertension and Vascular Research Center of Wake Forest University School of

Medicine were weaned at 3 weeks of age and allowed to acclimate in a temperature-

controlled, Association for Assessment and Accreditation of Laboratory Animal-

approved facility (12-hr light/dark cycle) with ad libitum food and water. The

experimental procedures were approved by Wake Forest University School of Medicine‟s

Animal Care and Use Committee. Rats were randomly divided into two groups at 4

weeks of age: bilateral oophorectomized (OVX; n=17) or Sham-operated (Sham; n=13),

which were performed under 2% isoflurane anesthesia, as previously described (10;12).

Once the rats reached 11 weeks of age, the OVX group was randomly divided to receive

(6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride [(tetrahydrobiopterin or BH4);

OVX+BH4; n=8; Schricks Laboratories, Jona, Switzerland] administered via an osmotic

minipump (Alzet Model 2ML4, 2.5 µL/hr, 4 weeks; DURECT Corporation, Cupertino,

CA) at a targeted dose of 10 mg•kg–1

•day–1

or saline (2.5 µL/hr) as a vehicle control

(OVX; n=9). This dose of BH4 has been shown to be therapeutically effective in

previous reports by other laboratories (22-24) . Weekly tail-cuff plethysmography

(NIBP-LE5001, Panlab, Barcelona, Spain) was used to measure blood pressure from 5 –

15 wk of age. Oophorectomy and depletion of circulating estradiol was confirmed using

a serum estradiol assay [5 pg/mL detection limit; Polymedco (Cortlandt Manor, NY)].

Echocardiographic evaluation

Cardiac function and structure were assessed prior to the protocol termination in

anesthetized (ketamine/xylazine: 80/12 mg/kg) animals using a Philips 5500

181

echocardiograph (Philips Medical Systems, Andover, MA) and a 12 MHz phased array

probe as previously described (10). Relative wall thickness (RWT) was calculated as: 2

× posterior wall thickness/left ventricular end diastolic dimension (PWT/LVEDD).

Measurement of total biopterin and BH4 levels

Cardiac tissue BH4 levels were measured by HPLC with fluorescence detection as

previously documented (25). Total biopterins [BH4, dihydrobiopterin (BH2) and oxidized

biopterin (B)] were measured following acid oxidation of the deproteinated cardiac

supernatant. BH2 plus B was determined by alkali oxidation, followed by iodine

reduction, and acidification. The resulting samples were centrifuged and small aliquots

injected into a 250-mm long, 4.6-mm inner diameter Spherisorb ODS-1 column (5 μm

particle size; Alltech Associates, Inc., Deerfield, IL). Fluorescence (350 nm excitation,

450 nm emission) was detected by a fluorescence detector (RF10AXL, Shimadzu Co.,

Columbia, MD). BH4 concentration was calculated by subtracting BH2 and B from total

biopterins.

Nitrite Measurements

Nitrite levels of were determined using a chemiluminescence-based Nitric Oxide

Analyzer (Sievers, Inc., GE Analytical Instruments, Boulder, CO) as previously

described (26). Standard curves were obtained and used for quantitative measurements.

Morphometry and Histopathology

182

Collagen deposition within the heart was evaluated using Picrosirius red (PSR)

stained, 4 µm, 4% paraformaldehyde-fixed, paraffin embedded sections as documented

previously (12). Additional sections stained with hematoxylin and eosin were used to

measure myocyte cross-sectional area in approximately 100 cardiomyocytes from 2

sections per rat (400× magnification) as previously described(12). Histological analysis

was completed using a Leica DM4000B microscope (Bannockburn, IL) with a tandem

Leica DFC digital camera and Simple PCI 6.0 software as well as an Olympus polarizing

microscope system (Center Valley, PA) equipped with a Digital SPOT RT, 3-pass

capture, thermoelectrically cooled charge-coupled camera (Sterling Heights, MI) and

SPOT® Advanced software. Adobe Photoshop Creative Suite 3 (Adobe Inc., San Jose,

CA) was used to calculate the ratios of collagen positive stained pixels to unstained pixels

in each photomicrograph (4 randomized quadrant field images per rat, magnified 200×).

In situ ROS production

Reactive oxygen species (ROS) was determined using dihydroethidium (DHE) as

previously described (12). The resulting images were analyzed at 400× using a

fluorescence microscope (Leica DM4000B, Bannockburn, IL; excitation = 510-550 nm,

emission = 590 nm for DHE; excitation = 330-380 nm, emission 420 nm for DAPI)

connected to a Leica DFC digital camera. The fluorescent signal intensities were

analyzed within the heart using Simple PCI 6.0 software. All data are presented as

average gray scale intensities ± SEM.

Statistical analysis

183

All values are expressed as mean ± SEM. For all endpoints, one-way ANOVA

evaluated significant effects among the groups. Significant interactions between the

groups were further characterized using Bonferroni post hoc tests. Analyses were

performed using GraphPad Prism 5 (GraphPad, San Diego, CA). Differences were

considered significant at p < 0.05.

Results

Cardiac Biopterin and BH4 Levels

Figure 1 depicts total cardiac biopterin and BH4 levels from Sham, OVX, and

OVX rats treated with BH4. Supplementation with BH4 (OVX+BH4) was associated with

more than a 3-fold increase in myocardial biopterin concentrations compared with

untreated Sham and OVX littermates (p < 0.001). This confirmed that the administered

BH4 reached the heart. As expected, estrogen depletion by surgical oophorectomy was

associated with a 3.2-fold reduction in cardiac BH4 when compared to levels in Sham-

operated littermates (p < 0.05). Exogenous BH4 supplementation attenuated this OVX-

associated fall in cardiac BH4 concentration, but did not restore levels to those measured

in Sham rats.

Physical characteristics and Hemodynamics

The effects of ovariectomy and BH4 supplementation on body weight and heart

weight are shown in Table 1. Prior to BH4 or vehicle treatment, there were no differences

in body weight or SBP among the three cohorts. At the end of the study, the OVX and

OVX+BH4 rats were 13% and 16% heavier, respectively, than Sham-operated controls (P

184

< 0.001 for each), corroborating previous reports from this and other laboratories that

deprivation of estrogens leads to increased body mass.(10;12;27;28) Likewise, the heart

weights from the OVX rats were substantially higher compared to the Sham group

(Sham: 830 ± 17 mg vs. OVX: 939 ± 35 mg; P < 0.01). BH4 had no effect on the OVX-

induced heart weight increase. Normalized heart weights (HW/BW) were similar among

the three groups.

Progressive increases in systolic blood pressure (SBP) occurred in Sham and

OVX rats from 5 - 11 wks (Figure 2). Similar to earlier findings,(11;12) substantial rises

in blood pressure were observed by week 6 in OVX rats as compared to intact littermates

( P < 0.0001) and this larger increase in SBP continued throughout the duration of the 15-

week study. While there was a tendency for BH4 administration to limit the exacerbated

hypertension, there were no significant differences between the ovariectomized groups

during the four-week supplementation period.

Heart Rate and Diastolic Function

Table 2 shows the effects of ovarian hormonal loss and BH4 supplementation on

heart rate (HR) and the conventional Doppler indices of diastolic function. While HR

and the more load-dependent, conventional measures of diastolic function were similar

among groups (Table 2), the loss of ovarian hormones led to modest reductions in

ventricular relaxation, (or e) and elevations in left ventricular filling pressure (or E/e) (p

< 0.001) when compared to Doppler indices from intact-littermates (Figure 3).

Importantly, four weeks of BH4 treatment restored ventricular lusitropy and reduced

filling pressures to that of Sham-operated littermates. Specifically, BH4 increased mitral

185

annular velocity (e) by 27% and reduced Doppler-derived filling pressures (E/e) by 20%

when compared to untreated OVX-rats.

Left Ventricular Structural Characteristics and Systolic Function

Figure 4a demonstrates a 1.6-fold increase in perivascular fibrosis in OVX

mRen2.Lewis rats compared to their estrogens-intact littermates (P < 0.005). OVX had

no effect on myocyte cross-sectional area (Figure 4b). Chronic BH4 supplementation in

OVX-mRen2.Lewis rats reversed or prevented the excess collagen deposition (37%, P <

0.001) (Figure 4a), without altering myocyte size (Figure 4b).

M-mode measurements of LV dimensions and wall thicknesses are summarized in

Table 3. The loss of ovarian hormones led to a 25% increase in posterior wall thickness

(P < 0.01) (Table 3), which combined with no alterations in the LVEDD yielded a robust

2-fold increase in relative wall thickness compared to Sham-operated rats (P < 0.001)

(Figure 4c). Treatment with BH4 reversed this effect as the RWT of the OVX+BH4

compared to OVX was 0.51 ± 0.04 cm vs 0.71 ± 0.05 cm, respectively (P < 0.01) (Figure

3c). Systolic function indicated as the % fractional shortening (FS) was similar among

the three groups (Table 3).

Reactive Oxygen Species and Nitrite Profile

Similar to our earlier findings,(12) dihydroethidium staining of the cardiac tissue

illustrated abundant O2- production within the myocytes of OVX rats compared to the

estrogens-intact mRen2.Lewis rats (Figure 5a). This increase in ROS generation was

associated with a 33% reduction in cardiac tissue nitrite availability, or NO release, in the

186

OVX group (P < 0.05) (Figure 5b). In hearts from OVX+BH4 rats, the intensity of the

fluorescent product, 2-hydroxyethidium, was diminished to a level found in hearts from

intact rats (Figure 5a). The decreased O2- production in the OVX+BH4 group

corresponded to a 1.7-fold increase in available nitrite content (Figure 4b).

Discussion

The major findings of this study are: 1) loss of estrogens by surgical ovariectomy

results in myocardial BH4 deficiency in mRen2.Lewis rats; 2) this deficiency is

associated with diastolic dysfunction, adverse LV remodeling, increases in cardiac O2-

production, and reduced cardiac NO release; and, 3) BH4 supplementation reverses all

these changes except the elevated heart weight induced by OVX. The observation that

the loss of estrogens is accompanied by increased body weight explains the presence of a

compensatory increase in cardiac mass likely due to excessive lengthening of cardiac

myocytes and ventricular dilation, (29) and/or from the loss of estrogen-stimulated cGMP

activity (30) Collectively, this study supports our original hypothesis that diminished

BH4 availability following the loss of ovarian hormones, and specifically estrogens, leads

to nNOS “uncoupling” within the heart of the OVX mRen2.Lewis rat. Under this

situation, O2-

rather than NO becomes the product of nNOS activity ultimately

contributing to the diastolic dysfunction phenotype (12). It is therefore plausible that

BH4 supplementation may represent a rational approach to the management of diastolic

dysfunction in women due to either surgically-induced or natural menopause. The

benefits of BH4 supplementation may be due to suppression of ROS generation alone or

in combination with enhanced NO release (12) and/ or a direct antioxidant action (31).

187

Extensive strides have been made to establish the cardioprotective role of

endothelial nitric oxide synthase (eNOS) in the regulation of cardiac function (18;32-35).

However, efforts targeting equivocal appreciation of cardiac (36;37) and vascular nNOS

(38) have been far more limited. It is important to note that nNOS compared to the other

NOS isoforms, is more likely to produce superoxide when the enzyme is uncoupled (39).

In an earlier report by Yamaleyeva et al. (40), renal eNOS decreased following OVX in

mRen2.Lewis rats while nNOS protein expression increased. In their study, selective

inhibition of nNOS attenuated the exacerbated increase in systolic blood pressure

characteristic of this model. Likewise, we observed overexpression of cardiac nNOS in

OVX-mRen2.Lewis rats compared to estrogens-intact littermates, but no difference in

cardiac eNOS between groups (12). Intrigued by the possibility that the excess nNOS

had a functional role in the model, we administered the specific nNOS inhibitor (L-

VNIO). The L-VNIO treatment led to cardioprotection in the OVX mRen2.Lewis model,

effects we proposed were due to blockade of the uncoupled nNOS activity, as ROS

generation was decreased while NO increased in the heart (12). Taken together with the

finding that cardiac BH4 levels were suppressed in OVX-rats compared to littermates

with intact estrogens (12), and that deficiency of estrogens has been reported to cause

decreased synthesis of BH4 by the de novo pathway (41) provides the rational basis for

the use of BH4 supplementation in our model. BH4 is not only an essential NOS cofactor,

it has in important role in maintaining the structure of all NOS isoforms by stabilizing the

active or “coupled” form of the enzyme, which leads to the production of NO rather than

superoxide, the end-product of nNOS uncoupling. These current data complement and

extend these prior observations by showing that BH4 supplementation halts the functional

188

and structural changes in the OVX-mRen2.Lewis hearts, even in the presence of

exacerbated increases in blood pressure.

Though the heart data are limited, the link between estrogens and BH4 is

highlighted in a report by Lam et al. (42) which showed decreased bioavailability of BH4

in the rat vasculature following ovariectomy. Another study has shown that estrogens

can directly increase BH4 through upregulation of GTPCH mRNA and activity in

cultured endothelial cells (18). Moreover, 17β estradiol therapy normalized aortic BH4

levels and suppressed O2-

generation (41). Although it is not known whether the

decreased concentration of cardiac BH4 in hearts from the OVX mRen2.Lewis rats was

due to the withdrawal of the regulation of estrogens on GTPCH, resetting the nNOS

system through BH4 supplementation was a sufficient means by which to normalize

diastolic function, attenuate remodeling, and restore myocardial antioxidant capacity to

that of littermates with intact estrogens.

Independent of the status of estrogens, equivalent doses of BH4 as used in the

present study have repeatedly been shown to preserve cardiac function and prevent

remodeling post-myocardial infarction (43;44). In isolated hearts from Dahl salt-

sensitive rats, administration of a BH4 donor increased left ventricular developed pressure

and recovery as measures of ischemia resistance (45). Additionally, in a pressure-

overload mouse model, BH4 reversed cardiac hypertrophy and improved heart function as

documented in improved sarcomere activity and calcium handling (46). The benefit of

BH4 extends beyond in vivo and in vitro experiments. In a pilot study designed to

establish a dose-response curve and determine the duration of oral BH4 therapy in

189

uncontrolled human hypertensive patients, BH4 significantly reduced systolic blood

pressure and improved flow-mediated vasodilation (47).

The first limitation of the present study is that these studies were conducted in a

rodent oophorectomized model, which is not an identical mimetic of naturally occurring

menopause. However, the phenotypic characteristics of the mRen2.Lewis following

depletion of ovarian hormones, including left ventricular hypertrophy and exacerbated

hypertension, are similar to the pathophysiological changes occurring in post-menopausal

women (48;49).

Secondly, the actual accumulated levels of BH4 detected within the myocardial

tissue of the BH4-treated OVX rats were not significantly increased compared to OVX.

These observations are likely due to the dual regulation of BH4 metabolism which can

lead to rapid oxidation of BH4 to BH2 and simultaneous recycling of BH4 from BH2 by

the enzyme dihydrofolate reductase. (50;51). Channon et al. (52) eloquently described

this phenomenon within the vasculature where they suggest that the overall availability of

BH4 within the vascular tissue is the summation of BH4 synthesis, BH4 oxidation, and

finally, its recycling from BH2. In our studies, the HPLC biopterin assay cannot

determine the rate of oxidation.

Finally, this study focused on proposed nNOS uncoupling as the source of

reactive oxygen species generation leading to diastolic dysfunction and LV remodeling.

Oxidative stress can also occur via alternative processes, including xanthine oxidase,

NADPH oxidases, or cytochrome P450. However, our rationale to explore the nNOS

uncoupling phenomenon in this model was based on the paradoxical increase in cardiac

nNOS expression following oophorectomy (12). This is an important consideration given

190

the fact that until recently, it had been assumed that nNOS was a positive contributor to

cardiovascular function through the production of NO as opposed to the maladaptive

characteristics (increased ROS and decreased NO) now demonstrated in this study.

Targeting individual components of the pathway allows for the progressive understanding

of this complex system and the extrapolation of potential clinical applications.

In summary, we show that BH4 supplementation led to significant improvements

in diastolic function, attenuation of cardiac remodeling, and reductions in oxidative stress

in the mRen2.Lewis female after ovarian hormonal loss. We propose that the restorative

effects are due to BH4 therapy recoupling the NOS activity which shifts the maladaptive

pathway of ROS generation back to coupled, adaptive catalytic activity. In light of the

controversy surrounding estrogen replacement therapy, it is plausible that BH4 may be a

viable therapeutic option for diastolic patients as it is currently used clinically for other

syndromes such as phenylketonuria (PKU). PKU patients who have either phenylalanine

hydroxylase (PAH) mutations or are BH4 deficient, therapeutically receive BH4, the

necessary cofactor for PAH activity, just as it is for NOS activity. In those patients that

are responsive to BH4, the level of circulating phenylalanine is significantly diminished

sparing the individuals from developmental disorders, cognitive impairment, and

seizures.(53-55) Importantly, these patients exhibit minimal side effects (56) providing

hope that BH4 supplementation could provide beneficial effects in patients with diastolic

dysfunction.

Acknowledgments

191

The authors extend sincere appreciation to Marina Lin, MS for her technical

contributions, Timothy T. Houle, PhD for his statistical expertise, and Addie Larimore

for her editorial assistance.

192

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Table 4.1. Body and Heart Weight Values

Variables Sham

N=13 OVX

N=9 OVX + BH4

N=10

Body weight, g

P <

239 ± 2 269 ± 7

0.001

277 ± 5

0.001

Heart weight, mg

P <

830 ± 17 939 ± 35

0.01

905 ± 20

Heart weight/Body weight,

mg/g

P <

3.47 ± 0.07 3.50 ± 0.13

3.27 ± 0.05

Data are expressed as mean ± SEM. P values are compared to Sham.

202

Table 4.2. Echocardiographic Indices of Diastolic Function

Variables Sham

N=13 OVX

N=9 OVX + BH4

N=10

Emax, cm/sec

P <

63 ± 2 64 ± 4 64 ± 3

Amax, cm/sec

P <

42 ± 2 40 ± 3 40 ± 2

E/A

P <

1.5 ± 0.1 1.6 ± 0.1 1.7 ± 0.1

Edectime, sec

P <

0.057 ± 0.002 0.062 ± 0.003 0.060 ± 0.003

Data are expressed as mean ± SEM. Emax = maximum early transmitral filling velocity;

Amax = maximum late transmitral filling velocity; E/A = early-to-late transmitral filling

ratio; Edectime = early-filling deceleration time.

203

Table 4.3. M-mode-derived measures of Left Ventricular Dimension, Wall

Thickness, and Systolic Function

Variables Sham

N=13 OVX

N=9 OVX + BH4

N=10

Heart Rate, bpm

P <

251 ± 8 245 ± 12 237 ± 6

LVEDD, cm

P <

0.68 ± 0.02 0.68 ± 0.01 0.67 ± 0.02

LVESD, cm

P <

0.35 ± 0.02 0.37 ± 0.02 0.39 ± 0.02

RWTed, cm

P <

0.37 ± 0.03 0.71 ± 0.05

0.0001

0.51 ± 0.04*

0.0001

PWTed, cm

P <

0.18 ± 0.01 0.23 ± 0.01

0.0001

0.20 ± 0.01

AWTed, cm

P <

0.17 ± 0.01 0.20 ± 0.01 0.20 ± 0.02

%FS

P <

47 ± 1 46 ± 1 43 ± 2

Data are expressed as mean ± SEM. P values are compared to Sham. * P < 0.05

compared to OVX. LVEDD = left ventricular end-diastolic dimension; LVESD = left

ventricular end-systolic dimension; RWTed = relative wall thickness; PWTed =

posterior wall thickness at end diastole; AWTed = anterior wall thickness at end

diastole; FS = fractional shortening.

204

Figure 4.1.

Cardiac concentrations of total biopterins and BH4 in Sham (n=8), OVX (n=8), and

OVX+BH4 (n=7). Data are mean ± SEM; * P < 0.001 compared to Sham, #

P < 0.001

compared to OVX.

Sham OVX OVX+BH40

2

4

6

8

0.0

0.5

1.0

1.5

2.0Total Biopterins *

*

,#

BH4

To

tal

Bio

pte

rin

(pm

ol/

mg

)

BH

4(p

mo

l/kg

)

205

Figure 4.2.

Tail-cuff systolic blood pressures in Sham (n=13), OVX (n=9), and OVX+BH4 (n=10)

conscious mRen2.Lewis rats. Values are means ± SEM. * P < 0.01 compared to Sham.

Systolic Blood Pressure

6 7 8 9 10 11 12 13 14 15

100

125

150

175OVX + BH4

*OVX

Sham

Age (Weeks)

mm

Hg

206

Figure 4.3.

A. Myocardial relaxation indicated by early mitral annular velocity (e´) in Sham (n=13),

OVX (n=9), and OVX+BH4 (n=10) mRen2.Lewis rats. Data are mean ± SEM; # P < 0.05

compared to OVX. B. Early transmitral filling velocity – to – mitral annular velocity

ratio (E/e´) in Sham (n=13), OVX (n=9), and OVX+BH4 (n=10) rats. Data are expressed

as mean ± SEM; * P < 0.05 compared to Sham; # P < 0.05 compared to OVX.

e'

Sham OVX OVX + BH40

1

2

3

4

5

*

cm

/sec

E/e'

Sham OVX OVX + BH40

10

20

30

*#

A.

B.

207

Figure 4.4.

A. Quantification and representative picrosirius red staining revealing perivascular

collagen in the hearts of Sham (n=9), OVX (n=9), and OVX+BH4 (n=10) rats. Values

are mean ± SEM; * P < 0.05 compared to Sham; # P < 0.05 compared to OVX.

Magnification = 200x. B. Myocyte cross-sectional areas of cardiac myocytes from Sham

(n=9), OVX (n=9), and OVX+BH4 (n=10) mRen2.Lewis hearts. Data are mean ± SEM.

C. Relative wall thickness of Sham (n=13), OVX (n=9), and OVX+BH4 (n=10)

mRen2.Lewis rats. Data are mean ± SEM; * P < 0.05 compared to Sham; # P < 0.05

compared to OVX.

Perivascular Fibrosis

Sham OVX OVX + BH40.00

0.02

0.04

0.06 *

#

Co

llag

en

Co

nte

nt

(sta

ine

d :

to

tal

pix

els

)

Myocyte Cross-sectional Area

Sham OVX OVX + BH40

200

400

600

800

1000

1200

m

2

Relative Wall Thickness

Sham OVX OVX + BH40.0

0.2

0.4

0.6

0.8

208

Figure 4.5.

A. Quantification and representative images illustrating the effect of OVX and BH4

supplementation on cardiac levels of fluorescent 2-hydroxyethidium from

dihydroethidium (DHE) staining in Sham (n=8), OVX (n=8), and OVX+BH4 (n=8) rats.

Fluorescent images are shown at 400x magnification where red indicates DHE staining

and blue indicates nuclei. Data are mean ± SEM; * P < 0.05 compared to Sham; # P <

0.05 compared to OVX. B. Cardiac concentrations of nitrite as an indicator of nitric

oxide availability from Sham (n=8), OVX (n=8), and OVX+BH4 (n=8) rat hearts. Data

are mean ± SEM; * P < 0.05 compared to Sham; # P < 0.05 compared to OVX.

Dihydroethdium Fluorescence

Sham OVX OVX + BH40

20

40

60

80

100

*

#

Ave

rag

e G

ray S

ca

le I

nte

nsit

y

Cardiac Nitrite

Sham OVX OVX + BH40

100

200

300

400

500

*

#

nM

209

CHAPTER FIVE: SUMMARY AND CONCLUSIONS

Cardiovascular disease remains the leading killer of women in the United States

even though it was long believed to primarily affect men (1). Experts in the field claim

that one in two women will die due to heart disease or stroke compared to one in twenty-

five that will succumb to breast cancer (2). There is not one precise reason why in the

past there existed such a blatant failure to recognize the substantial number of women

suffering from heart disease, hypertension, or strokes (3-9). One possibility is that the

onset of disease appears approximately ten years later in women than it does in men

(7;10;11). Additionally, the symptom profile varies dramatically between men and

women, whereby the symptoms in women are frequently disregarded as an alternative

ailment. Even caveats in the Framingham risk stratification lead to underestimation of

CVD risk in female patients (12;13). Unfortunately in the past, it has been stated that

women received less aggressive treatment for known heart disease and received fewer

diagnostic exams, which consequently led to exacerbated disease and poor prognosis

(4;14). This coincides with the observation that a detailed search of the PubMed database

on experimental publications showed a much higher proportion of male animal studies

compared to female models. Cardiovascular sex differences are not unique to humans, as

our efforts using the estrogen-sensitive mRen2.Lewis model demonstrates remarkable

differences in cardiac structure and function as previously discussed, even in relatively

young animals (15).

Fortunately, for the past decade extensive efforts have been made and are

continuing to dissect the complexities of the key physiologic systems involved in

cardiovascular health. A correlation between heart disease and estrogens was recognized

210

as far as the 1960s when estrogen therapy was considered by Robinson and Cohen (16-

19), for male patients with coronary heart disease following their observation of

estrogenic-protection in female patients. By the late 80s and early 90s, there seemed to

be an ubiquitous acceptance that the cardioprotective effects of estrogens could be

extended in postmenopausal women using hormone replacement therapy (HRT) (20-22).

We now know from the Women‟s Health Initiative (WHI) (23;24) and HERS (25-27)

studies that the answer is simply not to replace estrogens. The WHI is a 15-year

composite research program that was designed to address cardiovascular disease, cancer,

and osteoporosis in more than 160,000 postmenopausal women in terms of quality of life,

disability, and death. The program was divided into three main categories of

intervention: dietary modifications, vitamin D/calcium, and hormone replacement

therapy (HRT). The most interesting and surprising outcome was that in both HRT

groups, comprised of more than 27,000 participants receiving either conjugated equine

estrogen or conjugated equine estrogen plus medroxyprogesterone, there was an

increased risk for myocardial events and strokes to the extent that both intervention arms

of the study were halted early (24). Similarly, the HERS study found an increased risk

for thromboembolic events in a population of more than 2,700 postmenopausal women

with established coronary disease taking oral conjugated equine estrogen plus

medroxyprogesterone acetate (26;27). Fortunately, it does not appear that the increased

cardiovascular risks, including myocardial infarction, stroke, pulmonary embolism, and

veinous thrombosis risks persist once the hormone replacement was stopped in the WHI

study based on postintervention data 3 years in the estrogen plus progestin arm (28) and 5

years in the estrogen-only arm of the trial (29). Nevertheless, the data from WHI and

211

HERS underscore the need for further understanding of the dynamic relationship between

estrogens and the cardiovascular system in order to ascertain the appropriate composition

of estrogens, source, dosage, and the therapeutic window for perimenopausal women

(30;31).

I. Summary of Findings

Initial studies described in Chapter II characterized the effects of hypertension

and high tissue expression of renin angiotensin system components in male mRen2.Lewis

rats. Though there were published reports including assessment of hemodynamic data

and cardiac RAS components (32;33), specific data on the impact and characteristics of

the hypertension on cardiac function did not exist. To close this gap, male mRen2.Lewis

rats were followed from 5 weeks of age through the fifteenth week of life. At 15 weeks

of age, these rats exhibited severe hypertension, had left ventricular hypertrophy,

impaired ventricular relaxation, and augmented filling pressures, similar to what had been

observed in the parent strain (83). A parallel cohort of congenic male mRen2.Lewis rats

received dual AT1 receptor blockade and ACE inhibition at 5 weeks of age. The

combination therapy essentially prevented the development of extreme hypertension,

characteristic of the male mRen2.Lewis, resulting in the males having blood pressures

more similar to their female counterparts. The effects of this chronic Ang II blockade

and maintenance of lower blood pressure were associated with preservation of diastolic

relaxation, reduced filling pressures, and prevention of LV hypertrophy. Interestingly,

the beneficial effects of inhibition of Ang II synthesis and activity had no effect on

cardiac collagen deposition despite the substantial difference in blood pressure between

treated and untreated mRen2.Lewis rats. This finding in male mRen2.Lewis rats

212

contrasts with data reported in female mRen2.Lewis rats. In this study, oophorectomy

induced robust increases in cardiac perivascular collagen that were associated with

exacerbation of hypertension (34). We conclude in Chapter II that the lusitropic

protection of the combined renin angiotensin system blockade in male mRen2.Lewis rats

is derived from changes in vascular hemodynamics and/or improvements in calcium

homeostasis. These observations in the male were thought provoking as the data provide

yet another example of sex-based variation in the mRen2.Lewis model. These findings

may contribute to further the explanation of sex-differences in the etiology of diastolic

dysfunction.

Appreciating that similar mechanisms, governing the intricacies of estrogenic

cardioprotection, may be disrupted in diastolic dysfunction given its prevalence in

postmenopausal women, we characterized in Chapter III the effect of ovarian hormone

loss on cardiac function and structure in intact and OVX female mRen2.Lewis rats. This

was done in conjunction with selective nNOS inhibition due to the previous observations

that L-VNIO, given for 4-wks to OVX mRen2.Lewis rats, reversed salt-induced renal

damage and proteinuria, and indications that nNOS was elevated in cardiac and renal

tissues following OVX (35). Again, we confirmed that nNOS was increased in the heart

of OVX mRen2.Lewis rats. We observed that selectively inhibiting nNOS reduced

filling pressure and improved myocardial relaxation independent of significant effects on

blood pressure. These findings suggest that nNOS inhibition facilitates expression of

relaxant mechanisms that allow the heart to resume proper filling, which may be more

critical in this female model than direct effects on calcium handling since we did not

observe significant changes in SERCA2 (36). This is in contrast to the data in males

213

from Chapter II where we report that diastolic abnormalities were associated with

diminished SERCA2 activity without significant alterations in cardiac fibrosis (15).

Diastolic dysfunction in this model, similar to humans, is defined as a diminished

ability of the heart to properly relax, or a decrease in the distensibility of the

myocardium. The functional improvements in diastolic function with L-VNIO were

accompanied by decreases in perivascular collagen and cardiac hypertrophy, which

suggest a critical role of nNOS and/or its products NO or ROS in the progression of

myocardial remodeling. These findings support an alternative explanation to the

beneficial effects of nNOS inhibition whereby L-VNIO accelerates reverse remodeling of

the extracellular matrix. These alterations in collagen deposition are quite robust, but it is

not unusual for oxidant therapy such as a combination of vitamin C and E or fish oil

derivatives to either prevent or reverse fibrosis in rodent models (37;38). However,

duplicating the responses in humans is more difficult due to variations in lifestyle,

environment, and compliance (39). The relationship between the diastolic dysfunction

and perivascular fibrosis in the OVX female mRen2.Lewis is likely due to the decrease in

blood distribution throughout the myocardium as it is well understood that perivascular

fibrosis is in fact microvascular disease (40). Numaguchi et al. (41) reported that in

normotensive WKY rats, insufficient NO availability caused coronary microvascular

remodeling and cardiac hypertrophy through pressure-independent mechanisms.

Additionally, the presence of perivascular fibrosis increases intracoronary vascular

resistance, which can affect diastolic function by reducing the amount of blood entering

the myocardium during diastole. In keeping with this interpretation, there is significant

evidence of reduced myocardial blood flow reserve in hypertensive subjects (42;43).

214

Moreover, interstitial fibrosis will further impair diffusion of oxygen causing a chronic

state of hypoperfusion of the myocardium.

As previously discussed, the composition of the myocardial ECM includes

fibrillar proteins, proteoglycans, and basement membrane. It is well known that these

constituents are involved in diastolic dysfunction because pathologies that disrupt

diastolic function also change the composition of the ECM and collagen turnover

stabilization improves diastolic function, while experimentally provoked collagen

changes alter diastolic function (44-59). Fibrillar collagen homeostasis is influenced by

processes accounting for procollagen biosynthesis, post‐synthetic procollagen processing,

and fibrillar collagen degradation. In rabbit cultured vascular smooth muscle cells

Kolpakov et al. (60) showed that the NO-generating compounds S-nitroso-N-

acetylpenicillamine (SNAP) and sodium nitroprusside inhibited collagen synthesis in a

dose-dependent manner. Furthermore, in cultured mesangial cells SNAP suppressed the

expression and activity of TGF-β1 and the glucose-mediated increase in collagen

deposition (61). The shift in the nNOS activity induced by ovariectomy in part through

increased production of ROS may favor increased collagen synthesis or deposition.

These findings contrast with what we found in male mRen2.Lewis hypertensive rats

where blockade of expression or activity of Ang II improved ventricular lusitropic action

without altering the presence of cardiac fibrosis (15). The differences in the

hemodynamic and cardiac remodeling effects obtained in intact male mRen2.Lewis and

female mRen2.Lewis ovariectomized rats in terms of the cardiac collagen response

suggests that estrogens may be critical factors in regulating the effects of expression or

215

activity of NO on factors that modulate the synthesis or turnover of cardiac collagen.

These mechanisms appear to be independent of arterial blood pressure.

Another possible contributor to the collagen characteristics of the OVX

mRen2.Lewis females could be aldosterone, which has been identified as a profibrotic

factor. The critical studies completed by Weber and colleagues (62-64) showed that

infusions of exogenous aldosterone as well as experimental models of increased

aldosterone such as renovascular hypertension were associated with significant increases

in both perivascular and interstitial fibrosis within the left ventricle. Additionally, it was

later demonstrated in cultured adult rat cardiac fibroblasts that aldosterone stimulates

collagen synthesis (65). Antagonism of aldosterone receptors using spironolactone (66)

or eplerenone (67), results in a decrease of cardiac fibrosis in rat models. Clinically,

aldosterone receptor antagonism reduced morbidity and mortality in the Randomized

Aldactone Evaluation Study (RALES) and Eplerenone Post-Myocardial Infarction Heart

Failure Efficacy and Survival Study (EPHESUS) Trial independent of blood pressure

alterations (68;69). We know that OVX female mRen2.Lewis rats have significantly

higher circulating levels of aldosterone compared to their intact counterparts (34).

However, data comparing male and female mRen2.Lewis rats to normotensive Lewis rats

showed no differences in serum aldosterone (33). Therefore it could be that either

estrogen alone or the estrogenic effect on aldosterone could contribute to collagen

regulation in the OVX rats. Indeed, it has been shown that estrogen therapy decreases

adrenal aldosterone production following ovariectomy (70). These observations may

pave the way for future studies that will address more directly the mechanism by which

cardiac fibrosis results from the interplay between aldosterone and estrogens. With the

216

availability of selective mineralocorticoid receptor antagonists, these questions could

begin to be addressed. The confirmation of a positive interaction may broaden the use of

aldosterone receptor antagonists clinically given the fact that eplerenone appears to

exhibit enhanced tolerability compared to spironolactone, which had a less favorable side

effect profile due to its effects on potassium and gynecomastia.

A potential caveat of our model is that following oophorectomy, the female

mRen2.Lewis rats are left not only with depleted estrogens, but depleted progesterone

and possibly altered levels of estrone. Our studies specifically measured estradiol upon

sacrifice of the rats, which leaves us with the ability to only speculate on the possible role

of progesterone and estrone. This may call into question the contribution of progesterone

to cardiac structural and functional changes, because indeed progesterone receptors have

been detected in the heart (71;72). However, current data regarding the role of

progesterone are conflicting as some investigators report beneficial actions of

progesterone (73-75), while others document negative consequences of progesterone

therapy (76;77). Therefore, a critical role for progesterone influencing the outcome of

our studies is not supported by the current literature, where in the heart, progesterone was

not shown to affect NOS though estrogen led to stimulation of the enzyme (78).

Regarding estrone, since both the ovaries and adipose tissue secret estrone, ovariectomy

alone should not completely deplete estrone levels (79-81). Postmenopausal women

exhibit decreased levels of estrone, but to a lesser degree than estradiol (82). While

estrone itself is deemed to have limited biological activity, its conversion to estradiol via

estrone sulfate is the basis of considering its relevance during presumed estrogen

depletion. In contrast to estradiol, estrone is derived from aromatase activity on

217

androstenedione instead of testosterone by aromatase as is the case with estradiol.

Estrone is then converted to estrone sulfate, which becomes a storage molecule that can

be converted to estradiol as needed. Our data show that estradiol remains at the lower

limit of the assay‟s detectability so it seems that estrone is not playing a significant role in

this model or at least it is not substantially contributing to non-ovarian estradiol

production from sources such as adipose tissue. Highlighting the impact of estradiol on

fibrosis are data that show exposure to 17β-estradiol in female oophorectomized mice

reversed Ang II-induced hypertension associated with a substantial inhibition of

ventricular interstitial cardiac fibrosis (83). Since the beneficial effects of estrogen

replacement in this mouse model were attributed to inhibition of Ang II-induced

calcineurin activity, Pedram et al. (83) suggested that estrogens‟ stimulation of NO

production may play a critical role in the expression of cellular signaling mechanisms

that contribute to, or markedly regulate, cardiac interstitial collagen deposition. While

our studies are in general agreement with these conclusions, we show that estrogens have

a specific modulatory role in the expression of cardiac nNOS which acts as a profibrotic

agent in part through facilitating the generation of ROS. The data obtained from our

studies further suggest that the regulatory products resulting through NOS activity can

have selective, and as indicated by our findings, opposing effects on cardiac remodeling

and collagen deposition.

Though traditionally, nNOS was found to release NO, and NO characteristically

provides cardiovascular protection, more recently data shows that NOS enzymes exhibit

the ability to generate ROS in some pathological disease states (84-87). The actual ROS

that can be generated by NOS not only include the production of the rapid superoxide

218

intermediate (88), but peroxynitrite and in some situations, hydrogen peroxide (89).

Peroxynitrite has been shown to be the primary culprit leading to oxidative damage and

pathophysiological consequences (87;90;91). In a recent study by Silberman et al. (86),

nNOS uncoupling in a mouse model of diastolic dysfunction was shown to be primarily

responsible for increased cardiac oxidation. This phenomenon of “uncoupling” is the

most probable explanation of the paradoxical increase in nNOS following OVX in the

mRen2.Lewis. Indeed, we demonstrated increases in ROS generation, detected as

significant increases in fluorescent intensity of the 2-hydroxyethidium product following

dihydroethidium (DHE) staining in the heart tissue. These changes were associated with

a decrease in the overall content of cardiac NO in the OVX mRen2.Lewis, which were

reversed following chronic administration of L-VNIO. The uncoupling of nNOS

following OVX, would lead to a shift from the adaptive (NO-producing) NOS to a

maladaptive NOS (reactive oxygen species or ROS-producing). Uncoupling of

enzymatic eNOS has been vigorously described (87;92), but only recently has attention

been given to nNOS (86;93-95). This is likely due to the plethora of research activity

examining NOS, specifically eNOS activity in the vasculature in response to the growing

field of endothelial dysfunction. Our present data do not confirm if indeed nNOS is

uncoupled, which necessitates future studies that can determine if there is a

predominance of nNOS monomers following OVX. Attempts to do this using SDS-

PAGE and western blots have not been successful as the antibodies are not sufficiently

specific. However, there are reports detailing monomer to dimer interconversion of

eNOS using gel filtration chromatography (96;97) that may offer an alternative to

assessing nNOS monomers.

219

Until recently and prior to the localization of nNOS to myocytes, the significance

of nNOS within the heart was restricted to the investigation of transduction signalling,

heart rate control, and atrial localization (98-101). Regulation of nNOS in the heart

occurs through various means including protein phosphorylation, redox mechanisms, and

protein-protein interactions (102). Efforts by White and colleagues have clearly

demonstrated that nNOS is a critical component of coronary artery vascular smooth

muscle cells and it is through stimulation of nNOS that estrogens can either produce

vasodilation or vasoconstriction depending upon the coupled status of the enzyme (103).

This is in agreement with our data, which shows alterations in cardiac nNOS protein in

the absence of any changes in total or phosphorylated eNOS within the hearts of female

mRen2.Lewis

We can only speculate on the contribution of the decreased NO and/or increased

ROS toward OVX-elicited cardiac remodeling and diastolic derangement. Indeed, we

know from data in the mRen2.Lewis parent strain, [mRen2]27, that blockade of

mineralocorticoid receptors diminishes oxidative stress, which in turn attenuates the

collagen deposition in the heart, which was associated with diastolic dysfunction in male

rats (104). Similarly, cardiac oxidative stress due to Ang-II stimulation from the

activation of the renin angiotensin system in the [mRen2]27 model induces structural and

functional changes within the heart (105). We do not know if these same observations

hold true in the mRen2.Lewis rats. Aside from the potential consequences of an activated

renin angiotensin system, which is known to at least uncouple eNOS within the

vaculature (106), it is possible that in the present studies the diminished NO availability

provoked further collagen deposition. NO has been shown to regulate MMPs in the heart

220

(107;108), which are responsible for the intricate balance of collagen turnover. NO

appears to modulate this activity through activation of ERK and Akt pathways (109).

Alternative to direct NO actions, ROS have been implicated in collagen synthesis through

mediating MMP induction or stimulation, decreasing TIMP levels, and stimulating

collagen synthesis (110-112). Indeed, MMPs have been shown to be increased in age-

related diastolic dysfunction (113;114). Since decreased NO and increased oxidative

stress augments MMP release (109;115;116), these findings provide an additional

mechanism to account for the cardiac fibrosis found in the OVX mRen2.Lewis female

rat.

Our research progressed to exploring one specific identified phenomenon in the

OVX mRen2.Lewis rat based upon the blockade of nNOS improving diastolic function

and reversing cardiac fibrosis. Thus, Chapter III observations lead to the hypothesis that

the increase in ROS and decreased NO within the hearts of OVX mRen2.Lewis rats was

due to the lack of estrogenic regulation of BH4 synthesis. BH4, the necessary cofactor

for NO-producing NOS activity, is synthesized from either its de novo pathway from the

precursor GTP (117) or through its salvage pathway (118), which generates BH4 from

7,8-dihydrobiopterin (BH2) or sepiapterin using dihydrofolate reductase and sepiapterin

reductase, respectively (118;119). The rate limiting step toward BH4 synthesis is

conversion of GTP to dihydro neopterin triphosphate by the enzyme GTPCH.

Interestingly, GTPCH is regulated by estrogens (120-122), which provides the possibility

that in the OVX mRen2.Lewis rat, the depletion of estrogens impairs the synthesis of

BH4 through the lack of regulation on GTPCH. This would lead to uncoupling of NOS,

221

which given the elevations in nNOS protein expression and benefits of L-VNIO shown in

Chapter III, is most likely specifically the nNOS isoform.

In Chapters III and IV, we measured the cardiac tissue levels of BH4, which were

indeed found to be suppressed following OVX. The BH4 deficiency following depletion

of ovarian hormones was associated with impaired diastolic function and LV remodeling,

accompanied by the expected increases in ROS and decrease in tissue NO. BH4

supplementation restored the NO levels, while diminishing the ROS generation.

Additionally, BH4 improved cardiac function and reversed collagen deposition. The

mechanism(s) governing the cardioprotective role of BH4 are not completely clear and

until there is a better understanding as of how NOS, specifically in our model, nNOS,

becomes uncoupled in the first place, we can only speculate on the possibilities. As

previously mentioned, one scenario is that estrogen-depletion leads to decreased GTPCH,

which consequently diminishes BH4 since the rate-limiting step in the cofactor‟s

synthesis is GTPCH. Despite the presence of an alternative pathway for the generation of

BH4 (119), the “salvage” route may not provide for adequate compensation for the

reduction in GTPCH since BH4 levels remained significant low in the OVX rats, despite

having presumably functional components of the salvage pathway. Another possibility is

that our observations are a direct estrogen effect in that estrogens themselves afford the

cardioprotection, likely through their antioxidant properties. Once the estrogens are

removed, BH4, which is susceptible to oxidation, maybe become oxidized by the reactive

oxygen species leading to nNOS uncoupling and subsequent increase in O2- generation

(84). A third possibility is that the imbalance of NOS protein and BH4 may actually lead

to uncoupling of the nNOS isoform as the intricate relationship between the enzyme and

222

cofactor has been clearly documented such that if the balance is tilted, uncoupling occurs

(123). Finally, it is very likely that the benefit of BH4 supplementation is purely based

on the reduction in ROS generation. Since selective inhibition of nNOS with L-VNIO as

well as BH4 supplementation diminished ROS production, while only BH4 restored NO

levels, it seems plausible that excessive ROS release may be the driving force behind not

only the functional derangement, but the structural remodeling in the mRen2.Lewis

following OVX. It may be that adequately inhibiting ROS can prevent or reverse

diastolic function. Indeed, in multiple scenarios of ROS-provoked fibrosis both cardiac

function and structure are significantly impacted whether it be in terms of dysfunction

through sarcolemmal excitation-contraction coupling (124), contractility (125) and stress-

sensitive signaling proteins (126;127) or structure through provoked inflammation and

fibrosis by stimulating proinflammatory pathways (128;129). Inhibiting ROS can even

elicit profound effects on heavily cross-linked collagen. For example, radiation-induced

fibrosis typically results from activation of TGFβ leading to extracellular matrix

formation and fibroblast proliferation through its downstream target, connective tissue

growth factor (130). Animal‟s studies have shown that targeting oxidative stress can

ameliorate cardiac fibrosis following radiation therapy (131;132).

The studies described in this dissertation were performed in an experimental

model of tissue renin overexpression, a factor that needs to be considered as Ang II has a

definitive role in mediating oxidative stress. The sexual dimorphism of blood pressure

and target organ damage described in [mRen2]27 implicated a direct influence of

estrogen in the adaptation of the heart to the stimulus of increased renin angiotensin

system activity. Thus it would be remiss to ignore the potential interplay between the

223

renin angiotensin system and the cardiac nitric oxide system in contributing to our

findings. Expression of the renin gene in the heart of the mRen2.Lewis‟ parent strain, the

transgenic [mRen2]27 hypertensive rat, has been documented and this is associated with

high prorenin levels (133). The presence and potential functionality of prorenin in

experimental models of hypertension, diabetes, and human disease has been extensively

documented by us (134) and others (135-138) previously. In the study reported by

Langheinrich et al. (137) in [mRen2]27 transgenic hypertensive rats, unchanged or even

suppressed concentrations of active renin, Ang I, and Ang II were associated with very

high concentrations of circulating prorenin. As reviewed by Paul (139) tissue expression

of renin, such as in the heart, interacts with the blood-borne renin angiotensin system in

modulation of organ function. The more recent demonstration of the existence of a

prorenin/renin receptor (Pro/renin receptor) in tissues including the heart and the kidneys

has provided a new level of understanding as to the potential role of renin in inducing

intracellular formation of Ang II (140-145). The demonstration that isolated adult

cardiomyocytes internalize unglycosylated prorenin, which is followed by the generation

of angiotensins, provides direct evidence for an intracrine mechanism for Ang II

synthesis (133). Importantly, this pathway is apparently functional in [mRen2]27 rats as

in Peter‟s studies they showed enhanced levels of unglycosylated renin within

intracellular compartments in the heart (133). It is reasonable to expect that the same

characteristics would be present in the mRen2.Lewis rat as they were derived from the

same strain and the data currently available have not identified marked differences in the

two models. While the studies described in this dissertation did not address a direct role

of renin, prorenin, and the Pro/renin receptor on the observed anti-fibrotic effects of BH4

224

supplementation, it is pertinent to note that the flanking region of human and rat renin

genes contain regulatory sequences of glucocorticoid, estrogen, progesterone, and cAMP

(146-149). In support of the possibility that estrogen can directly affect the expression

and transcription of the renin gene, Mansego et al. (150) reported that polymorphisms of

the renin gene were associated with blood pressure levels and risk of hypertension in

women over 40 years old. Further studies are needed to explore the potential interactions

between NO and renin gene expression.

Our results have the potential to add to the treatment repertoire or therapeutic

approach for diastolic dysfunction. The viability of BH4‟s clinical potential is based

upon its current usage in treating phenylketonuria (PKU) in patients that have the

inability to metabolize phenylalanine due to a genetic defect in phenylalanine

hydroxylase or inadequate amounts of BH4 (151;152). BH4 supplementation elicited

favorable responses in patients by reducing the circulating phenylalanine levels with

minimal side effects (153-156). Though promising, further studies are required to

examine the long-term toxicity of BH4 since the drug has not been used chronically in

patients. The use of BH4 in an effort to restore diastolic function and limit

cardiovascular remodeling holds promise, particularly in light of the current debate

surrounding hormone replacement therapy.

Some potential limitations of our model that merit further discussion is that just as

we do not know the status of progesterone and estrone following ovariectomy, it is not

known what impact dietary estrogen consumption has on our observations. One would

think that the phytoestrogens available in the standard rat chow diet would actually

improve or offer a degree of cardioprotection. It may be that if we were to eliminate the

225

extraneous sources that could augment estrogens, the diastolic and cardiac remodeling

phenotype may worsen. Nevertheless, this issue underscores the necessity of including

not only controls in terms of estrogen status, but also control subjects in terms of

treatment. In a separate collaborative study looking at cynomolgus monkeys whose diet

was casein based instead of standard soy based diet, the diastolic phenotype persisted. In

fact, estradiol supplementation following OVX in these middle-aged monkeys resulted in

small, but significant improvements in diastolic function (157). We do not yet know the

structural phenotype, but in terms of function, the monkey data are similar to our

observations in the mRen2.Lewis. Whether or not these observations would exist in the

presence of an alternative diet remains to be determined.

A potential second limitation of our studies is that our model is an angiotensin II-

dependent model of hypertension with an activated renin angiotensin system. Therapies

that block the activity of the renin angiotensin system have not shown significant benefit

in terms of diastolic dysfunction. However, there are no long-term studies, in which

morbidity and mortality have been specifically evaluated in patients with pure diastolic

dysfunction as the clinical data currently available is based upon subjects with heart

failure with preserve systolic function (158). Furthermore, the CHARM study did not

specifically investigate outcomes as a function of sex a point that is of considerable

importance given the fact that in the SENIORS Trial the greatest benefits with the novel

beta blocker, nebivolol, were found in female heart failure patients (159). Our model is

not a model of heart failure and exhibits only the mechanical abnormalities that constitute

diastolic dysfunction.

226

Finally, we previously discussed the limitation of knowing if nNOS

monomerization participated in these outcomes. Another caveat to mention is that we do

not know the zinc status following ovariectomy in our model. Similar to BH4 being

required for nNOS to produce NO, the Zn2+

is an additional requirement for normal NOS

activity. In fact, within cardiac myocytes, Zn2+

can enhance antioxidant properties by

upregulating metallothionein 1 and through activating metal-responsive transcription

factor 1 (160-163). Any alteration in Zn 2+

availability following OVX could impact

overall release of ROS in our model.

II. Concluding Remarks

In summary, the key findings outlined in this dissertation illustrate that in

estrogen-depleted mRen2.Lewis rats, inhibition of nNOS leads improved diastolic

function through diminished ROS generation and attenuated LV remodeling.

Furthermore, this work characterized the deficiency in myocardial BH4 and NO

availability following OVX. Supplementation of BH4 restores cardiac NO release and

attenuates ROS generation while reversing the OVX-induced exacerbations in diastolic

dysfunction and limiting cardiac remodeling. This evidence adds to our current

knowledge and understanding regarding the mechanisms responsible for cardiac diastolic

dysfunction, and what link estrogens have that influence the pathophysiological process.

The contrasting effects between male and female mRen2.Lewis rats in terms of

hemodynamic responses and collagen deposition stresses further the differential

mechanisms regulating cardiac remodeling and diastolic function.

227

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253

CURRICULUM VITAE

JEWELL A. JESSUP

BORN: May 25, 1980

Mount Airy, North Carolina

EDUCATION:

2005-Present Wake Forest University Graduate School of Arts and Sciences

Winston-Salem, North Carolina

Doctor of Philosophy

Graduate Program: Physiology and Pharmacology

Laboratory Advisor: Leanne Groban, M.D.

Department of Anesthesiology

1998-2002 Salem College

Winston-Salem, North Carolina

Bachelor of Science, Biology

1997-1998 Surry Community College

Dobson, North Carolina

Certified Nursing Assistant, North Carolina CNA licensee

EMPLOYMENT:

2005-Present Graduate Fellow

Physiology and Pharmacology Graduate Program

Wake Forest University School of Medicine

Winston-Salem, North Carolina

2009 - Present Executive Secretary / Webmaster

Inter-American Society of Hypertension

Winston-Salem, North Carolina

Website: www.iashonline.org

2007 - 2008 Editorial Assistant

Therapeutic Advances in Cardiovascular Disease

Sage Publications, Inc.

London, England

2006 - 2007 Program Coordinator

Inter-American Society of Hypertension

XVIIth

Scientific Sessions held in Miami, Florida

254

2002 – 2005 Laboratory Technician III

The Hypertension & Vascular Disease Center

Wake Forest University Health Sciences

Winston-Salem, North Carolina

2002 - 2002 Research Laboratory Technician

Piedmont Triad Community Research Center

Winston-Salem State University

Winston-Salem, North Carolina

2001 - 2002 Research Laboratory Student Internship

The Hypertension & Vascular Disease Center

Wake Forest University School of Medicine

Winston-Salem, North Carolina

2002 Student Intern

Department of Pathology/Forensics and Autopsy Services

Wake Forest University Baptist Medical Center

Winston-Salem, North Carolina

2001 American Physiological Society Undergraduate Research Fellow

The Hypertension & Vascular Disease Center

Wake Forest University School of Medicine

Winston-Salem, North Carolina

2000 - 2001 Research Laboratory Student Intern-Independent Study

The Hypertension & Vascular Disease Center

Wake Forest University School of Medicine

Winston-Salem, North Carolina

2001 Student Intern

Department of Pediatric Hematology/Oncology

Brenner Children‟s Hospital

Winston-Salem, North Carolina

2000 Research Laboratory Assistant

The Hypertension & Vascular Disease Center

Wake Forest University School of Medicine

Winston-Salem, North Carolina

2000 Student Intern

The Hypertension & Vascular Disease Center

Wake Forest University School of Medicine

Winston-Salem, North Carolina

255

1999 Research Laboratory Assistant

The Hypertension & Vascular Disease Center

Wake Forest University School of Medicine

Winston-Salem, North Carolina

1997 Research Laboratory Assistant

Pulmonary and Critical Care Medicine

Bowman Gray School of Medicine

Winston-Salem, North Carolina

HONORS AND AWARDS:

2009 American Federation of Aging Research‟s GE Healthcare Junior

Investigator Award for Excellence in Aging-Heart Research,

AFAR Scientific Conference- The Aging Heart: A Roadmap to

Cardiac Independence, New York, New York

2009 American Physiological Society Travel Award APS Conference,

Sex Steroids and Gender in Cardiovascular-Renal Physiology and

Pathophysiology, Broomfield, Colorado

2009 Wake Forest University Graduate School of Arts of Sciences

Alumni Student Travel Award, APS Conference, Sex Steroids and

Gender in Cardiovascular-Renal Physiology and Pathophysiology,

Broomfield, Colorado

2008 American Heart Association Council for High Blood Pressure

Research Young Investigator Travel Award, VIIth

International

Symposium on Vasoactive Peptides, Ouro Preto, Brazil

2007 Wake Forest University Graduate School of Arts of Sciences

Alumni Student Travel Award, 61th

Annual American Heart

Association Council for High Blood Pressure Research

Conference, Tucson, Arizona

2006 Wake Forest University Graduate School of Arts of Sciences

Alumni Student Travel Award, 60th

Annual American Heart

Association Council for High Blood Pressure Research

Conference, San Antonio, Texas

2001 American Physiological Society Undergraduate Research Fellow

Functional Assessment of Kidney Angiotensin Receptors

Hypertension and Vascular Research Center, Wake Forest

University School of Medicine, Winston-Salem, North Carolina

256

2001 First Place in Research Category, Salem College Research Day,

“AT1 Receptor Inhibition in the Solitary Tract Nucleus”

Third Place in Clinical Category, Salem College Research Day,

“Pediatric Oncology at Brenner Children‟s Hospital”, Salem

College, Winston-Salem, North Carolina

2000 First Place in Research Category, Salem College Research Day,

“AT1 Receptor Inhibition and the Baroreceptor Reflex”, Salem

College

Winston-Salem, North Carolina

2000 Induction into the Salem College Premedical Honor Society,

Alpha Epsilon Delta, Salem College, Winston-Salem, North

Carolina

2000 Induction into the Salem College Chapter of the National

Biological Honor Society, Beta Beta Beta, Salem College,

Winston-Salem, North Carolina

PROFESSIONAL AFFILIATIONS:

2007-present American Society of Pharmacology and Experimental

Therapeutics

2006-present Inter-American Society of Hypertension

2006-present American Heart Association

2001-present Consortium for Southeastern Hypertension Control

2001-present American Physiological Society

COMMITTEES:

2006 - 2007 Physiology/Pharmacology Representative

Graduate Student Association

Wake Forest University Graduate School of Arts & Sciences

Winston-Salem, North Carolina

PROFESSIONAL DEVELOPMENT:

2009 American Federation of Aging Research Scientific Conference -

The Aging Heart: A Roadmap to Cardiac Independence, New

York, New York

“GPR30 Receptor Activation Improves Cardiac Function in Intact

Female mRen2.Lewis Rats” (poster)

2009 Inter-American Society of Hypertension XVIIIth

Scientific

Sessions, Belo Horizonte, Brazil, “GPR30 Receptor Activation

257

Improves Cardiac Function in Intact Female mRen2.Lewis Rats”

(poster)

2009 American Physiological Society Conference, Sex Steroids, and

Gender in Cardiovascular-Renal Physiology and Pathophysiology,

Broomfield, Colorado, “GPR30 Receptor Activation Improves

Cardiac Function in Intact Female mRen2.Lewis Rats” (oral and

poster)

2009 Invited presentation, Joint Meeting of the German Societies for

Pharmacology and Clinical Pharmacology and the British

pharmacological Society - New Drugs in Cardiovascular Research,

Dresden, Germany “Counterbalancing the Effect of Estrogen on

Nitric Oxide Dependent Mechanisms: Interactions with the Renin

Angiotensin System” (invited lecture)

2008 American Heart Association‟s Basic Cardiovascular Sciences

Conference 2008, Keystone, Colorado, “Combination RAS

Blockade Improves Diastolic Function Without Affecting Cardiac

Fibrosis or the Cardiac RAS in the Hypertensive mRen2.Lewis

Rat” (poster)

2008 International Society of Heart Research - 2008 North American

Section Meeting, Cincinnati, Ohio, “Combination RAS Blockade

Improves Diastolic Function Without Affecting Cardiac Fibrosis or

the Cardiac RAS in the Hypertensive mRen2.Lewis Rat” (poster)

2008 VIIth

Biennial International Symposium on Vasoactive Peptides,

Ouro Preto, Brazil, “Localization of the Novel Angiotensin

Peptide, Proangiotensin-12 [Ang-(1-12)], in the Heart and Kidney

of Hypertensive and Normotensive Rats” (poster)

2007 15th

Annual Division of Surgical Sciences Fellows‟ and Students‟

Research Day, Wake Forest University School of Medicine,

Winston-Salem, North Carolina, “Localization of the Novel

Angiotensin Peptide, Proangiotensin-12 [Ang-(1-12)], in the Heart

and Kidney of Hypertensive and Normotensive Rats” (poster)

2007 61th

Annual Fall Conference and Scientific Sessions of the Council

for High Blood Pressure Research, Tucson, Arizona, “Localization

of the Novel Angiotensin Peptide, Proangiotensin-12 [Ang-(1-12)],

in the Heart and Kidney of Hypertensive and Normotensive Rats”

(poster)

2006 60th

Annual Fall Conference and Scientific Sessions of the Council

for High Blood Pressure Research, San Antonio, Texas, Impaired

258

Counter-regulatory Response of Angiotensin Converting Enzyme 2

(ACE2) and Angiotensin-(1-7) [Ang-(1-7)] in the Heart of a New

Congenic Model of Hypertension Derived from [mRen2]27

Transgenic Rats” (poster)

2005 American Society of Hypertension 20th

Annual Scientific Meeting

and Exposition, San Francisco, California, “ACE2 Gene

Expression is Differentially Regulated in the Kidney and Heart of

Normotensive Rats” (oral and poster)

2003 11th

Annual Division of Surgical Sciences Fellows‟ and Students‟

Research Day, Wake Forest University School of Medicine,

Winston-Salem, North Carolina, “Effects of Angiotensin II

Receptor Antagonist and Statin on the Renin Angiotensin System

After Myocardial Infarction” (poster)

2002 Federation of American Societies for Experimental Biology

Annual Experimental Biology Meeting, New Orleans, Louisana,

“Characterization of Angiotensin AT1 Receptors in Fetal Sheep

Brain During Development” (poster)

2001 9th

Annual Division of Surgical Sciences Fellows‟ and Students‟

Research Day, Wake Forest University School of Medicine,

Winston-Salem, North Carolina, “Characterization of Angiotensin

AT1 Receptors in Fetal Sheep Brain During Development”

(poster); “AT1 Receptor Inhibition in the Solitary Tract Nucleus by

Antisense Oligonucleotides versus an AT1 Antagonist in

Transgenic Hypertensive Rats” (poster)

2001 Consortium for Southeastern Hypertension Control, 8th

Annual

National Scientific Session, Charleston, South Carolina,

“Functional Assessment of Kidney Angiotensin Receptors”

(poster); “Effect of Pre-versus Postsynaptic Inhibition of the

Nucleus Tractus Solitarii AT1 Receptors on Cardiac Baro- and

Chemoreflexes” (poster)

2001 Federation of American Societies for Experimental Biology

Annual Experimental Biology Meeting, Orlando, Florida, “Effect

of Pre-versus Postsynaptic Inhibition of the Nucleus Tractus

Solitarii AT1 Receptors on Cardiac Baro- and Chemoreflexes”

(poster)

2001 Women in Science Exchange Conference (WISE), Salem College

Women in Science and Mathematics Program, Winston-Salem,

North Carolina

259

I. “Angiotensin II and the Baroreflex” (oral presentation); “AT1

Receptor Inhibition in the Solitary Tract Nucleus” (poster);

“Pediatric Oncology” (poster)

2000 8th

Annual Division of Surgical Sciences Fellows‟ and Students‟

Research Day, Wake Forest University School of Medicine,

Winston-Salem, North Carolina, “AT1 Receptor Inhibition in the

Solitary Tract Nucleus by Antisense Oligonucleotides versus an

AT1 Antagonist in Transgenic Hypertensive Rats” (poster)

1999 7th

Annual Division of Surgical Sciences Fellows‟ and Students‟

Research Day, Wake Forest University School of Medicine,

Winston-Salem, North Carolina, “AT1 Receptor Inhibition in the

Solitary Tract Nucleus by Antisense Oligonucleotides to Reveal

Actions of Angiotensin II on the Baroreflex” (poster co-authored

with Munira Siddiqui, MSII)

COMMUNITY SERVICE:

2000 - 2008 Health on Wheels, various events

Hypertension and Vascular Disease Center

Wake Forest University School of Medicine

Winston-Salem, North Carolina

2003 - 2007 Habitat for Humanity

Habitat Forsyth Chapter

Winston-Salem, North Carolina

2003 2003 Fall Games

North Carolina Special Olympics

Winston-Salem, North Carolina

2002 - 2003 American Red Cross, various events

Northwest North Carolina Chapter

Winston-Salem, North Carolina

2002 2002 Fall Games

North Carolina Special Olympics

Winston-Salem, North Carolina

2001 - 2002 Spanish Interpreter Office & Medical Records (120 hours)

Downtown Health Plaza

Wake Forest University Baptist Medical Center

Winston-Salem, North Carolina

260

2001 – 2001 Operating Room (60 hours)

Wake Forest University Baptist Medical Center

Winston-Salem, North Carolina

2000 – 2001 Emergency Department (70 hours)

Wake Forest University Baptist Medical Center

Winston-Salem, North Carolina

1999 – 1999 Pediatric Bedside (43 hours)

Brenner Children‟s Hospital

Winston-Salem, North Carolina

1998 Women‟s Ways of Leading Conference

Salem College

Winston-Salem, North Carolina

BIBLIOGRAPHY:

BOOKS:

Ferrario CM, Jessup JA. Renin Inhibitor Pharmacotherapy for Hypertension.

Summit Communications, New York, NY. December 2007.

BOOK CHAPTER:

Ferrario CM, Jessup JA, Trask AJ, Varagic J. Basic Science of Hypertension.

In. The Year in Hypertension, Volume 7. R Townsend ed. Clinical Publishing,

London. February 2008; 1478-0194; 288pp.

MANUSCRIPTS:

Diz DI, JA Jessup, BM Westwood, SM Bosch, S Vinsant, PE Gallagher, and

DB Averill. Angiotensin peptides as neurotransmitters/neuromodulators in the

dorsalmedial medulla. Clinical & Experimental Pharmacology Physiology

2002; 29:473.

Ferrario CM, Jessup J, Chappell MC, Averill DB, Brosnihan KB, Tallant EA,

Diz DI, Gallagher PE. Effect of angiotensin converting enzyme inhibition and

angiotensin II receptor blockers on cardiac angiotensin converting enzyme 2.

Circulation 2005;111(20):2605-10.

Ferrario CM, Jessup J, Gallagher PE, Averill DB, Brosnihan KB, Tallant EA,

Smith RD, Chappell MC. Effects of renin-angiotensin system blockade on

renal angiotensin-(1-7) enzymes and receptors. Kidney International

2005;68(5):2189-2196.

261

Ferrario CM, Trask AJ, Jessup JA. Advances in biochemical and functional

roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation

of cardiovascular function. Am J. Physiol Heart Circ Physiol 2005;

289:H2281-H2290.

Jessup JA, Gallagher PE, Averill DB, Brosnihan KB, Tallant EA, Chappell

MC, Ferrario CM. Effect of Angiotensin II Blockade on a New Congenic

Model of Hypertension Derived from Transgenic Ren-2 Rats. Am J Physiol

Heart Circ Physiol 2006 Nov; 291(5):H2166-72.

Ferrario CM and Jessup JA. Interplay Between ACE2 and Angiotensin-(1-7)

in the Regulation of Blood Pressure. Current Hypertension Reviews 2007; 3,

97-104.

De Mello WC, Ferrario CM, Jessup JA. Beneficial versus harmful effects of

Angiotensin (1-7) on impulse propagation and cardiac arrhythmias in the

failing heart. J Renin Angiotensin Aldosterone Syst. 2007; Jun;8(2):74-80.

Jessup JA, Brosnihan KB, Gallagher PE, Chappell MC, and Ferrario CM.

Differential Effect of Low Dose Thiazide on the Renin Angiotensin

System in Genetically Hypertensive Rats. J Am Society of Hypertension

2008;2(2):106-115.

Trask AJ, Jessup JA, Chappell, MC, Ferrario CM. Angiotensin-(1-12) is an

alternate substrate for angiotensin peptide production in the heart. Am J Physiol

Heart Circ Physiol. 2008;294:H2242-H2247.

Jessup JA, Trask AJ, Chappell MC, Nagata S, Kato J, Kitamura K, Ferrario

CM. Localization of the novel angiotensin peptide, angiotensin -12[An-(1-12)],

in heart and kidney of hypertensive and normotensive rats. Am J Physiol Heart

Circ Physiol. 2008;294:H2614-8.

Varagic J, Trask AJ, Jessup JA, Chappell MC and Ferrario CM. New

angiotensins. J Mol Med 2008;86(6):663-71.

Jessup JA, Westwood BM, Chappell MC, Groban L. Combination Renin

Angiotensin System ImprovesVentricular Lusitropy without Affecting Cardiac

Fibrosis or Cardiac RAS in the Hypertensive mRen2.Lewis Rat. Therapeutic

Advances in Cardiovascular Disease; 2009:3(4):245-257.

Pulgar V, Hong J, Jessup JA, Massmann A, Diz D, Figueroa J. Mild chronic

hypoxemia modifies expression of brainstem Angiotensin peptide receptors

and reflex responses in fetal sheep. Am J Physiol Reg, Integrative, Comp Phys;

2009;297(2):R446-52.

Jessup JA, Lindsey SH, Chappell MC, Groban L. GPER Activation Improves

262

Cardiac Function in Intact Female mRen2.Lewis Rats. PLoS One. 2010 Nov

3;5(11):e15433

Sergeant S, McQuail JE, Riddle DR, Chilton FH, Ortmeier S, Jessup JA,

Groban L, Nicolle MM. Dietary Fish Oil Attenuates Diastolic Dysfunction,

but Has No Effect on Spatial Memory and Brain Measures of Inflammation in

Aged Rats. J. Gerontol – Biol Sci, 2011; 66(5):521-33.

Jessup JA, Zhang L, Chen AF, Presley TD, Kim-Shapiro DB, Chappell MC,

Wang H, Groban L. nNOS Inhibition Improves Diastolic Function and

Reduces Oxidative Stress in Ovariectomized-mRen2.Lewis Rats. Menopause

2011; 18(6):698-708.

Jessup JA, Zhang L, Chen AF, Presley TD, Kim-Shapiro DB, Wang H,

Groban L. Tetrahydrobiopterin Repletion Prevents Diastolic Dysfunction in

Ovariectomized-mRen2.Lewis Rats. Endocrinology 2011; 152(6):2428-2436.

ABSTRACTS:

Jessup JA, Westwood BM, Tommasi EN, Ferrario CM, Ganten D, Averill DB,

and Diz DI. Effect of Pre- versus Postsynaptic Inhibition of Nucleus Tractus

Solitarii AT1 Receptors on Cardiac Baro- and Chemoreflexes. FASEB Journal

15:5, 2001.

Jessup JA, Figueroa J, Rose J, Averill DB, Diz DI. Characterization of

Angiotensin AT1 and AT2 Receptors in Fetal Sheep Brain During Development.

FASEB Journal 16:4, 2002.

Hong JK, Jessup JA, Rose J, Averill DB, Figueroa J, Diz DI. Effects of

Hypoxia on Angiotensin Peptide Receptors in Fetal Sheep Brainstem.

Proceedings of the XVth

Inter-American Society of Hypertension; 100. 2003.

Averill DB, Jessup JA, Ishiyama Y, Ferrario CM. Augmented ACE2

Expression in Ischemic Cardiomyopathy. American Journal of Hypertension;

18:118A (P-310). 2005.

Jessup JA, Gallagher, PE, Chappell, MC, Ferrario, CM. Impaired Counter-

regulatory Responses of Angiotensin Converting Enzyme 2 and Angiotensin-(1-

7) in the Heart of a New Congenic Model of Hypertension Derived from

[mRen2]27 Transgenic Rats. Hypertension; 26:48(4) lE74. 2006.

Jessup JA, Chappell MC, Nagata S, Kato J, Kitamura K, Ferrario CM.

Localization of the Novel Angiotensin Peptide, Proangiotensin-12, in the Heart

and Kidney of Hypertensive and Normotensive Rats. Hypertension; 50(4): E101.

2007.

263

Chappell MC, Westwood BM, Pendergrass KD, Jessup JA, Ferrario CM.

Distinct Processing Pathways for the Novel Peptide Angiotensin-(1-12) in the

Serum and Kidney of the Hypertensive mRen2.Lewis Rat. Hypertension; 50(4),

E139. 2007.

Trask AJ, Jessup JA, Ferrario CM. Angiotensin-(1-12) is a Precursor for

Processing of Cardiac Tissue Angiotensin Peptides. Hypertension; 50(4), E154.

2007.

Jessup JA, Westwood BM, Chappell MC, Groban L. Combination Renin

Angiotensin System Improves Ventricular Lusitropy Without Affecting Cardiac

Fibrosis or Cardiac RAS in the Hypertensive mRen2.Lewis Rat. Circulation

Research; 103(5) p. e35. 2008.

Trask AJ, Jessup JA, Tallant EA, Chappell MC, Ferrario CM. Renin-

Independent Processing of Angiotensin-(1-12) in the Rat Heart and Isolated

Myocytes. Hypertension; 52:e34-e131. 2008.

Groban L, Jessup J, Register T. Early estrogen replacement prevents lusitropic

impairment following ovariectomy in middle-aged cynomolgus monkeys. J Am

Coll Cardiol. 2010; 55(10A):A38

Kassik K, Lin M, Carter C, Jessup JA, Groban L. Low-dose ACE-inhibition

late-in-life modulates cardiac reactive oxygen species to retard diastolic

dysfunction in the BNF344 rat model of normative aging. Anesthesiology; In

Press. 2010.