Post on 11-Jun-2022
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.
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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
on
ten
t(s
tain
ed
: t
ota
l p
ixels
)
A
B Sham - Veh Sham –
L-VNIO
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 *
#
#
DH
E F
luo
rescen
ce
(Avera
ge G
rayscale
In
ten
sit
y)
A
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
180
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
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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.