age-associated alterations in the f344xbn rat heart and
TRANSCRIPT
Marshall UniversityMarshall Digital Scholar
Theses, Dissertations and Capstones
1-1-2011
Age-Associated Alterations in the F344XBN RatHeart and the Efficacy of Chronic AcetaminophenIngestion as a Therapeutic ApproachSunil K. KakarlaMarshall University, [email protected]
Follow this and additional works at: http://mds.marshall.edu/etdPart of the Circulatory and Respiratory Physiology Commons, and the Genetic Processes
Commons
This Dissertation is brought to you for free and open access by Marshall Digital Scholar. It has been accepted for inclusion in Theses, Dissertations andCapstones by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected].
Recommended CitationKakarla, Sunil K., "Age-Associated Alterations in the F344XBN Rat Heart and the Efficacy of Chronic Acetaminophen Ingestion as aTherapeutic Approach" (2011). Theses, Dissertations and Capstones. Paper 681.
AGE-ASSOCIATED ALTERATIONS IN THE F344XBN RAT HEART AND THE
EFFICACY OF CHRONIC ACETAMINOPHEN INGESTION AS A
THERAPEUTIC APPROACH
Dissertation submitted to
the Graduate College of
Marshall University
In partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
in Biomedical Sciences
by
Sunil Kumar Kakarla
Approved by
Eric R. Blough, Ph.D., Committee Chairperson
Todd Green, Ph.D.
Elsa I. Mangiarua, Ph.D.
Nalini Santanam, Ph.D.
Robert Harris, Ph.D.
Department of Physiology, Pharmacology and Toxicology
Joan C Edwards School of Medicine
Marshall University, Huntington, WV
May, 2011
ABSTRACT
AGE-ASSOCIATED ALTERATIONS IN THE F344XBN RAT HEART AND THE
EFFICACY OF CHRONIC ACETAMINOPHEN INGESTION AS A
THERAPEUTIC APPROACH
By
Sunil K. Kakarla
Despite advances in therapeutic and diagnostic strategies cardiovascular disease
still remains the leading cause of death in older people. Recent studies suggest that age-
related elevations in oxidative stress are associated with increases in cardiac apoptosis
and compensatory hypertrophy of the remaining cardiomyocytes. Acetaminophen has
been shown to exhibit cardioprotective effects following ischemia-reperfusion injury by
acting as an antioxidant. Here, we investigate the molecular mechanisms underlying
aging associated cardiac remodeling in male F344XBN rats and examine the efficacy of
chronic acetaminophen ingestion as a therapeutic approach.
Aging in male F344XBN rats is characterized by a significant increase in heart
size, heart weight, heart weight to body weight ratio and cardiomyocytes fiber cross
sectional area. Our findings suggest that aging-associated cardiac hypertrophy in
F344XBN rats is predominantly mediated via activation of the extracellular regulated
kinase 1/2 (ERK1/2) and protein kinase B (Akt) signaling pathways resulting in increased
protein translational signaling and ultimately increased protein synthesis.
To determine the efficacy of chronic acetaminophen treatment in preventing age-
related alterations in cardiac structure and function, male F344XBN rats (27-mo; n=6)
were treated with acetaminophen (30mg/kg/day p.o.) for six months and underwent serial
electrocardiography before and after the completion of the study. Compared to 6-month
and 33-month age matched control rats, chronic acetaminophen ingestion diminished
age-related increases in cardiac ROS levels and the number of TUNEL positive nuclei.
These changes were accompanied by improvements in the Bax/Bcl2 ratio, diminished
evidence of caspase-3 activation and increased phosphorylation of Akt, ERK1/2, p70S6K
and GSK-3β. In addition, acetaminophen treatment diminished age-related increases in
the incidence of arrhythmias and these alterations were associated with diminished
fibrosis and changes in cardiac miRNA expression. Taken together, these results suggest
that acetaminophen may attenuate the age associated deterioration in cardiac structure
and function possibly via diminishing age associated elevation in reactive oxygen species
production.
iv
ACKNOWLEDGEMENTS
Though this dissertation is an individual effort, it would have not been possible
without spiritual, psychological, scientific, financial and technical efforts of lot many
people. I would like to express many thanks to one and all who helped me in realizing
this dissertation.
First of all I would like to express my deepest gratitude to my mentor Dr. Eric R.
Blough for his supervision, advice, and guidance from the very early stage of this
research as well as believing in me throughout the work. His infectious enthusiasm and
unlimited zeal have been major driving forces through my graduate career at Marshall
University. Dr. Blough has been very helpful and easily accessible with a single knock on
his office door to discuss at length about my research. Often times, it surprises me that he
used to keep his work aside to discuss about my research and I would never forget warm
support and affection he showed towards me. Dr. Blough has created an indelible
impression on my personal and professional career. I can’t ask you anymore Eric; thanks
a lot for everything and I am very much proud to have a mentor like you.
I would like to thank my other committee members Dr. Nalini Santanam, Dr. Elsa
Mangiarua, Dr. Todd Green and Dr. Robert Harris for their critical thinking on my
research, valuable comments and suggestions on the dissertation.
It is the most apt situation to express my greatest gratitude to my parents Venkata
Subbaiah Kakarla and Masthanamma Kakarla, my brother Suresh Babu Kakarla for
v
everything they gave. Without their help, support, motivation and confidence this
dissertation would never have realized.
I am extraordinarily fortunate to have my wife Venkata Surekha Kakarla and my
wonderful kids Asmitha Chowdary Kakarla and Venkata Subodh Chowdary Kakarla who
are very understandable and never complained when I am late to home while I work on
my research. Their love, affection and motivation at home drove me this much distance in
my life thus so far. I would like to thank my sister-in-law Lakshmi Praveena, my niece
Dhatri Kakarla and my nephew Samhith Kakarla for their love and affection. I would like
to take this opportunity to thank my in-laws Prasada Rao Ravipati and Radha Ravipati
who always stood by me in difficult times.
It is my immense pleasure to thank my esteemed colleagues Dr. Kevin Rice, Dr.
Arun Kumar, Dr. Miaozong Wu, Devashish Desai, Anjaiah Katta, Jacqueline Fannin,
Sriram Prasad Mupparaju, Anil Gutta, Satyanarayana Paturi, Madhukar Kolli, Ravi
Arvapalli, Murali Gadde, Siva Nalabotu, Brent Kidd, Srinivas Thulluri, Sudarsanam
Kundla, Eli Shleiser, Hari Addagarla, Nandini Manne, Geeta Nandyala, Radhakrishna
Para and Sravanthi Bodapati who were very friendly and helpful in many ways in
successful completion of my research work. I would like to extend my thanks to Lucy
Dornon who is very helpful in collecting the data that I used for echocardiography
studies.
I would also like to convey my thanks to family friends and well-wishers Manjula
Mupparaju, Kiran Kodali, Satish Adusumilli, Dhanvanthari Emmadi, Dr. Micheal Dyer
vi
and Dr. Kelly Pinkston who were very helpful in my personal life. Finally, I once again
thank Dr. Blough for the grant support.
vii
DEDICATION
To my loving Brother Suresh Babu Kakarla who extended tremendous support to
materialize my dream of pursuing extraordinary career in the USA; has been greatly
helpful in every shade of both my personal and professional life and he always
encouraged me to achieve the best in my life with his valuable suggestions and
motivational thoughts.
viii
TABLE OF CONTENTS
ABSTRACT ....................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................ iv
DEDICATION................................................................................................................. vii
LIST OF FIGURES ......................................................................................................... xi
ABBREVIATIONS ........................................................................................................ xiv
CHAPTER 1 .......................................................................................................................1
INTRODUCTION......................................................................................................... 1
SPECIFIC AIMS .......................................................................................................... 5
CHAPTER II ......................................................................................................................7
REVIEW OF THE LITERATURE ............................................................................ 7
2.1 Aging..................................................................................................................... 7
2.2 Physiological changes with aging ......................................................................... 9
2.3 Cardiac aging ...................................................................................................... 10
2.3.1 Age associated alterations in cardiac function ................................................. 11
2.3.2 Cardiac aging in animal models ....................................................................... 12
2.3.3 Role of oxidative stress in cardiac aging ......................................................... 14
2.3.3.1 Signaling mechanisms underlying cardiac oxidative-nitrosative stress........ 15
2.3.4 Age-associated cardiac apoptosis .................................................................... 17
2.3.4.1 Signaling mechanisms underlying cardiac apoptosis ................................... 19
2.3.5 Age-associated cardiac hypertrophy ................................................................ 20
2.3.5.1 Signaling mechanisms underlying cardiac hypertrophy ............................... 21
2.3.6 Age-associated myocardial fibrosis ................................................................. 23
2.3.6.1 Signaling mechanisms underlying cardiac fibrosis....................................... 24
2.4 Regulation of miRNA in aged hearts .................................................................. 25
ix
2.5 Acetaminophen ................................................................................................... 27
2.5.1 Mode of Action ................................................................................................ 28
2.5.2 Pharmacology .................................................................................................. 29
2.5.3 Cardioprotective value of acetaminophen ....................................................... 29
CHAPTER III ..................................................................................................................33
PAPER 1 ...........................................................................................................................33
Mechanisms of age-related cardiac hypertrophy in the F344XBN rat model ...........34
Abstract ........................................................................................................................ 35
Introduction ................................................................................................................. 36
Materials and Methods ............................................................................................... 38
Results .......................................................................................................................... 42
Aging in the F344XBN rats is associated with cardiac hypertrophy ........................ 42
Aging associated cardiac hypertrophy in F344XBN rats is associated with increases
in the activation of the Ras/Raf/MEK/ERK signaling cascade ................................ 43
PI3K/Akt signaling in aging associated cardiac hypertrophy in the F344XBN rat .. 43
Age-related cardiac hypertrophy in F344XBN rats is associated with increased
protein translational signaling ................................................................................... 45
Discussion..................................................................................................................... 46
PAPER 2 ...........................................................................................................................61
Chronic acetaminophen attenuates age-associated increases in cardiac ROS and
apoptosis in the Fischer Brown Norway rat ..................................................................62
Abstract ........................................................................................................................ 63
Introduction ................................................................................................................. 64
Materials and Methods ............................................................................................... 66
Data Analysis ............................................................................................................... 70
Results .......................................................................................................................... 71
Acetaminophen decreases indices of cardiac ROS ................................................... 71
x
Acetaminophen decreases age-associated cardiomyocyte apoptosis ........................ 72
Acetaminophen attenuates age-associated alterations in cell survival signaling ...... 73
Discussion..................................................................................................................... 75
PAPER 3 ...........................................................................................................................91
Anti-arrhythmic effect of chronic acetaminophen treatment in the aging F344XBN
rat involves diminished myocardial fibrosis and altered miRNAs regulation ...........92
Abstract ........................................................................................................................ 93
Introduction ................................................................................................................. 94
Materials and Methods ............................................................................................... 97
Data Analysis ............................................................................................................. 101
Results ........................................................................................................................ 102
Aging is associated with increased incidence of cardiac arrhythmias .................... 102
Aging is associated with greater myocardial fibrosis ............................................. 102
Aging is associated with diminished connexin-43 levels ....................................... 103
Aging is associated with alterations in cardiac miRNA expression ....................... 103
Acetaminophen treatment is associated with alterations in miRNA expression .... 104
Discussion................................................................................................................... 105
CHAPTER IV.................................................................................................................119
GENERAL DISCUSSION ............................................................................................119
Age-related alterations in cardiac structure and function of F344XBN rats .......... 120
Cardioprotective properties of acetaminophen in aged F344XBN rats .................. 122
Regulation of miRNA with aging and acetaminophen treatment in F344XBN rats
................................................................................................................................. 126
SUMMARY ....................................................................................................................127
FUTURE DIRECTIONS ...............................................................................................128
REFERENCES ...............................................................................................................130
xi
CURRICULUM VITAE ................................................................................................148
LIST OF TABLES
Table 1. Total body weight (BW), heart weight (HW) and heart weight to body weight
ratio (HW/BW %) of 6-, 33-month control and 33-month acetaminophen treated
male F344XBN rats.. ................................................................................................ 80
LIST OF FIGURES
Figure 1. Molecular structure of acetaminophen .............................................................. 28
Figure 2. 1. Heart size, heart weight, body weight, heart weight to body weight ratio
altered with age in F344XBN rats. ........................................................................... 52
Figure 2. 2. Aging in F344XBN rats is associated with cardiomyocyte hypertrophy. ..... 53
Figure 2. 3. Aging is associated with higher phosphorylation of ERK1/2, ERK5 and total
calcineurin protein in F344XBN rat hearts. .............................................................. 54
Figure 2. 4. Aging is associated with higher phosphorylation of Akt in F344XBN rat
hearts. ........................................................................................................................ 55
Figure 2. 5. Aging is associated with higher phosphorylation of GSK3β and PTEN in
F344XBN rat hearts. ................................................................................................. 56
Figure 2. 6. Aging is associated with higher phosphorylation of mTOR in F344XBN rat
hearts. ........................................................................................................................ 57
xii
Figure 2. 7. Aging lowers the phosphorylation of TSC2 and is associated with higher
expression of Raptor in F344XBN rat hearts. ........................................................... 58
Figure 2. 8. Aging is associated with higher phosphorylation of 4EBP1 and rpS6 in
F344XBN rat hearts. ................................................................................................. 59
Figure 2. 9. Proposed scheme depicting the possible molecular mechanisms underlying
age-associated cardiac hypertrophy in F344XBN rats. ............................................. 60
Figure 3. 1. Effect of aging and acetaminophen treatment on indices of cellular ROS. ... 81
Figure 3. 2. Acetaminophen treatment decreases the incidence of TUNEL positive nuclei
in aged cardiac muscle. ............................................................................................. 82
Figure 3. 3. Acetaminophen treatment alters the ratio of Bax / Bcl-2 in aged cardiac
muscle. ...................................................................................................................... 83
Figure 3. 4. Acetaminophen treatment decreases the age-associated activation of caspase-
3................................................................................................................................. 84
Figure 3. 5. Acetaminophen treatment increases Akt phosphorylation in aged cardiac
muscle. ...................................................................................................................... 85
Figure 3. 6. Acetaminophen treatment increases mTOR phosphorylation in aged cardiac
muscle. ...................................................................................................................... 86
Figure 3. 7. Acetaminophen treatment increases ERK1/2 phosphorylation in aged cardiac
muscle. ...................................................................................................................... 87
Figure 3. 8. Acetaminophen treatment increases p70S6K phosphorylation in aged cardiac
muscle. ...................................................................................................................... 88
Figure 3. 9. Acetaminophen treatment increases GSK-3β phosphorylation in aged cardiac
muscle. ...................................................................................................................... 89
xiii
Figure 3. 10. Proposed scheme for the effects of chronic acetaminophen treatment in
attenuating aging-associated cardic ROS and apoptosis. .......................................... 90
Figure 4. 1. Chronic acetaminophen treatment decreases incidence of cardiac
arrhythmias. ............................................................................................................ 113
Figure 4. 2. Chronic acetaminophen attenuates myocardial fibrosis in aged rat hearts. . 114
Figure 4. 3. Chronic acetaminophen preserves myocardial membrane Cx43. ............... 115
Figure 4. 4. Aging decreases myocardial miR-1 and miR-133 expression. ................... 116
Figure 4. 5. Aging decreases myocardial miR-30a and miR-30d expression. ................ 117
Figure 4. 6. Chronic acetaminophen treatment alters myocardial miR-214 expression. 118
xiv
ABBREVIATIONS
4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1
4-HNE 4-hydroxy-2-nonenal
•O2-
Superoxide anions
•OH Hydroxyl radicals
AAALAC Association for assessment and accreditation of laboratory animal care
ANOVA One-way analysis of variance on ranks
Apaf-1 Apoptotic protease activating factor-1
APAP N-acetyl p-aminophenol
ASK-1 Apoptosis-signaling kinase 1
Bax Bcl-2-associated X protein
Bcl2 B-cell lymphoma 2
BSA Bovine serum albumin
Cx43 Connexin 43
COX3 Cyclooxygenase 3
CTGF Connective tissue growth factor
ECL Enhanced chemiluminescence
xv
ERK Extracellular signal-regulated kinase
F344XBN Fisher 344 Brown Norway hybrid
FAK Focal adhesion kinase
GSK-3β Glycogen synthase kinase 3 beta
JNK Jun-N-terminal kinase
KRB Krebs-Ringers Buffer Solution
MAPK Mitogen-activated protein kinase
mTOR Mammalian target of rapamycin
NFAT Nuclear factor of activated T-cells
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
p70S6K p70S6 kinase
PBS Phosphate buffered saline
PBST Phosphate buffered saline with 0.5% tween
PI3K Phosphatidylinositol 3-kinases
PKB Protein kinase B
PKC Protein kinase C
PTEN Phosphatase and tensin homolog
xvi
ROS Reactive oxygen species
rpS6 Ribosomal protein S6
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
TBS Tris buffered saline
TBST Tris buffered saline with 0.5% tween
TGF-β Transforming growth factor beta
TNF-α Tumor necrosis factor-α
TSC2 Tuberous sclerosis protein 2
TUNEL TdT-mediated dUTP nick end labeling
1
CHAPTER 1
INTRODUCTION
The number of elderly, especially in developed countries, continues to grow at a
rapid pace due to availability of better healthcare facilities and improved standards of
living. Epidemiological studies suggest that one in every four citizens of the United
States will be over the age of 65 by the year 2030. This is thought to represent an increase
of 52 million elderly individuals. Furthermore, the population of people over the age of
85 years is expected to increase by 389%, leaving a tremendous burden on the health care
system, as the incidence of cardiovascular diseases increases linearly with age [1].
Indeed, recent data has suggested that older patients account for up to a quarter of all
emergency department visits and that cardiovascular disease remains one of the frequent
presenting complaints [2]. In the United States, cardiovascular disease, e.g.,
atherosclerosis and hypertension that lead to heart failure and stroke, is the leading cause
of mortality, accounting for over 40 percent of deaths in those aged 65 years and above
[3]. Apart from the medical challenge, this impending demographic shift also poses a
major economical challenge for the nation, as the cost of providing health care for elderly
individuals is three to five times greater than the cost for young people [4]. As such, the
development of newer therapeutic agents and methodologies for preventive care is a top
priority. To achieve this goal, it is imperative that we better understand how aging affects
cardiovascular physiology and further, that we increase our knowledge of the underlying
molecular mechanism(s) involved.
2
As we age, numerous physiological and environmental factors increase the risk of
cardiac damage. One such factor is increases in oxidative stress, which has been shown to
cause alterations in the structure and function of major organs, including the heart. It is
well established that the aged heart has a diminished functional and adaptive reserve
capacity, an increased susceptibility to incur damage (e.g., as a result of ischemia), and a
limited practical ability for repair/regeneration [5]. Aging of the heart is mainly
characterized by an increase in the loss of cardiomyocytes and hypertrophy of the
remaining cardiomyocytes, ultimately leading to diastolic heart failure [1]. Throughout
life, the heart is exposed to minor, highly controlled episodes of ischemia followed by
reperfusion, an innate mechanism that helps condition the heart gradually to prevent
injury from such episodes of larger magnitude taking place later in life. This process is
known as ischemic preconditioning of the myocardium [6]. As we age, the capability of
the myocardial tissue to handle oxidative stress decreases progressively. This finding has
led some to postulate that antioxidants may be useful for the treatment and / or prevention
of age-associated cardiac dysfunction.
Cardiac structure and function are remarkably similar among mammalian species,
and the use of animal models has been extremely helpful in developing treatment
strategies for alleviating heart disease in humans. Extending animal studies beyond young
adult ages to very old ages may provide similar benefits to cardiovascular health of the
growing aged population. Multiple experimental models have been used to assess the
effects of aging on reactive oxygen species and cardiovascular disease (CVD), including
dogs, the Fisher 344 (F344), the Fischer 344/NNiaHSd X Brown Norway/BiNia
3
(F344XBN) aging rat models, and other species [7-12]. A recent review of aging rat
models concluded that the F344XBN rat strain seems to live longer while also exhibiting
“physiological aging” to a greater degree than others [13-15]. Recent investigation using
the F344XBN rat model has demonstrated that aging in these animals is associated with
an increased incidence of cardiac arrhythmias as well as impairment of systolic and
diastolic function [16, 17]. In addition, other data has shown that the aged F344XBN
heart exhibits increased expression of oxidative-nitrosative stress markers [18]. Reactive
oxygen species (ROS) such as superoxide anions (•O2-) and hydroxyl radicals (•OH) have
been implicated in cardiac dysfunction, remodeling and in cardiomyocyte apoptosis [19].
Recent data has also suggested that aging in the F344XBN heart is associated with
increases in cardiac apoptosis and further that this increase in cell death is associated with
elevations in cardiac ROS [20]. Whether strategies designed to diminish ROS levels are
efficacious in improving age-related cardiac function is not well understood.
Acetaminophen (N-acetyl p-aminophenol) is an analgesic, antipyretic agent
introduced into western medicine over 100 years ago. Although its precise mechanisms
of action are not known, acetaminophen has been shown to cause dose-dependent
reductions in the amount of circulating prostaglandins, to inhibit myeloperoxidase
activity and decrease the oxidation of lipoproteins. In addition, this agent also appears to
confer cardioprotection by blocking the effects of hydroxyl radical, peroxynitrite, and
hydrogen peroxide [21]. Until recently, most investigations on acetaminophen have
focused on its analgesic/antipyretic properties and hepatotoxicity, while the effects of the
drug on other mammalian organ systems, including the heart and circulation, have been
4
largely ignored. In the last few years, studies have begun to shed light on the antioxidant
properties of acetaminophen and its potential cardioprotective value. Being a phenolic
compound [22], acetaminophen exhibits antioxidant properties [23] and it has been
shown to protect the heart against ischemia-reperfusion injury [24-27]. Other data has
suggested that acetaminophen exhibits potent anti-oxidant activity and that an acute bolus
of acetaminophen is effective in preventing ROS-associated cardiac damage in the rat and
dog heart [28, 29]. These findings are important as they suggest that this molecule may
also have application for the prevention of myocardial infarction, which is one of the
leading causes of severe morbidity and death in the aged. Moreover, as the use of
acetaminophen in the aged is increasing [21], additional research is needed to investigate
the effects of this agent on the cardiovascular system. Herein, we propose to (i.)
Investigate the molecular mechanism(s) underlying aging-associated alterations in the
structure and function of F344XBN rat hearts and (ii.) To determine the efficacy of
chronic acetaminophen treatment in preventing age-associated cardiac dysfunction in the
F344XBN rat heart.
5
SPECIFIC AIMS
Advancing age is associated with increased risk of cardiovascular disease and a
progressive decrease in cardiac function [30]. Recent investigation using the Fischer 344
x Brown Norway F1 hybrid (F344XBN) rat model has demonstrated cardiovascular
changes similar to that seen in aging humans including an increased incidence of cardiac
arrhythmias as well as impairment of systolic and diastolic function [16, 17]. In addition,
other data has shown that the aged F344XBN heart exhibits increased expression of
oxidative-nitrosative stress markers [18]. Reactive oxygen species (ROS) such as
superoxide anions (•O2-) and hydroxyl radicals (•OH) have been implicated in cardiac
dysfunction, remodeling and in cardiomyocyte apoptosis [19]. Recent data has also
suggested that aging in the F344XBN heart is associated with increases in cardiac
apoptosis and that this finding is correlated with elevated cardiac ROS levels [20]. Aging
has been shown to be associated with compensatory hypertrophy in humans and animals;
however, the underlying molecular mechanisms remain unclear.
Our long-term goal is to improve our understanding of the molecular mechanisms
underlying aging associated alterations in the structure and function of the heart and to
develop therapeutic strategies to prevent or reverse such age associated cardiac
dysfunction. The central hypothesis of this dissertation is that chronic acetaminophen
ingestion will be associated with decreases in cardiac oxidative stress that will, in turn, be
strongly associated with the prevention of cardiomyocyte cell death and dysfunction.
We plan to test our central hypothesis and accomplish the objective of this study
by pursuing the following three specific aims:
6
SPECIFIC AIM I: To investigate the different signalling mechanisms underlying aging
associated cardiac hypertrophy in F344XBN rats.
SPECIFIC AIM II: To determine if chronic acetaminophen ingestion is able to diminish
age-related increases in indices of oxidative and nitrosative stress and the effect of these
changes, if present, on the incidence of age-related increases in cell death and apoptotic
signaling.
SPECIFIC AIM III: To investigate age associated alterations in the cardiac rhythm in
F344XBN rats and to determine if chronic acetaminophen ingestion is able to preserve
cardiac structure and diminish cardiac arrhythmias.
7
CHAPTER II
REVIEW OF THE LITERATURE
The following chapter presents a review of the current literature concerning the
present study. Specifically, the following areas will be addressed: aging, physiological
changes with aging in different organ systems, cardiac aging, age-related alterations in
cardiac structure and function, and the potential role of oxidative stress, apoptosis,
cardiac hypertrophy, myocardial fibrosis and miRNA during cardiac remodeling. Finally,
the chapter will conclude with a review on the effects of acetaminophen on cardiac
structure and function.
2.1 Aging
Aging, or the process of growing old, is defined as the gradual deterioration in the
function of cells (mitotic cells, such as fibroblasts and post-mitotic cells, such as neurons,
cardiomyocytes etc.) and structural components (such as bone and muscle) which
consequently have a direct impact on the functional ability of organs, biological systems
and ultimately the organism as a whole [31]. Epidemiological studies indicate that the
elderly population of the world, and especially those in developed countries, continue to
grow rapidly due to availability of better healthcare facilities and improved standard of
living [32]. Due to a decline in fertility rate and old age mortality, in recent years there
has been a shift in the age distribution of the population toward older ages [3]. For
instance, a recent report from the United Nations, World Population Ageing 2009,
indicates that the proportion of older persons, aged 60 years or older has been rising
steadily, from 8 % in 1950 to 11 % in 2009, and is expected to reach 22 % in 2050 [33].
8
According to the U.S. Department of Health and Human Services, the older population
aged 65 years or above numbered 38.9 million in 2008. This was an increase of 4.5
million or 13.0% since 1998 [33]. Current estimates project a further increase of 31% by
2030. At present, one in every eight individuals is aged 65 or more with an additional life
expectancy of 18.6 years [33]. Interestingly, the current intensive biomedical anti-aging
studies may further extend the healthy and productive period of human life in the future
[34]. While increased life expectancy is a success story for mankind, at the same time it
poses a great challenge for our health care system due to the growing burden of chronic
illnesses, injuries, and disabilities. As the aging population increases, the prevalence of
disability, frailty, and chronic diseases (Alzheimer’s, cancer, cardiovascular
and cerebrovascular diseases, etc.) is expected to increase dramatically [33, 35].
Chronic diseases, which affect older adults disproportionately, contribute to
disability, diminished quality of life, and increased long term health care costs [36]. In
the United States, approximately 80% of all persons aged >65 years have at least one
chronic condition, and 50% have at least two [33, 37]. In 2001, the leading causes of
death in developed countries were primarily cardiovascular diseases and cancer, followed
by respiratory diseases and injuries [35]. The health-care cost per capita for persons
aged >65 years in the United States and other developed countries is three to five times
greater than the cost for persons aged <65 years, and the rapid growth in the number of
older persons, coupled with continued advances in medical technology, is expected to
create upward pressure on health- and long-term-care spending [4]. Three-quarters of the
total health-care expenditures in the United States is spent on treating chronic disease
($1·7 trillion per year) and 76% of Medicare expenditure goes to beneficiaries with more
9
than five chronic conditions. The rising prevalence of these diseases has also resulted in
bigger out-of-pocket expenses for senior citizens. In 2008, on average, older Americans
spent $4,605 towards out-of-pocket health care expenditures, an increase of 57% since
1998 [37].
2.2 Physiological changes with aging
Aging is inevitable, and as life expectancy increases it becomes more important to
understand the physiological mechanisms associated with the normal aging process.
Cross-sectional studies have indicated that the prevalence of high body weight or obesity
increases with age up to 60 years and then tends to decline due to decreases in lean body
mass, while the increase in body circumference seen with advanced age is mostly due to
redistribution of body fat to the abdominal cavity [38]. Age-related decreases in lean
body mass, which is also known as sarcopenia, is characterized by decreased muscle
mass and muscle strength, and increased muscle fatigability [39]. Sarcopenia contributes
significantly to the morbidity, affects quality of life, and increases health care costs in the
elderly [40]. Decrease in whole body protein turnover, mixed muscle protein synthesis,
myosin heavy chain synthesis, and mitochondrial protein synthesis have been implicated
in age-related sarcopenia [40]. Such age-related decreases in active cellular mass, i.e., fat
free mass or lean body mass, and a concomitant increase in body visceral fat lead to
decreased body metabolic rate [41]. The free radical theory of aging proposed by Harman
has suggested a possible mechanistic link between metabolic rate and aging [31]. He
proposed that normal oxygen consumption inevitably results in the production of oxygen
free radicals, which, in turn, damage important biological molecules (nucleic acids,
10
proteins, and lipids, among others). This damage, if allowed to proceed unchecked,
oftentimes results in death of the organism [42].
Recently, the free radical theory of aging has evolved into what has been termed
the oxidative-stress theory of aging, which is currently the most widely accepted
mechanistic explanation of aging [43]. Different tissues and organs display different
responses to aging, with more oxidative tissue generally having more age-related
changes. For instance, age-related changes in pulmonary function include but are not
limited to increases in residual volume, decreased elasticity of structures, diminished
compliance, decreased number of airway-supporting structures, increase in closing
volumes and increased risk for atelectasis [44, 45]. The structure, function and
morphology of kidneys also changes markedly with age ultimately leading to decreased
glomerular filtration [46]. Aging has been shown to be associated with structural and
functional changes in the cardiovascular system including the remodeling of the arteries,
heart and the microcirculation [30]. Remodeling of arteries produces a thickening of the
intima-media inducing an increase of the pulse wave and aortic impedance, with greater
resistance to ventricular ejection that in turn induces the remodelling of the left ventricle
and ultimately leading to systolic and diastolic dysfunction [47].
2.3 Cardiac aging
The aged (65+ years of age) now comprise the most rapidly growing segment of
the United States population [33]. Cardiovascular disease is the leading cause of
mortality accounting for over 40 percent of deaths in this population [3, 48]. Apart from
other risk factors like smoking, diabetes, obesity, alcohol, etc., aging by itself represents a
11
major risk factor for cardiovascular disease as it is associated with structural and
functional alterations in the heart [49]. Age-related myocardial structural changes include
loss of myocytes, a progressive increase in myocyte cell volume per nucleus, hypertrophy
of the remaining cardiomyocytes, a reduction in the number of conduction cells and wall
thickening [1]. These structural changes are associated with alterations in cardiac
function including diastolic dysfunction, increased systolic arterial pressure and pulse
pressure, decreased maximal heart rate, maximal cardiac output and maximal VO2 [1]. In
addition, the aged myocardium is prone to inadequate oxygenation as the capillary
density does not increase in parallel with the remodeling myocardium [50].
2.3.1 Age associated alterations in cardiac function
It is established that the aged heart becomes slightly hypertrophic and
hyporesponsive to sympathetic (but not parasympathetic) stimuli, so that the exercise-
induced increases in heart rate and myocardial contractility are blunted in older hearts
[49]. Gerstenblith et al. performed echocardiograms on 105 male participants in the NIA
volunteer Longitudinal Study Program and reported that aging in the normal male is
associated with altered LV diastolic filling, increased aortic root diameter and left
ventricle hypertrophy but little change in contractile ability in the resting state [2]. In
another study, using 2-dimensional echocardiograms, Downes et al. examined the effect
of aging on the pattern of LV diastolic filling in 11 elderly subjects (68 +/- 5 years, mean
+/- standard deviation) and 15 normal young adults (31 +/- 7 years) without coronary
artery disease, systemic hypertension, LV hypertrophy or abnormality of LV systolic
function. Their findings suggested that early diastolic filling is reduced in normal aged
12
subjects, even in the absence of coronary artery disease and systolic dysfunction [51].
Bikkina et al. examined the prognostic implications of asymptomatic ventricular
arrhythmias by performing ambulatory electrocardiographic (ECG) monitoring in 2425
men without clinically evident coronary heart disease, 302 men with coronary heart
disease, 3064 women without disease and 242 women with disease. Their findings
suggested that increased incidence of ventricular arrhythmias is associated with a two
fold increase in the risk for all-cause mortality and myocardial infarction or death due to
coronary heart disease [52]. Haider et al. examined the associations of LV mass and LV
hypertrophy with sudden death risk in a large group of middle-aged and elderly subjects
enrolled in the Framingham Heart Study. Their work demonstrated that LV hypertrophy
is an independent risk factor for sudden death [53]. Morales et al. performed quantitative
echocardiography in seventy-eight subjects (47 men; mean age, 51 years; age range, 23-
87 years) without apparent cardiovascular and systemic disease and reported that
advancing age is associated with changes in myocardial structure that are characterized
by a greater echogenicity and loss in tonal organization, possibly due to increased
collagen content within the fibers [54]. Taken together, these data suggest that cardiac
aging in humans is characterized by alterations in LV diastolic filling, ventricular
dimensions, diastolic function and increased incidence of arrhythmias.
2.3.2 Cardiac aging in animal models
Cardiac structure and function are remarkably similar among mammalian species,
and the use of animal models have provided new approaches to study the effects of aging
on the cardiovascular system [55]. Anversa et al. investigated the effect of age on
13
functional characteristics of the heart in Fischer 344 rats at 4, 12, 20, and 29 months of
age, they found that mean arterial pressure, LV systolic and end-diastolic pressures, its
first derivative (dP/dt), heart rate and stroke volume were comparable in rat groups up to
20 months, while LV end-diastolic pressure was elevated and dP/dt and stroke volume
were decreased during the interval from 20 to 29 months, indicating a significant
impairment of ventricular function with senescence [56, 57]. Boluyt et al. performed an
echocardiographic assessment of age-associated changes in chamber dimensions and
systolic and diastolic properties of the female Fischer 344 (F344) rat hearts. They found
that, compared with young adult 4-month rats, there is a significant decrement in the
resting systolic function of the LV, an increase in isovolumic relaxation time (25.0 +/- 7.6
vs. 17.2 +/- 4.4 ms) and decreases in the tissue Doppler peak E waves at the septal
annulus and at the lateral annulus of the mitral valve in 30-month female F344 rats [58].
In recent years, the Fischer 344/Brown Norway F1 (F344/BNF1) rat has been
recommended for age-related studies by the National Institutes on Aging because this
hybrid rat lives longer and has a lower rate of pathological conditions than other inbred
rat strains [17]. Hacker et al. used echocardiographic and hemodynamic analyses to
determine standard values for LV mass, LV wall thickness, LV chamber diameter, heart
rate, LV fractional shortening, mitral inflow velocity, LV relaxation time, and aortic/LV
pressures in male Fischer 344/ Brown Norway F1 hybrid rats from adulthood to the very
aged (n = 6 per 12-, 18-, 21-, 24-, 27-, 30-, 33-, 36-, and 39-month old group). Their
findings suggested that aged rats had an increased LV mass-to-body weight ratio and
deteriorated systolic function. Histological analysis revealed a gradual increase in fibrosis
14
and a decrease in cardiomyocyte volume density with age [16]. Walker et al. performed
two-dimensional ECHO measurements, two-dimensional guided M-mode, Doppler M-
mode, and other recordings from parasternal long- and short-axis views on male Fischer
344/Brown Norway F1 (F344/BNF1) rats aged 6-, 30-, and 36-month and revealed
persistent cardiac arrhythmias in 72% (13/18) of 36-month rats, 10% (1/10) of 30-month
rats and none in 6-month rats [17]. Taken together these findings suggest that aging in
rodents, like humans, is also associated with increased incidence of cardiac arrhythmias,
decrease in ventricular compliance and impairments in systolic and diastolic function.
2.3.3 Role of oxidative stress in cardiac aging
A growing body of emerging evidence suggests that age-related declines in
cardiac function may be associated with age-related increases in oxidative-nitrosative
stress [59]. Oxidative stress is most commonly defined as an imbalance between the
production and scavenging of reactive oxygen species (ROS) [60]. This imbalance is
associated with measurable increases in ROS. It is well established that elevation of
cardiac ROS levels with age have been shown to influence several important factors
including cardiac hypertrophy, structure and function of blood vessels, inflammation,
apoptosis and alteration of the extracellular matrix profile [61-64]. Indeed, Asano et al.
examined the influence of aging on multiple markers of oxidative-nitrosative stress in the
hearts of 6-, 30- and 36-month F344XBN rats and found that indices of oxidative
(superoxide anion [O2•–], 4-hydroxy-2-nonenal [4-HNE]) and nitrosative (protein
nitrotyrosylation) stress were significantly increased in 36-month hearts and these
findings were highly correlated with increases in LV wall thickness [18]. In a follow-up
15
study, Kakarla et al. showed that aging in F344XBN rats is associated with increased
cardiomyocyte apoptosis suggesting the possible role of oxidative-nitrosative stress [20].
Increases in oxidative stress can also elicit oxidative modification of various cellular
components, such as lipids, proteins, DNA and antioxidant enzymes [65, 66]. Being a
post-mitotic tissue, the myocardium is more vulnerable to oxidative stress as it has lesser
ability to up-regulate antioxidant defenses and / or to repair accumulated oxidative
damage compared to other tissues with greater proliferative capacity such as the liver
[67]. Recent work has demonstrated that mitochondrial ROS generation increases in the
heart with advanced age [68-70]. Additional sources of ROS also include the
peroxisomes, cytochrome P-450, xanthine oxidase and nicotinamide dinucleotide
phosphate (reduced) (NADPH) oxidase, although the quantitative significance of those
pathways in generating ROS and causing cellular senescence remains unclear [71]. Apart
from its role in aging, oxidative-nitrosative stress has also been implicated in chronic
cardiovascular diseases, cardiac injury, cell death, and dysrhythmia [72]. The exact
molecular mechanisms underlying age-related increases in cardiac oxidative-nitrosative
stress and whether pharmacological intervention can be used to counteract such changes
remain to be elucidated.
2.3.3.1 Signaling mechanisms underlying cardiac oxidative-nitrosative stress
Reactive oxygen species (ROS)-dependent modulation of intracellular signaling is
implicated in the development of cardiac structural and functional changes including
cardiomyocyte hypertrophy, interstitial fibrosis, contractile dysfunction and cell death
[73]. Growing evidence suggests that ROS-induced signaling pathways play a key role in
16
the development of cardiac hypertrophy either in response to neurohumoral stimuli or
chronic pressure overload [74]. Experimentally, cardiac hypertrophy has been induced in
cultured cardiomyocytes by angiotensin II, endothelin 1, norepinephrine and tumor
necrosis factor-α in an ROS intracellular signaling dependent manner and can be
inhibited by antioxidants [74]. Angiotensin II (AT II) has been shown to induce cardiac
hypertrophy via a G-protein–linked pathway involving ROS-associated activation of
several downstream signals, including the mitogen activated protein kinases (MAPKs),
while inhibition of ROS by antioxidants blocked ATII-mediated cardiac hypertrophy [75,
76]. Several in vitro and animal studies have also implicated the involvement of protein
kinase C (PKC), p38-MAPK, JNK-MAPK, and ERK1/2-MAPK; PI3K; Akt; several
tyrosine kinases (e.g., src and focal adhesion kinase (FAK)); NF-κB, and calcineurin in
ROS-dependent cardiac hypertrophy [77, 78]. Similarly, the redox-sensitive apoptosis-
signaling kinase 1 (ASK-1) is strongly activated by ROS, which in turn activates p38-
MAPK and JNK-MAPK, while deletion of ASK-1 attenuates p38 and JNK activation and
the cardiac hypertrophic response to ATII [79].
Increasing evidence indicates that ROS can mediate apoptosis by a variety of
mechanisms depending upon the level of ROS produced [80]. For instance, in adult
cardiomyocytes, relatively low levels of H2O2 are associated with the activation of
ERK1/2 MAPK and the stimulation of protein synthesis. Conversely, a higher level of
H2O2, while still activating ERK1/2, also activated the JNK, p38 MAPKs and Akt and
induced apoptosis [80]. Using isolated cardiac cells and different reactive oxygen species
generating systems, researchers have shown that different ROS may use distinct apoptotic
17
pathways [81]. Moreover, the age-related accumulation of ROS-induced biomolecules
are thought to play a major role in the development of cardiovascular disease [82].
However, the signaling mechanisms underlying age-related oxidative stress-induced
cardiac pathologies remain not well understood. Further animal studies involving a wide
range of age groups and the use of different anti-oxidants will no doubt fill the gaps in
understanding of the signaling mechanisms underlying age-related oxidative stress
associated cardiac pathologies.
2.3.4 Age-associated cardiac apoptosis
Apoptosis is a specialized form of programmed cell death that is characterized by
nuclear chromatin condensation and DNA fragmentation. Once death occurs, cellular
debris undergo endocytosis by neighboring cells with no spillage of intracellular content
and no inflammatory response [77]. During the last few years, a growing body of
evidence in both humans and animal models suggests that increases in apoptosis due to
varying stimuli play an important role in the pathological remodeling of the heart [83].
Nevertheless, aging itself has been shown to predispose cardiomyocytes to apoptosis
[84]. Olivetti et al. examined the effects of aging on the human myocardium in 67 hearts
obtained from individuals between the ages of 17-90 years who died from causes other
than cardiovascular disease. Regression analysis of their results demonstrated that the
aging process was characterized by a loss of 38 million and 14 million myocyte nuclei/yr
in the left and right ventricular myocardium, respectively [85]. In another study Melissari
et al. measured cardiac dimensions with respect to body weight, and the number of
myocyte nuclei in the ventricles of 45 males and 22 females (17 to 90 years old), who
18
died from causes independent of cardiovascular disease. These researchers found that
aging was characterized by increases in total heart weight, while LV and inter ventricular
septum weights decreased significantly (r = 0.44; p < 0.001). At the structural level, the
number of myocyte nuclei within the left ventricle LV decreased (r = 0.45; p < 0.001),
whereas myocyte cell volume per nucleus increased (r = 0.30; p >0.05) [86]. Anversa et
al. examined the effect of aging in 3, 10 to 12, and 19 to 21 month old Fischer 344 rat
myocardium and reported that between 3 and 10 to 12 months of age, the mean myocyte
cell volume per nucleus increased by 53 % in the left ventricle while the total number of
myocyte nuclei remained constant [87]. Kajstura et al. determined the effect of aging on
cardiomyocyte cell death in Fischer 344 rats aged 3, 7, 12, 16, and 24 months and
reported that apoptosis is more prevalent than necrosis in the later stages of aging. This
study also showed that apoptosis in the left ventricle increased by more than 200% in 24-
month-old compared to 16-month old rats, with no change in necrotic cell death,
indicating an accelerated rate of apoptosis at a later age [88]. Recently, using the
F344XBN aging rat model, our laboratory has demonstrated that compared to young 6-
month hearts, aged 30-month and very aged 36-month hearts exhibited increased TdT-
mediated dUTP nick end labeling positive nuclei (TUNEL+ nuclei), caspase-3 activation,
caspase-dependent cleavage of alpha-fodrin and diminished phosphorylation of protein
kinase B/Akt (Thr 308) [20]. Taken together, these studies suggest that aging in
mammalian hearts is associated with increases in cardiomyocyte apoptosis and, in an
attempt to compensate the functional loss, the remaining myocytes undergo hypertrophy.
19
2.3.4.1 Signaling mechanisms underlying cardiac apoptosis
The factors regulating cardiac apoptosis are not well understood. Oxidative stress
has been shown to activate mitogen-activated protein kinases (MAPKs), which are
thought to regulate, at least in part, the fate of the cell to go through proliferation,
differentiation, or cell death [89-91]. Different studies have shown that the suppression of
ERK and activation of JNK and p38 MAPKs play a role in apoptosis in different cell
lines [90, 91]. An increased expression of JNK- and p38-MAPKs has been shown to
cause apoptosis in H9c2 cells [92], perfused rat hearts [93], as well as ischemia-
reperfusion rat heart [94]. The role of MAPKs signaling in age-related cardiomyocyte
apoptosis remains largely unknown.
In-vitro and in-vivo experimental models using various apoptotic stimuli have
demonstrated that upregulation of proapoptotic proteins Bax, caspase-3 and p53, as well
as downregulation of antiapoptotic protein Bcl-2, control the apoptotic machinery in the
heart [95-98]. Bax is a key pro-apoptotic regulator that can insert itself into the outer
mitochondrial membrane and promote the release of mitochondrial apoptogenic factors
like cytochrome C [98, 99]. Conversely, Bcl-2 is an anti-apoptotic factor that is thought
to block the membrane insertion of Bax by forming Bax/Bcl-2 heterodimers [100-102].
It is postulated that the ratio of Bax to Bcl-2 plays an important role in regulating cellular
apoptosis with cell death being favored as the balance shifts toward Bax [103]. Cytosolic
cytochrome-c couples with apoptotic protease activating factor-1 (Apaf-1), which leads
to the activation of caspase-9. Activated caspase-9 in turn has been shown to cleave
caspase-3 resulting in caspase-3 activation and proteolytic disassembly of the cell [104,
20
105]. On the other hand, inhibition of the phosphatidylinositol-3-kinase (PI-3 kinase)/Akt
pathway has been shown to induce apoptosis in both in vivo animal models and in vitro
cultured cardiomyocytes [106, 107]. Our laboratory has previously reported that the
increase in cardiac apoptosis observed in aging F344XBN rats is associated with
increased activation of proapoptotic proteins JNK1, JNK3, p38 and Bax, and decreased
expression of anti apoptotic proteins Bcl2, Akt and ERK1/2 [18, 20]. Whether the chronic
administration of antioxidants has an effect on the age-related cardiac remodeling is
currently unknown.
2.3.5 Age-associated cardiac hypertrophy
It is thought that aging in both humans and animal models is associated with
alterations in cardiac structure and function including myocyte dropout and a
compensatory hypertrophy of the remaining myocytes [16, 17, 108]. Although, the
increased heart size is initially a compensatory mechanism, sustained hypertrophy can
ultimately lead to decompensation, arrhythmias and contractile dysfunction, and thus
representing an independent risk factor for heart failure [109]. Gerstenblith et al.
performed echocardiograms on 105 male participants (25-84 years of age) and reported
that even in the absence of arterial hypertension there is a moderate increase in the
thickness of the LV wall, suggesting that aging in the normal male is associated with LV
hypertrophy [2]. In another study, Badano et al. performed echocardiograms on 104
physically active, normal, community-dwelling volunteers (68 men and 36 women),
ranging in age from 18 to 84 years and having no evidence of hypertension or
cardiovascular disease. Their data revealed that LV end-diastolic volume (r = 0.25), wall
21
thickness (r = 0.30) and mass (r = 0.37) increased with age [110]. To determine the age-
related changes in normal human hearts during the first 10 decades of life, Kirtzman et al.
measured heart weights, ventricular wall thicknesses, and valve circumferences in 765
autopsy specimens in the hearts of persons 20 to 99 years old. Their findings suggested
that the ratio of septal to LV free wall thicknesses was significantly elevated after the
seventh decade of life [111]. Similar to humans, echocardiographic studies in F344XBN
rats revealed that intrinsic cardiac aging in these rodent models is also associated with LV
hypertrophy, fibrosis, and diastolic dysfunction [16, 17]. The molecular mechanisms
underlying aging associated cardiac hypertrophy remain to be elucidated.
2.3.5.1 Signaling mechanisms underlying cardiac hypertrophy
The signaling mechanisms underlying cardiac hypertrophy have been widely
studied in models of physiological and pathological cardiac hypertrophy due to exercise,
hypertension, work overload and loss of myocytes following ischemic damage [112].
Characteristics of hypertrophic cardiac myocyte growth include an increase in myocyte
cross-sectional area, increased mRNA content, and protein synthesis [113]. Varying
extracellular stimuli have been shown to induce cardiomyocyte growth including peptide
and amine hormones (Ang II, norepinephrine (NE), endothelin-1, and thrombin),
cytokines (interleukin-1b (IL-1b)), interleukin-6, tumor necrosis factor-α (TNF-α), and
mitogens (fibroblast growth factor and epidermal growth factor) [77]. It is thought that
these agonists bind to cell-surface receptors where they activate multiple signal
transduction cascades that link receptor activation to the regulation of hypertrophic
22
growth, including tyrosine kinases (src, focal adhesion kinase), MAPK (ERK1/2, p38,
JNK, ERK5), PKC, calcineurin, PI3-kinase)/Akt, and NF-kB [114-116].
Several previous studies employing in vivo and in vitro models emphasized that
ERK1/2 MAPK plays a key regulatory role in promoting cardiac hypertrophy through the
Ras/Raf/MEK/ERK signaling pathway [110, 116]. Similar to ERK1/2 MAPK, the
activation of ERK5 in cardiomyocytes has also been shown to increase in response to a
variety of hypertrophic stimuli [117, 118]. Using a transgenic mice model, Sanna et al.
demonstrated that the MEK1–ERK1/2 pathway induces cardiac hypertrophy in vivo, at
least in part, by enhancing the transcriptional activity of nuclear factor of activated T
cells (NFAT) [119]. Calcineurin is a calcium-calmodulin activated serine/threonine-
specific phosphatase that is activated by sustained elevations in intracellular calcium and
is thought to mediate the hypertrophic response through its downstream transcriptional
effector NFAT [120]. Furthermore, chronic stimulation of the PI3K/Akt signaling
pathway in response to pressure overload in the rodent heart and following the incubation
of cultured cardiomyocytes with growth factors has also been shown to induce
myocardial hypertrophy [121-125]. In neonatal rat ventricular myocytes (NRVM),
H2O2 has been shown to induce hypertrophy via activation of src, PKC, and p38,
ERK1/2, and JNK MAP kinases and PI3-kinase signaling proteins, suggesting a
possible link between oxidative stress and cardiac hypertrophy [126-128]. Whether there
is a direct relationship between the age-related oxidative stress and cardiac hypertrophy is
currently unclear. Recently, the mammalian target of rapamycin (mTOR) has emerged as
a potentially important regulator of cardiac hypertrophy by promoting protein
23
translational signaling [129, 130]. Rapamycin, an inhibitor of mTOR activity has been
shown to attenuate cardiac hypertrophy caused by aortic banding induced pressure
overload [131], while it has also been shown to block skeletal muscle hypertrophy [132].
Mounting evidence indicates that mTOR induces hypertrophy by increasing protein
synthesis through the control of a number of downstream signaling proteins that regulate
mRNA translation [133] such as the translational repressor proteins 4E-binding protein-1
(4E-BP1) and the ribosomal protein S6 kinase (rp6k) [123]. Whether age-related
cardiomyocyte hypertrophy involves the same molecular mechanisms that have been
documented with varying hypertrophic stimuli in different models of hypertrophy or
involves different mechanisms remains unclear and warrants further experimentation.
2.3.6 Age-associated myocardial fibrosis
Extensive evidence, derived from both clinical and experimental studies, suggests
that aging in both humans and rodents is characterized by a progressive decrease in the
number of cardiomyocytes and the development of compensatory LV hypertrophy and
fibrosis leading to diastolic dysfunction and heart failure with preserved systolic function
[85, 87, 134, 135]. Previous reports suggest that age-related myocardial fibrosis and
ventricular remodeling leads to increased wall stress, decreased elasticity, impaired early
diastolic filling, and reduced rate of ventricular shortening [136-138]. Excessive fibrosis
can also cause decreases in myocardial compliance and may be linked to the development
of ventricular dysfunction and arrhythmias [139]. Age-related cardiomyocyte loss due to
apoptosis and necrosis leads to an increase in interstitial fibrosis in aged hearts [140].
Myocardial fibrosis is mainly due to progressive accumulation of collagen and other
24
extracellular matrix (ECM) proteins [141]. Eghbali et al. examined the total amount and
structure of myocardial collagen as a function of age in the hearts of male Fischer 344
rats and reported that collagen content in the left ventricle increased from 5.5% of total
protein in young Fischer 344 rats to approximately 12% in senescent animals [142].
Other studies have also shown a significant increase in collagen content in old
normocholesterolemic rabbits [143] and in senescent mice [144].
2.3.6.1 Signaling mechanisms underlying cardiac fibrosis
Although it is well established that cardiac fibrosis plays a key role in
deterioration of cardiac function in advanced age, the underlying molecular mechanisms
remain poorly understood. Recent findings suggest that the renin–angiotensin system and
the generation of reactive oxygen species (ROS) may be involved in age-associated
cardiac fibrosis [140]. Mitochondria isolated from senescent rats showed significant
increases in superoxide radical production [145], while over expression of catalase
targeted to mitochondria diminished cardiac hypertrophy and fibrosis and preserved
diastolic function [145]. These findings indicate an important role for ROS in the
pathogenesis of age-associated fibrotic cardiac remodeling. Siwiki et al. exposed
neonatal and adult rat cardiac fibroblasts in vitro to H2O2 or the superoxide-generating
system xanthine plus xanthine oxidase and demonstrated that oxidative stress regulates
the quantity and quality of extracellular matrix by modulating both collagen synthesis and
metabolism [146]. In another study, Cheng et al. treated cultured neonatal rat cardiac
fibroblasts with angiotensin II (Ang II) to examine the role of ROS mediated signaling in
cardiac fibroblasts and reported that ROS are involved in Ang II-induced proliferation
25
and endothelin-1 (ET-1) gene expression [147]. In a parallel experiment, they also
demonstrated that the combination of AT (I) and ET (A) receptor antagonists plus
antioxidants may be beneficial in preventing the formation of excessive cardiac fibrosis
[147]. Previous studies in both humans and animals have shown ROS-mediated up
regulation of MCP-1/CCL2 in ischemic cardiac fibrosis [36, 37]; however, its
involvement in aging-associated cardiac remodeling needs further investigation [148,
149]. Both ROS and angiotensin II have been shown to activate transforming growth
factor (TGF)-b/Smad2/3 signaling pathways and induce pro-inflammatory mediator
expression leading to cardiac fibrosis. In particular, activation of TGF-/Smad2/3
signaling promotes myofibroblast transdifferentiation and collagen deposition [150-155].
2.4 Regulation of miRNA in aged hearts
MiRNAs (miRNAs) are small ~ 22-nucleotide noncoding RNAs that inhibit
transcription or translation by interacting with the 3′ untranslated region (3′UTR) of
target mRNAs [156]. MiRNAs were first discovered in 1993 while conducting a study of
the gene lin-14 in C. elegans development [157]. Immediately, several researchers began
to shed light on the function of miRNAs and revealed that they play fundamental roles in
diverse biological and pathological processes, including cell proliferation, differentiation,
apoptosis, and carcinogenesis in species ranging from C. elegans and D. melanogaster to
humans [158-161]. Rooji et al. induced cardiac remodelling in mice by transverse aortic
constriction or expression of activated calcineurin and examined the expression of
distinctive miRNAs in diseased cardiac tissue. These researchers reported that >12
miRNAs were up- or down-regulated compared to normal mice [162]. Later, several
26
genetic studies conducted in mice implicated the regulatory role of miRNAs in cardiac
remodeling, growth, conductance and contractility [163-168]. Ikeda et al. measured the
expression of 428 miRNAs in three different types of human heart disease (ischemic
cardiomyopathy, dilated cardiomyopathy, and aortic stenosis). Their data suggest that
expression of many miRNAs is altered in heart disease and that different types of heart
disease are associated with distinct changes in miRNA expression [169]. In this context,
several miRNAs including miR-1, 21, 29, 30, 195, 133, 206 and 208, were shown to play
an important role in the process of cardiac remodeling, putatively by regulating changes
in gene expression that accompany pathological cardiac hypertrophy and contractile
dysfunction [162, 164, 165, 170]. Interestingly, muscle specific miR-1, miR-206 and
miR-133 expression has been shown to be upregulated, downregulated, and unchanged
during cardiac remodeling [164-166]. Furthermore, miR-1 has been implicated in the
etiology of ventricular arrhythmias as it is thought that this miRNA post-transcriptionally
represses the expression of connexin 43 (Cx43) [166]. Similarly, miR-206 has also been
postulated to play a role in cardiac remodeling as miR-206 levels have been shown to be
increased in a rat model of cardiac infarction [171] and in regulating the expression of
Cx43 [166]. Recent data suggest that increased miR-133 resulted in the suppression of
protein synthesis and inhibition of hypertrophic growth in cultured neonatal cardiac
myocytes [164]. MiR-133a has also been shown to act as an antiapoptotic factor by
repressing the translation of caspase-9 [172]. Conversely, miR-133 has also been
implicated in the pathogenesis of cardiac arrhythmias as it has been shown that this
molecule can regulate the ether-a-go-go related gene (ERG), which encodes a key portion
of the cardiac potassium channel [173]. Elevated miR-21 has been linked to increased
27
cardiac fibrosis and increased ERK1/2 MAPK activity [174], and its expression is
relatively low in adult cardiomyocytes compared to neonatal cardiomyocytes [174].
Alternatively, data from other studies suggest that miR-21 has an anti-apoptotic effect
and that it is upregulated during cardiomyocyte hypertrophy [175]. Recently, Duisters et
al. investigated the regulation of connective tissue growth factor (CTGF), and showed
that the expression of miR-133 and miR-30 was inversely related to the amount of CTGF
in rodent models of heart disease and in human patients with pathological LV
hypertrophy [176]. The disparities between results among different studies may likely
reflect differences in disease models, extent of disease, myocardial regions sampled,
assay platforms, normalization methods, sample size, control samples, and statistical
analyses. The factors that regulate the expression of miRNA are not clear; however,
recent studies have suggested that alterations in cellular ROS levels may be involved
[172, 177, 178]. Interestingly, the effect of antioxidants on the expression of miRNA
largely remains unclear and warrants further investigation. Similarly, how aging may
affect the expression of miR-1, miR-206 and miR-133 and their role in age-related
cardiac remodeling and arrhythmias remain unclear.
2.5 Acetaminophen
Acetaminophen (N-acetyl-p-amino-phenol; also known as paracetamol; Figure 1)
is one of the most widely used of all drugs and is well established as a standard
antipyretic and analgesic for mild to moderate pain states [179]. It was first synthesized
at Johns Hopkins University in 1877 and was used in clinical medicine in 1893 by Von
Mehring [180]. Since 1955 acetaminophen has been available over the counter without
28
prescription in the United States [180]. During the 1960s and 1970s, increasing concern
was raised about the toxicity of nonprescription analgesics, but in normal use
paracetamol exhibited a consistent safety profile [22]. In the 1980s, when aspirin was
linked to Reye's syndrome, acetaminophen became the mainstay analgesic and antipyretic
for children with a subsequent reduction in the incidence of Reye’s syndrome [179].
Currently, acetaminophen is a first-line drug of choice for pain management and
antipyresis in a variety of patients, including children, pregnant women, the elderly and
those with osteoarthritis, simple headaches, and noninflammatory musculoskeletal
conditions.
Figure 1. Molecular structure of acetaminophen
2.5.1 Mode of Action
Although acetaminophen has been in use for more than 50 years, its precise
mechanism of action remains unclear. It is thought to inhibit prostaglandin synthesis by
inhibition of cyclooxygenase 3 (COX3) in the central nervous system leading to the
antipyretic and analgesic action. It has little effect on the peripheral cyclooxygenase,
explaining its weak anti-inflammatory properties [181, 182].
29
2.5.2 Pharmacology
Acetaminophen is rapidly absorbed from the GI tract. In the liver, acetaminophen
undergoes glucuronidation or sulfation. Part of the drug is hydroxylated to N-acetyl-
benzoquinone-imine (NABI), which is a highly reactive metabolite [183]. Under normal
circumstances, NABI reacts with the sulfhydryl group on glutathione and is excreted in
urine. At very large doses (~7 times the therapeutic dose), glutathione in the liver is
depleted and large amounts of NABI bind covalently with SH groups on hepatic proteins,
permanently impairing their activity [184, 185]. This decrease in activity, if it persists,
can lead to hepatic necrosis, which may be life threatening [186, 187]. Unconjugated
metabolites can also cause renal tubular necrosis [188].
2.5.3 Cardioprotective value of acetaminophen
Despite decades of wide spread use, little is known about the potential
cytoprotective properties of acetaminophen. Until recently, most acetaminophen
investigations have focused on its analgesic/antipyretic properties and hepatotoxicity
while the effects of this drug on other mammalian organ systems, including the heart and
vasculature, have been largely ignored.
When used to block the synthesis of prostaglandin, acetaminophen caused the
concentration-dependent decrements in the production of prostacyclin [189]. Chou et al.
found that by attenuating the activity of myeloperoxidase, which is an important marker
of heart disease in humans [190], acetaminophen significantly reduced the oxidation of
low-density lipoprotein (LDL) in macrophages [191]. With all these preliminary results,
acetaminophen has begun to be considered as an effective antioxidant because of its
30
ability to protect from membrane lipid oxidation by scavenging peroxyl radicals and
peroxynitrite [192]. Nakamoto et al. examined the effect of acetaminophen on acute
gastric mucosal injury induced by ischemia-reperfusion in rats and showed that
acetaminophen (300 and 500 mg/kg) administered intraperitoneally 90 min before the
ischemia significantly reduced the total area of erosions measured 60 minutes after
reperfusion compared to control animals. The drug also inhibited the increase in lipid
peroxide levels induced by ischemia-reperfusion in gastric tissue and the increase in lipid
peroxidation caused by hydroxyl radicals in vitro. With these results, they concluded that
acetaminophen protects the gastric mucosa by averting the damaging effects of oxygen
radicals [193]. Merrill et al. have shown that acetaminophen protects isolated guinea pig
hearts from the damaging effects of myocardial ischemia and reperfusion, that it
preserves myofibrillar ultrastructure, and was associated with the attenuated production
of both hydroxyl radicals and peroxynitrite [29]. In an another study, Merrill et al.
investigated the effect of acetaminophen on pentobarbital pretreated isolated guinea pig
hearts that underwent ischemia-reperfusion and reported that acetaminophen
(0.35mmol/l) significantly reduces the frequency of pentobarbital induced arrhythmias
[27]. Collectively these findings suggest that under selective conditions (e.g., those
causing release of free radicals and other oxidants) acetaminophen can act as an
antioxidant and is able to prevent ventricular arrhythmias [27, 29]. Merrill et al. has
further investigated the cardioprotective efficacy and antioxidant mechanisms of
acetaminophen in global, low-flow myocardial ischemia and reported that acetaminophen
reduced ischemia-mediated protein oxidation. Furthermore, acetaminophen was able to
reduce ventricular dysfunction caused by exogenously administered hydrogen peroxide
31
indicating that antioxidant mechanisms were responsible for its cardioprotective
properties during postischemia-reperfusion [25]. Golfetti et al. showed that this was true
even if acetaminophen was administered chronically or at the onset of reflow [28].
Furthermore, acetaminophen has shown to attenuate peroxynitrite-mediated activation of
matrix metalloproteinase-2 (MMP-2) and subsequent cleavage of troponin I (TnI) in
ventricular myocytes following myocardial ischemia-reperfusion in isolated guinea pig
hearts, and to preserve contractile function [194-196]. Experiments directed at
investigating the effect of acetaminophen treatment on non-ischemic, hypoxic and
reoxygenated myocardium revealed that acetaminophen treated hearts retained a greater
fraction of mechanical function and preserved myocardial ultrastructure compared to the
vehicle treated hearts [24]. In the dog, acetaminophen treatment has been shown to
reduce infarct size following ischemia-reperfusion [197]. In another study, the same
authors investigated the antiarrhythmic properties of acetaminophen in dogs by inducing
arrhythmias either by regional myocardial ischemia and reperfusion or injecting toxic
levels of ouabain. Their results indicated that acetaminophen has previously unreported
antiarrhythmic properties and that the mechanism appears to involve either protection of
mitochondrial Na+-K
+ pumps or protection of function of the pumps at the sarcolemma
and other locations [198]. Hadzimichalis et al. reported that in guinea pig hearts,
ischemia/reperfusion causes a significant increase in mitochondrial permeability
transition pore (MPTP) opening and mitochondrial cytochrome c release, and that
acetaminophen treatment during injury significantly and completely blocks these effects
leading to a significant decrease in late stage apoptotic myocytes [199]. Taken these
studies together, there is cumulative evidence suggesting that acetaminophen confers
32
functional cardioprotection following cardiac insult, including ischemia-reperfusion,
hypoxia-reoxygenation, exogenous hydrogen peroxide and peroxynitrite administration in
different animal models. The mechanistic pathway(s) underlying acetaminophen
mediated cardioprotection may involve its ability to scavenge hydroxyl radical [25],
hydrogen peroxide [200], peroxynitrate [197], myeloperoxidase [191], cyclooxygenase
[189], and other peroxides [193]. Whether the chronic ingestion of acetaminophen is
efficacious in preventing or reducing age-associated cardiovascular dysfunction has not
been investigated.
33
CHAPTER III
The following chapter includes three different research papers describing in detail the
research experiments conducted to test our hypotheses set forth for the specific aims I, II
and III of this dissertation project.
PAPER 1
The following paper corresponds to the specific aim I
34
Mechanisms of age-related cardiac hypertrophy in the F344XBN rat model
Sunil K. Kakarla1,3
, Sudarsanam Kundla2,3
, Madhukar Kolli1,3
, Anjaiah Katta1, Siva K.
Nalabotu1,3
, Emily Whitt3, Kevin M. Rice
2,3,4 and
Eric R. Blough
1,2,3,4
1 Department of Pharmacology, Physiology and Toxicology, Marshall University,
Joan C. Edwards School of Medicine 2
Department of Biological Sciences, Marshall University 3 Center for Diagnostic Nanosystems, Marshall University
4 Department of Exercise Science, Sport and Recreation, College of Education and
Human Services, Marshall University, Huntington WV
Key words: Aging, Myocardial hypertrophy, protein translational signaling, ERK1/2,
Akt
Running Title: Age-related cardiac hypertrophy is associated with increased
ERK/Akt/mTOR signaling.
35
Abstract
Aims: The aging heart undergoes well characterized changes in cardiac structure and
function though the underlying molecular mechanisms remain obscure. Recent studies
suggest that age-related increases in oxidative stress are associated with increases in
cardiac apoptosis and compensatory hypertrophy of the remaining cardiomyocytes. Here
we examine the possible molecular mechanisms underlying age-related cardiac
hypertrophy in 6, 27, 30, 33 and 36-month F344XBN rat hearts. Methods and results:
Compared to 6-months, the heart to body weight ratio remain unchanged in 27 and 30-
month rats, but was significantly increased by 26% and 37% in 33- and 36-month old
rats, respectively (P < 0.05). Immunohistochemical staining for the sarcolemmal protein
dystrophin and cytoskeletal protein actin revealed robust increases in cardiomyocyte fiber
cross sectional area by 85% and 187% in very aged 33- and 36-month animals (P <
0.05). Increases in cardiomyocyte size was accompanied by the hyperphosphorylation of
several different signaling molecules including ERK1/2, ERK5, Akt (Ser473 and
Thr308), GSK3β, PTEN, mTOR (Ser2448), 4EBP1 (Thr 37/46) and rpS6 (Ser235/236)
(P < 0.05). Conclusion: Taken together, these data suggest that aging in the F344XBN
rat heart is associated with the activation of the ERK1/2 and Akt signaling pathways.
(Word Count: 189)
36
Introduction
Cardiovascular disease remains the leading cause of death in the elderly
population and it is estimated that by 2035, nearly one in four individuals in the United
States will be sixty-five years of age or older [201]. The aged heart undergoes well
characterized structural changes leading to a diminished functional and adaptive reserve
capacity, an increased susceptibility to incur damage and a limited practical ability for
repair / regeneration [5]. Cellular and molecular mechanisms that have been implicated in
age-associated changes in myocardial structure and function in humans have been studied
largely in rodents. The Fischer 344/Brown Norway F1 (F344/BNF1) rat is recommended
for age-related studies by the National Institutes on Aging because this hybrid rat lives
longer and has a lower rate of pathological conditions than inbred rats [202]. According
to the probability of aging curves generated by National Institute on Aging, the aged 30-
month and very aged 36-month rats correspond roughly to humans in their sixth, and
eighth decades of life, respectively [17]. We and others have performed
echocardiographic studies in F344XBN rats and have reported that intrinsic cardiac aging
in these rodent models closely recapitulates age-related cardiac changes in humans,
including left ventricular hypertrophy, fibrosis, and diastolic dysfunction [16, 17].
Previous data from our laboratory has also suggested that aging in F344XBN rat hearts is
associated with increases in oxidative-nitrosative stress and apoptosis [18, 20, 203].
Recent studies in the aged heart have shown that to compensate for the functional loss
due to cardiomyocyte death that the remaining cardiomyocytes undergo compensatory
hypertrophy [87].
37
Although, the increased heart size is initially a compensatory mechanism,
sustained hypertrophy can ultimately lead to decompensation, arrhythmias, contractile
dysfunction and thus represents an independent risk factor for heart failure [109]. It is
well established that increased protein synthesis is a key feature of cardiac hypertrophy
and likely underlies the increased cell and organ size observed under this condition [204].
Previous studies have shown that the extracellular signal-regulated kinase 1/2 (ERK1/2)
plays a central regulatory role in promoting cardiac hypertrophy through
Ras/Raf/MEK/ERK signaling cascade both in vivo and in vitro [205-209] . Other studies
suggested a significant role for protein kinase B or Akt, a serine threonine kinase in
promoting cardiac hypertrophy to varying stimuli [123, 210]. Recently, mammalian
target of rapamycin (mTOR) has emerged as potentially important regulator
of cardiac
hypertrophy by promoting protein translational signaling [129, 130]. The molecular
mechanisms underlying cardiac hypertrophy have been very well investigated in models
of physiological and pathological cardiac hypertrophy due to exercise, hypertension,
work overload and loss of myocytes following ischemic damage [112]. However, the
molecular mechanisms underlying aging associated cardiac hypertrophy are not well
understood. A fundamental understanding of the molecular events underlying age-related
cardiac hypertrophy will provide an additional dimension to studies of aged myocardial
biochemistry and function and may be useful in elucidating mechanisms of excitation-
contraction coupling in the aging heart. Using F344XBN rat model, here we investigated
the molecular signaling profile underlying age-related cardiac hypertrophy and report that
very aged hearts exhibit activation of multiple signaling mechanisms during the
hypertrophic response.
38
Materials and Methods
Animals
Animal care and procedures were conducted in accordance with the Animal Use
Review Board of Marshall University using the criteria outlined by the American
Association of Laboratory Animal Care (AALAC) as proclaimed in the Animal Welfare
Act (PL89-544, PL91-979, and PL94-279). Young (6 months, n = 6), adult (27 months, n
= 6), aged (30-months, n = 6), and very aged (33- and 36 months, n = 6) male F344XBN
rats were obtained from the National Institute on Aging. Rats were housed two per cage
in an AALAC -approved vivarium. Housing conditions consisted of a 12 hour: 12 hour
dark – light cycle with temperature maintained at 22 ± 2°C. Animals were provided food
and water ad libitum. Rats were allowed to acclimate to the housing facilities for at least
two weeks before experimentation began. During this time, the animals were carefully
observed and weighed weekly. None of the animals exhibited signs of failure to thrive,
such as precipitous weight loss, disinterest in the environment, or unexpected gait
alterations.
Tissue collection
Rats were anesthetized with a ketamine-xylazine (4:1) cocktail (50 mg/kg, I/P)
and supplemented as necessary for reflexive response before tissue collections. After
midline laparotomy, the heart was removed and placed in Krebs–Ringer bicarbonate
buffer containing; 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2
mM MgSO4, 24.2 mM NaHCO3, and 10 mM D-glucose (pH 7.4) equilibrated with 5%
CO2 / 95% O2 and maintained at 37°C. Isolated hearts were quickly massaged to remove
39
any blood from the ventricles, cleaned of connective tissue, weighed, and immediately
snap frozen in liquid nitrogen.
Tissue homogenization and determination of protein concentration
Cardiac muscles were homogenized in a Pierce Tissue Protein Extraction Reagent
(T-PER) (10 mL/g tissue; Rockford, IL, USA) that contained protease inhibitors (P8340,
Sigma-Aldrich, Inc., St. Louis, MO, USA) and phosphatase inhibitors (P5726, Sigma-
Aldrich, Inc., St. Louis, MO, USA). After incubation on ice for 30 min, the homogenate
was collected by centrifuging at 12,000 g for 5 min at 4◦C. The protein concentration of
homogenates was determined via the Bradford method (Fisher Scientific, Rockford, IL,
USA). Homogenate samples were boiled in a Laemmli 2× sample buffer (Sigma-Aldrich,
Inc., St. Louis, MO, USA) for 5 min.
SDS-PAGE and immunoblotting
Fifty micrograms of total protein from each sample was separated on a 10%
PAGEr Gold Precast gel (Lonza, Rockland, ME, USA) and then transferred onto a
nitrocellulose membrane using standard conditions [211]. Membranes were stained with
Ponceau S, and the amount of protein quantified by densitometric analysis to confirm
successful transfer of proteins and equal loading of lanes as described previously [212-
214]. For immunodetection, membranes were blocked for 1 h at room temperature in
blocking buffer (5% non-fat dry milk in TBS-T (20mM Tris-base, 150mM NaCl, 0.05%
Tween-20), pH 7.6), serially washed in TBS-T at room temperature, then probed with
antibodies for the detection of Akt (#9272), phospho-Akt (Ser473) (#9271), phospho-Akt
40
(Thr308) (#9275), mTOR (#2972), phospho-mTOR (Ser2448) (#2971), rpS6 (#2217),
phospho-rpS6 (Ser235/236) (#4858), 4E-BP1 (#9452), phospho 4E-BP1 (Thr37/46)
(#9459), eEF2 (#2332), phospho-eEF2 (Thr56) (#2331), Tuberin/TSC2 (#3612),
phospho-Tuberin/TSC2 (Thr1462) (#3617), PTEN (#9552), phospho-PTEN
(Ser380/Thr382/383) (#9552), GSK-3β (#9315) and phospho- GSK-3β (#9336s) (from
Cell Signaling Technology, Inc., Beverly, MA, USA). Membranes were incubated
overnight at 4◦C in primary antibody buffer (5% BSA in TBS-T, pH 7.6, primary
antibody diluted 1:1000), followed by washing in TBS-T (3X 5 min each), and
incubation with HRP- conjugated secondary antibody (anti-rabbit (#7074) or anti-mouse
(#7076), Cell Signaling Technology, Inc., Danvers, MA, USA) in blocking buffer for 1 h.
After removal of the secondary antibody, membranes were washed (3X 5 min each) in
TBS-T and protein bands visualized on reaction with ECL reagent (Amersham ECL
Western Blotting reagent RPN 2106, GE Healthcare Bio-Sciences Corp., Piscataway, NJ,
USA). Target protein levels were quantified by AlphaEaseFC image analysis software
(Alpha Innotech, San Leandro, CA, USA) and normalized to glyceraldehyde 3-phosphate
dehydrogenase (GAPDH).
Immunohistochemistry
Briefly, sections (8 µm) were washed three times with phosphate-buffered saline
(PBS) containing 0.05% Tween-20 (PBS-T), pH 7.4. After incubation for 30 min in a
blocking solution (5% BSA in PBS-T), sections were incubated with specific antisera
diluted in 3% BSA in PSB-T (antibody dilution of 1:100) for 1 h at 24°C in a humidified
chamber and washed three times with PBS-T prior to incubation with FITC labeled anti-
41
rabbit IgG (1:200) for 30 min at 24°C in a humidified chamber. Experiments performed
in parallel omitting either the primary or the secondary antibody was utilized to confirm
specificity and background immunoreactivity. DAPI was included in the secondary
antibody solution at a concentration of 1.5 µg/ml in order to visualize cell nuclei. After a
final PBS wash and mounting, specimens were visualized by epifluorescence using an
Olympus fluorescence microscope (Melville, NY, USA) fitted with 209 and 409
objectives. Images were recorded digitally using a CCD camera and the integrated optical
density determined using the AlphaEase software package (Alpha Innotech, Santa Clara,
CA, USA).
Data analysis
Results are presented as mean ± SEM. Data were analyzed by using the SigmaStat
3.5 statistical program. A one-way analysis of variance was performed for overall
comparisons with the Student–Newman–Keuls post hoc test used to determine
differences between groups. The level of significance accepted a priori was P ≤ 0.05.
42
Results
Aging in the F344XBN rats is associated with cardiac hypertrophy
Compared to 6-month animals, heart size was visibly larger with age in 27-month
animals and appears to be same in 30-month animals, but then appeared significantly
bigger in 33- and 36-month animals (Figure 2.1A). Compared to 6-month animals, the
body weight was significantly increased until 27-months and then gradually decreased
with age in 30-, 33- and 36-month animals but still significantly higher compared to
young 6-month animals (Figure 2.1B). In a similar fashion, the heart weight was
significantly increased with age in 27-month animals and then tends to decline slightly
yet significantly in 30-month animals, while significantly higher in 33- and 36-month
animals compared to 6-month animals (Figure 2.1B). Whereas, the heart weight to body
weight ratio remained unchanged between 6-, 27- and 30-month animals, while it was
significantly higher in 33- and 36-month animals compared to 6-, 27-, and 30-month
animals (Figure 2.1B) (P < 0.05).
To investigate whether increases in heart mass were associated with larger cardiac
myocytes we next measured the muscle fiber cross sectional area (CSA). Because the
myofibres in different areas of the heart are orientated in different directions we imaged
only those areas of the left ventricle where all the muscle fibers were orientated in a
transverse direction. This task was accomplished by using immunofluorescence after
labeling for dystrophin (FITC-green), actin (rhodamine phalloidin-Red) and DAPI (blue)
on tissue cross sections obtained from 6-, 27-, 30-, 33- and 36-month rat hearts.
Compared to that observed in the 6-month animals, our analysis indicated that the
43
average muscle fiber CSA was 20%, 33%, 85% and 187% higher in the 27-, 30-, 33- and
36-month rat hearts, respectively (Figure 2.2) (P < 0.05).
Aging associated cardiac hypertrophy in F344XBN rats is associated with increases
in the activation of the Ras/Raf/MEK/ERK signaling cascade
Several studies have implied that the extracellular signal-regulated kinase 1/2
(ERK1/2) may play a central regulatory role in promoting cardiac hypertrophy through
the Ras/Raf/MEK/ERK signaling cascade [209]. Herein, our immunoblotting results also
suggest that age-related cardiac hypertrophy in the F344XBN rat is associated with a
significant increase in the phosphorylation of ERK1/2. Compared to that observed in the
hearts obtained from 6-month animals, the phosphorylation of ERK1/2 was 19% and
23% lower in 27- and 30-month hearts before increasing by 50% and 263% in the very
aged 33- and 36- month animals (Figure 2.3A) (P < 0.05). In addition to ERK1/2, the
extracellular signal-regulated kinase ERK5 and the serine/threonine-specific phosphatase
calcineurin (CaN) have also been shown to be activated in cardiomyocytes during the
hypertrophic process [117, 119]. Similar to that observed for ERK1/2, it appears that
ERK5 and CaN exhibit a bimodal signaling response with aging as the expression of
these molecules first decreases with age before returning to levels similar to that seen in
the 6-month adult hearts (Figures 2.3B, 3C) (P < 0.05).
PI3K/Akt signaling in aging associated cardiac hypertrophy in the F344XBN rat
Compared to 6-month animals, phosphorylation of Akt at Ser 473 decreased was
31%, 63%, 56% lower and unchanged in 27-, 30-, 33- and 36-month hearts, respectively
(Figure 2.4) (P < 0.05). Similarly, the phosphorylation of Akt at Thr 308 decreased
44
significantly by 21%, 18%, unchanged in 27-, 30-, and 33-month animals before
becoming increased in the very aged 36-month old animals (Figure 2.4) (P < 0.05).
Consistent with our Akt findings, the phosphorylation of GSK-3β was 17% and 31%
lower in 27- and 30-month old animals, unchanged in 33-month animals before
increasing to a level 26% higher than that seen in the 6-month animals at 36-months.
(Figure 2.5A) (P < 0.05). Compared to 6-month animals, the phosphorylation of PTEN
significantly decreased with age in 27-, 30- and 33-month hearts by 45%, 53% and 30%,
respectively while it remain unchanged in the very aged 36-month animals (Figure 2.5B)
(P < 0.05).
The mammalian target of rapamycin (mTOR) has recently emerged as potentially
important regulator of cardiac hypertrophy [130]. Phosphorylation of mTOR at Ser 2448
significantly decreased with age in 27- and 30-month hearts by 30% and 30%, while it
was unchanged and 73% higher in the hearts obtained from 33- and 36-month old
animals (Figure 2.6) (P < 0.05). Compared to 6-month animals, the amount of Tuberin /
TSC2 decreased significantly with aging by an amount of 22%, 23%, 33% in 27-, 30-,
and 33-month animals and unchanged in the very aged 36-month animals (Figure 2.7A)
(P < 0.05). The amount of phosphorylated Tuberin / TSC2 remain was 14%, 26% and
56% lower in 30-, 33- and 36-month animals compared to 6-month animals (Figure 2.7A)
(P < 0.05). When compared to 6-month animals, the cardiac expression of raptor was
24%, 48%, 33% and 27% lower in 27-, 30-, 33- and 36-month animals (Figure 2.7B) (P
< 0.05).
45
Age-related cardiac hypertrophy in F344XBN rats is associated with increased
protein translational signaling
Increase in phosphorylation of mTOR leads to activation of its downstream
targets S6K1 and 4EBP-1 [215]. Phosphorylated 4EBP1 becomes inactive and binds to
and inactivates eIF4E thereby increasing protein translation [216]. Similar to our findings
for mTOR, the phosphorylation of 4EBP1 was 15%, 33%, 32% lower in 27-, 30- and 33-
month animals, while it remained unchanged in the very aged 36-month animals (Figure
2.8A) (P < 0.05). Upon activation by mTOR, S6K1 in turn phosphorylates ribosomal
protein S6 (rpS6) leading to increased ribosomal biogenesis and ultimately protein
translation [217]. Compared to 6-month animals, the phosphorylation of rpS6 was 24%,
67%, 13% and 93% higher in 27-, 30-, 33- and 36-month animals (Figure 2.8B) (P <
0.05).
46
Discussion
In spite of advances in treatment and early diagnostic approaches, cardiovascular
disease still remains the leading cause of death in the elderly. Studies in both humans and
animal models have indicated that aging is associated with alterations in cardiac structure
and function including myocyte dropout and a compensatory hypertrophy of the
remaining myocytes [16, 17, 108]. Similar to that seen in humans, previous work using
the aging F344XBN rat model, has demonstrated aging in these rats is associated with
increases in cardiac oxidative-nitrosative stress, and apoptosis [18, 20]. Here we examine
the signaling processes that may be associated age-related cardiomyocyte hypertrophy.
Heart size and heart weight gradually increased with age until 27-months,
declined slightly in 30-month animals and then regained in 33- and 36-month animals,
while the body weight increased significantly until 27-months and then tends to decline
gradually in 30-, 33- and 36-month rats (Figure 2.1). Whereas the heart to body weight
ratio remain unchanged up to 30 months before increasing significantly and peaking at
36-months of age (Figure 2.1). These findings suggest that the heart seems to grow in a
parallel fashion with body weight until 27-months of age and then declining at 30-months
of age before undergoing pathological hypertrophy in very aged 33- and 36-months of
animals. The decline in heart weight we observed in 30-month animals compared 27-
month animals might be due to increase in cardiomyocyte apoptosis with age, which we
have reported earlier in F344XBN rats [20]. To investigate whether the increases in heart
weight were due to cardiomyocyte hypertrophy or to increases in fibrosis or some other
alteration, we measured the cardiomyocyte cross sectional area. Our data showed that the
47
cardiomyocyte fiber cross sectional areas tends to increase rapidly from 30 – 36 months
before reaching a maximal diameter at 36 months that is nearly three times larger than
that observed in the 6-month adult animals (Figure 2.2). These data are in agreement with
the heart to body weight ratio findings discussed above indicating that the
cardiomyocytes grow in size in a parallel fashion to body weight until 30-months of age
and then rapidly growing in size due pathological cardiac hypertrophy in very aged 33-
and 36-month animals.
The factors regulating cardiac hypertrophy are not fully understood however
several reports have demonstrating a role for the Raf/MEK/ERK signaling pathway [123,
204, 208, 209, 218, 219]. The data of the present study support this contention as ERK1/2
was robustly increased in the 33- and 36-month rat hearts (Figure 2.3A). Similar findings
were also observed for ERK5 (Figure 2.3B). Like ERK1/2, the activation of ERK5 in
cardiomyocytes has been shown to increase in response to a variety of hypertrophic
stimuli [117, 118]. Using a transgenic mice model, Sanna et al., demonstrated that
MEK1–ERK1/2 pathway induces cardiac hypertrophy in vivo, at least in part, by
enhancing the transcriptional activity of NFAT [119]. Calcineurin is a calcium-
calmodulin activated serine/threonine-specific phosphatase that is activated by sustained
elevations in intracellular calcium and is thought to mediate the hypertrophic response
through its downstream transcriptional effector nuclear factor of activated T cells
(NFAT) [120]. Similar to our findings for the activation of ERK1/2 and ERK5, we also
observed a decrease in the amount of calcineurin with age in 27- and 30-month animals
and then tend to recover in very aged 33- and 36-month rat hearts (Figure 2.3C).
48
It is well accepted that the chronic stimulation of the PI3K/Akt signaling pathway
participates in the development of myocardial hypertrophy [121-123]. Indeed, PI3K/Akt
signaling has been shown to be activated in response to pressure overload in the rodent
heart and following the incubation of cultured cardiomyocytes with growth factors [124].
PI3K inhibitors have also been shown to block ligand-induced hypertrophy in cultured
cardiomyocytes [220] while the over-expression of a constitutively active Akt (caAkt) in
the heart has been reported to increase heart weight and cell size [221], [222]. Our data
are in agreement with these findings. Specifically, we observed decreased
phosphorylation (activation) of Akt at Ser 473 and Thr 308 with age in 27- and 30-month
animals, while significantly recovered in 33- and 36-month animals (Figure 2.4). The
decrease in the amount of calcineurin and the phosphorylation of Akt we observed in 30-
month animals compared to 6- and 27-month animals might be due increases in
cardiomyocyte apoptosis in 30-month F344XBN rats that we have reported before and
the underlying molecular mechanisms awaits future investigations [20]. The GSK3β is a
downstream substrate of Akt that is inhibited by Akt-mediated phosphorylation. GSK3β
functions to negatively regulate the activity of GATA4, β-catenin, c-Myc and NFAT
[223] which leads to a blunting of the hypertrophy response [224]. In agreement with this
data, we note an increased phosphorylation GSK3β in very aged and hypertrophied 36-
month F344XBN rat hearts (Figure 2.5A). Phosphatase and tensin homolog (PTEN), has
been shown to function as a negative modulator of PI3K/Akt/mTOR pathway that is
inactivated upon phosphorylation [225]. Consistent with our Akt data, the
phosphorylation of PTEN was found to be decreased with age in 27- and 30-months of
age and then recovered significantly in the very aged 33- and 36-month animals (5B).
49
Taken together, our data suggests that the PI3K/Akt pathway may be involved in
regulating age-associated cardiac hypertrophy in F344XBN rats.
Like Akt, a growing body of evidence suggests that mTOR may play a key role in
regulating cell growth and protein synthesis [129, 226]. Rapamycin, an inhibitor of
mTOR activity has been shown to attenuate pressure overload cardiac hypertrophy
induced by aortic banding [131] and also blocked skeletal muscle hypertrophy [132].
Here we demonstrate a large increase in the amount of mTOR phosphorylation in the
very aged 36-month animals (Figure 2.6). To examine the upstream signaling events that
may be associated with this age-associated activation of mTOR, we next investigated the
effect of aging on the regulation of TSC2 and Raptor. The tuberous sclerosis complex
TSC2 inhibits mTOR when unphosphorylated while the phosphorylation of this molecule
is thought to lead to increased mTOR activity [227]. Unexpectedly, we found a gradual
decrease in the phosphorylation of TSC2 with aging (Figure 2.7A). As decreased TSC2
phosphorylation is hypothesized to decrease mTOR activity, it is possible that the aging-
associated activation of mTOR we observed is not due to alterations in TSC2 regulation.
In addition, why aging-related increases in the phosphorylation of Akt are not associated
with higher levels of TSC2 phosphorylation is currently unclear. Given that recent studies
have suggested a cross talk between Ras/ERK signaling and mTOR during cardiac
hypertrophy [228, 229], the increased phosphorylation of mTOR we see with aging may
be ERK1/2-dependent or due to some other factor. Besides TSC2, it is thought that
Raptor may also function to influence mTOR activity [230]. Unlike our findings for
TSC2, the amount of Raptor was found to decrease with age until 30-months of age and
50
then tend to recover significantly in the very aged 33- and 36-month animals (Figure
2.7B). Whether this increase in Raptor is directly linked to the increased phosphorylation
of mTOR is currently unclear. Future studies directed to investigate the link will no doubt
provide clues for better understanding of the molecular mechanisms that underlie age-
related cardiac hypertrophy.
It is thought that mTOR may be involved in controlling a number of downstream
signaling proteins that regulate mRNA translation [133] such as the translational
repressor proteins 4E-binding protein-1 (4E-BP1) and the ribosomal protein S6 kinase
(rp6k) [123]. Consistent with our findings of an increased mTOR phosphorylation, here
we found that the phosphorylation of 4EBP1 decreased significantly with age until 33-
months before recovering to the level of 6-month animals in very aged 36-month animals
(Figure 2.8A). The phosphorylation of rpS6 was increased significantly in the very aged
36-month animals (Figures 2.8B). Taken together, our current and previous findings [17,
18, 20, 203] suggest that aging in the F344XBN rat heart is associated with diminished
cardiac function, elevations in oxidative stress, increased cardiomyocyte apoptosis and
cardiomyocyte hypertrophy.
In summary, findings from this study suggest that aging in F344XBN rats is
associated with cardiac hypertrophy and the underlying molecular mechanisms probably
involve increased activation of the ERK1/2 and Akt signaling pathways (Figure 2.9). As
shown in schematic diagram (Figure 2.9), advanced aging in F344XBN rat hearts is
associated with increased activation of ERK1/2-Akt signaling pathways leading to
increased activation of mTOR associated signaling mechanisms thereby resulting in
51
increased protein synthesis and ultimately cardiac hypertrophy. How these events and
processes are interrelated is still unclear and will require further investigation. Given the
high prevalence of age-associated heart disease worldwide and the ever growing
expansion of the elderly population it is anticipated that a greater understanding of the
molecular mechanisms associated with age-related cardiac hypertrophy could lead to
valuable improvements in the ability to manage heart disease.
Funding
This study was supported by Department of Energy Grant DE-PS02-09ER09-01 to
E.R.B.
52
Figures
Figure 2. 1. Heart size, heart weight, body weight, heart weight to body weight ratio altered with age in F344XBN rats. A. Image series depicting hearts immediately after excision from the rats. B. Heart weight and heart weight to body weight ratio significantly increased in very aged 33- and 36-month animals compared to 6-, 27- and 30-month animals. Data are mean ± SE (n = 6). abc: Groups without the same letter are significantly different (P < 0.05).
53
Figure 2. 2. Aging in F344XBN rats is associated with cardiomyocyte hypertrophy. Aging in F344XBN rats is associated with cardiomyocyte hypertrophy. A. Immunofluorescence labeling of dystrophin (FITC-green), actin staining (rhodamine phalloidin-red) and nuclei (DAPI-blue) in 6-, 27-, 30-, 33- and 36-month F344XBN rat heart cross sections. Bar = 50µm. B. Muscle fiber cross sectional area (µm2/fiber). Number of fibers measured for 6-, 27-, 30-, 33- and 36-month rats was 640, 423, 311, 235 and 190 respectively. Data are mean ± SE (n = 4). abcd: Groups without the same letter are significantly different (P < 0.05).
54
Figure 2. 3. Aging is associated with higher phosphorylation of ERK1/2, ERK5 and total calcineurin protein in F344XBN rat hearts. Protein isolates of hearts excised from 6-, 27-, 30-, 33- and 36-month rats were analyzed by immunoblotting for changes in total and phosphorylated ERK1/2 (A), ERK5 (B) and total protein content of calcineurin (C). Ponceau S staining of the nitrocellulose membrane along with densitometric analysis of protein present was done to verify equivalent protein loading between the lanes (data not shown). Data are mean ± SE (n = 6). abcde: Groups without the same letter are significantly different (P < 0.05).
55
Figure 2. 4. Aging is associated with higher phosphorylation of Akt in F344XBN rat hearts. Protein isolates of hearts excised from 6-, 27-, 30-, 33- and 36-month rats were analyzed by immunoblotting for changes in total and phosphorylated Akt (Ser 473, Thr 308). Ponceau S staining of the nitrocellulose membrane along with densitometric analysis of protein present was done to verify equivalent protein loading between the lanes (data not shown). Data are mean ± SE (n = 6). abcd: Groups without the same letter are significantly different (P < 0.05).
56
Figure 2. 5. Aging is associated with higher phosphorylation of GSK3β and PTEN in F344XBN rat hearts. Protein isolates of hearts excised from 6-, 27-, 30-, 33- and 36-month rats were analyzed by immunoblotting for changes in total and phosphorylated GSK3β (A) and PTEN (B). Ponceau S staining of the nitrocellulose membrane along with densitometric analysis of protein present was done to verify equivalent protein loading between the lanes (data not shown). Data are mean ± SE (n = 6). abcd: Groups without the same letter are significantly different (P < 0.05).
57
Figure 2. 6. Aging is associated with higher phosphorylation of mTOR in F344XBN rat hearts. Protein isolates of hearts excised from 6-, 27-, 30-, 33- and 36-month rats were analyzed by immunoblotting for changes in total and phosphorylated mTOR (Ser 2448). Ponceau S staining of the nitrocellulose membrane along with densitometric analysis of protein present was done to verify equivalent protein loading between the lanes (data not shown). Data are mean ± SE (n = 6). abcd: Groups without the same letter are significantly different (P < 0.05).
58
Figure 2. 7. Aging lowers the phosphorylation of TSC2 and is associated with higher expression of Raptor in F344XBN rat hearts. Protein isolates of hearts excised from 6-, 27-, 30-, 33- and 36-month rats were analyzed by immunoblotting for changes in total and phosphorylated TSC2 (A) and total protein content of Raptor (B). Ponceau S staining of the nitrocellulose membrane along with densitometric analysis of protein present was done to verify equivalent protein loading between the lanes (data not shown). Data are mean ± SE (n = 6). abcde: Groups without the same letter are significantly different (P < 0.05).
59
Figure 2. 8. Aging is associated with higher phosphorylation of 4EBP1 and rpS6 in F344XBN rat hearts. Protein isolates of hearts excised from 6-, 27-, 30-, 33- and 36-month rats were analyzed by immunoblotting for changes in total and phosphorylated 4EBP1 (A) and rpS6 (B). Ponceau S staining of the nitrocellulose membrane along with densitometric analysis of protein present was done to verify equivalent protein loading between the lanes (data not shown). Data are mean ± SE (n = 6). abcde: Groups without the same letter are significantly different (P < 0.05).
60
Figure 2. 9. Proposed scheme depicting the possible molecular mechanisms underlying age-associated cardiac hypertrophy in F344XBN rats. Molecules shown in the blue were experimentally assessed in this study, while the molecules in the other circles are postulated to be involved in inducing cardiac hypertrophy to varying stimuli in the previous studies. Aging associated hypertrophy in F344XBN rats is predominantly mediated via ERK/Akt/mTOR signaling.
61
PAPER 2
The following paper corresponds to the specific aim II
62
Chronic acetaminophen attenuates age-associated increases in cardiac ROS and
apoptosis in the Fischer Brown Norway rat
Sunil Kakarla1, Jacqueline C. Fannin
1, Saba Keshavarzian
2, Anjaiah Katta
1,
Satyanarayana Paturi2, Siva K. Nalabotu
1, Miaozong Wu
2, Kevin M. Rice
2, Kamran
Manzoor5, Ernest M. Walker Jr.
3, and Eric R. Blough
1, 2, 4
1Department of Pharmacology, Physiology and Toxicology,
2Department of Biological
Sciences, 3Department of Pathology,
4 Department of Exercise Physiology, Marshall
University, Huntington, WV. 5Charleston Area Medical Center, Charleston, WV.
Keywords: acetaminophen; aging; reactive oxygen species; apoptosis; caspase-3;
bax/bcl2 ratio
Running title: Efficacy of chronic acetaminophen ingestion in preventing age-associated
cardiac dysfunction.
Basic Res Cardiol. 2010 Jul; 105(4):535-44
63
Abstract
There is a growing need for pharmacological agents to manage cardiovascular disease in
the rapidly growing elderly population. Here we determine if acetaminophen is
efficacious in decreasing age-related increases in cardiac reactive oxygen species (ROS)
and apoptosis in aging Fischer344XBrown Norway rats. Compared to 6-month control
animals, indices of oxidative (superoxide anion [O2•–] and 4-hydroxy-2-nonenal [4-
HNE]) and nitrosative (protein nitrotyrosylation) stress were markedly increased in 33-
month old rat hearts. Thirty three month animals that had been treated with
acetaminophen (30mg/kg/day p.o. for six months) exhibited diminished age-related
increases in cardiac ROS levels and TUNEL positive nuclei and these changes were
accompanied by improvements in the Bax/Bcl2 ratio, diminished evidence of caspase-3
activation and increased phosphorylation of protein kinase B (Akt), ERK1/2, p70S6K and
GSK-3β. Taken together these results suggest that acetaminophen may attenuate the age
associated increases in cardiomyocyte apoptosis, possibly via diminishing age associated
elevation in ROS production.
64
Introduction
Cardiovascular disease (CVD) is responsible for more than 30% of all deaths
worldwide and heart failure is the leading cause of death in individuals over age 65 [231].
Large cross-sectional epidemiological studies have demonstrated that aging, by itself,
confers a greater risk for CVD than do the other major risk factors such as plasma lipid
levels, smoking, diabetes, or sedentary lifestyle [232, 233]. Although not well
understood, it has been hypothesized that the profound impact of age on the risk of the
occurrence, severity, and prognosis of cardiovascular disease is related to age-associated
changes in cardiovascular structure and/or function [234].
Similar to humans, advanced aging in the Fischer 344 X Brown Norway
(F344XBN) rat is characterized by cardiac remodeling and an impairment of cardiac
function [32]. The molecular mechanisms responsible for these changes are presently
unclear; however recent data have suggested that age-associated increases in cardiac
apoptosis could play a role in the pathological remodeling of the heart and heart failure
[235, 236]. It is thought that this increased apoptotic activity may be regulated, at least in
part, by the increased production of reactive oxygen species (ROS) [237]. Whether
pharmacological intervention during aging is able to diminish age-associated increases in
cardiac ROS and cardiac apoptosis has not been fully elucidated.
Acetaminophen ((N-acetyl p-aminophenol) is a non-narcotic analgesic and
antipyretic agent with weak anti-inflammatory activity that has been used therapeutically
for several decades [22]. Recent work has suggested that acetaminophen exhibits potent
anti-oxidant activity and that an acute bolus of acetaminophen is effective in preventing
65
ROS-associated cardiac damage in the rat and dog heart [28, 29]. On the basis of these
reports and previous data demonstrating that chronic acetaminophen (30 mg/kg body
weight / day) ingestion can be safely (e.g., in the absence of heptatoxicity) used to
decrease age-associated increases in skeletal muscle ROS levels [238], we tested whether
acetaminophen treatment could also decrease cardiac ROS levels and the incidence of
age-related apoptosis.
66
Materials and Methods
Animals
All procedures were performed in accordance with the Marshall University
Institutional Animal Care and Use Committee (IACUC) guidelines, using the criteria
outlined by the American Association of Laboratory Animal Care (AALAC). Fischer
344/NNiaHSD x Brown Norway/BiNia (F344XBN) rats aged 6 and 27 months, were
purchased from the National Institute on Aging colony in Harlan. Rats were housed two
per cage in an AAALAC approved vivarium with a 12 hour light-dark cycle and
temperature maintained at 22 ± 2°C, and fed ad libitum. All animals were allowed to
acclimatize for 2 weeks before initiation of any treatment or procedures. All animals were
examined for precipitous weight loss, failure to thrive or unexpected gait alterations and
animals with apparent abnormalities were removed from the study. Periodic weight
measurements were taken throughout the duration of the study.
Acetaminophen treatment and tissue collection
The F344XBN rats (27-month) were given acetaminophen (30 mg/kg body
weight/day, Sigma-Aldrich, Inc., St. Louis, MO, USA) for 6 months in drinking water.
Age-matched F344XBN rats were maintained as controls. Rats were anesthetized with a
ketamine–xylazine (4:1) cocktail (50 mg/kg, I/P) and supplemented as necessary for
reflexive response before tissue collections. After midline laparotomy, the heart was
removed and placed in Krebs-Ringer bicarbonate buffer (KRB) containing; 118 mM
NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24.2 mM
67
NaHCO3, and 10mM α-D-glucose, (pH 7.4) equilibrated with 5% CO2 / 95% O2 and
maintained at 37°C. Isolated hearts were quickly massaged to remove any blood from the
ventricles, cleaned of connective tissue, weighed, and immediately snap frozen in liquid
nitrogen.
Dihydroethidium staining
Cardiac muscles were serially sectioned (8 μm) using an IEC Microtome cryostat
and the sections were collected on poly-lysine coated slides. The fluorescent superoxide
indicator dihydroethidium [hydroethidine (HE)] was used to evaluate superoxide levels as
outlined elsewhere [239]. Upon oxidation, dihydroethidium is intercalated within DNA
exhibiting a bright fluorescent red [240]. Briefly, frozen tissue sections were washed with
phosphate-buffered saline (PBS) for 5 min and then incubated with 200 µl of 10 μm of
dihydroethidium (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature.
After washing with PBS (3 × 5 min), fluorescence was visualized under Texas red filter
using an Olympus BX51 microscope (Olympus America, Melville, NY, USA) equipped
with Olympus WH 20× widefield eyepieces and an Olympus UPlanF1 40×/0.75 objective
lens.
Immunohistochemistry
Immunostaining for nitrotyrosine (#9691, Cell Signaling Technology, Beverly,
MA, USA) and 4-HNE (#MHN-020P, JaICA, Shizuoka, Japan) was visualized by
immunofluorescence as outlined by the antibody manufacturer. Briefly, sections were
washed three times with phosphate-buffered saline (PBS) containing 0.5% Tween-20
68
(PBS-T), pH 7.4. After incubation for 30 min in a blocking solution (5% BSA), sections
were incubated with specific antisera diluted in 3% BSA in PSB-T (antibody dilution of
1:100) for 1 h at 24°C in a humidified chamber. After washing three times with PBS-T,
sections were incubated with FITC anti-rabbit IgG (1:200) for 30 min at 24°C in a
humidified chamber. Experiments performed in parallel omitting either the primary or
secondary antibody were utilized to confirm specificity and background
immunoreactivity. DAPI was included in the secondary antibody solution at a
concentration of 1.5 μg/ml in order to visualize cell nuclei. After a final PBS wash and
mounting, specimens were visualized by epifluorescence using an Olympus fluorescence
microscope (Melville, NY, USA) fitted with 20x and 40x objectives. Images were
recorded digitally using a CCD camera and the integrated optical density determined
using the AlphaEase software package (Alpha Innotech, Santa Clara, CA).
TUNEL staining
DNA fragmentation after staining for dystrophin was determined by TdT-
mediated dUTP nick end labeling (TUNEL) according to the manufacturer’s
recommendations (In Situ Cell Death Detection Kit, Roche Diagnostics, and Mannheim,
Germany). TUNEL staining was performed on tissue sections (8 μm) obtained from 6
month control (n=4), 33 month control (n=4), and 33 month treated (n=4) hearts, which
were fixed with 4% paraformaldehyde, washed with PBS (pH 7.4), and then
permeabilized with 0.1% sodium citrate and 0.1% Triton-X. Cross-sections from each
muscle were treated with DNase I to induce DNA fragmentation as a positive control.
Heart sections were blocked with 3% BSA and incubated with anti-dystrophin antibody
69
(NCL-DYS2, Novocastra Vector Laboratories, Burlingame, CA) at a dilution of 1:200 to
visualize the cell membrane. Nuclei were counter stained using DAPI (VECTASHIELD
HardSet Mounting Medium, Vector Laboratories, Burlingame, CA). Muscle cross
sections were visualized by epifluorescence using an Olympus fluorescence microscope
(Melville, NY) fitted with 20X and 40X objectives and images were recorded digitally
using a CCD camera (Olympus, Melville, NY). The number of TUNEL positive nuclei
was counted in three randomly selected regions in each slide. Four different animals were
counted from each group.
Immunoblotting analysis
Portions of individual cardiac muscles homogenized in buffer (T-PER, 2 mL/g
tissue; Pierce, Rockford, IL) containing protease (P8340, Sigma-Aldrich, Inc., St. Louis,
MO, USA) and phosphatase inhibitors (P5726, Sigma-Aldrich, Inc., St. Louis, MO,
USA). After incubation on ice for 30 min, the homogenate was collected by centrifuging
at 12,000 g for 5 min at 4◦C. Protein concentration of homogenates was determined via
the Bradford method (Fisher Scientific, Rockford, IL, USA). Homogenate samples were
boiled in a Laemmli 2X sample buffer (Sigma-Aldrich, Inc., St. Louis, MO, USA) for 5
min. Fifty micrograms of total protein for each sample was separated on a 10% PAGEr
Gold Precast gel (Lonza, Rockland, ME, USA) and then transferred to nitrocellulose
membranes. Gels were stained with a RAPID Stain protein stain reagent (G-Biosciences,
St. Louis, MO, USA) to verify transfer efficiency to membrane. Membranes were stained
with Ponceau S and the amount of protein quantified by densitometric analysis to confirm
successful transfer of proteins and equal loading of lanes as detailed somewhere else [20].
70
Membranes were blocked with 5% milk in Tris Buffered Saline (TBS) containing 0.5%
Tween- 20 (TBST) for 1 h and then incubated with primary antibody overnight at 4◦C.
After washing with TBST, the membranes were incubated with the corresponding
secondary antibodies conjugating with horseradish peroxidase (HRP) (anti-rabbit (#7074)
or anti-mouse (#7076), Cell Signaling Technology, Danvers, MA, USA) for 1 h at room
temperature. Protein bands were visualized following reaction with ECL reagent
(Amersham ECL Western Blotting reagent RPN 2106, GE Healthcare Bio-Sciences
Corp., Piscataway, NJ, USA). Target protein levels were quantified by AlphaEaseFC
image analysis software (Alpha Innotech, San Leandro, CA, USA). Primary antibodies
against Caspase-3 (#9662), cleaved Caspase-3 (#9661S), Bax (#2772), Bcl2 (#2870S) ,
Akt (#9272), p-Akt (Thr308#9275S/Ser473#9271L), mTOR (#2972), p-mTOR (Ser2448,
#2971S), p70 S6K (#9202), p-p70 S6K (Thr421/Ser424) (#9204S), ERK1/2 (#9102) and
p-ERK1/2 (#4377S) were purchased from Cell Signaling Technology (Beverly, MA,
USA).
Data Analysis
Results are presented as mean ± SEM. Data were analyzed by using the
SigmaStat 3.5 statistical program. A one-way analysis of variance (ANOVA) was
performed for overall comparisons with the Student-Newman-Keuls post hoc test used to
determine differences between groups. The level of significance accepted a priori was ≤
0.05.
71
Results
Acetaminophen decreases indices of cardiac ROS
Compared to 6-month animals, body weight was significantly increased with age
(Table 1). Acetaminophen treatment significantly attenuated age-associated increases in
heart weight and heart / body weight ratio (n=6; P < 0.05). Compared to 6-month control
and 33-month treated animals, dihydroethidium reactivity was visibly increased in 33-
month control hearts (Figure 3.1). Because superoxide can also react with nitric oxide to
form other reactive oxygen species such as peroxynitrite, we also examined heart sections
for the presence of protein nitrosylation using an antibody that recognizes proteins and
peptides containing nitrated tyrosine residues. With aging, the amount of immunoreactive
nitro-tyrosine signal was increased (Figure 3.1). Similar to our findings with
dihydroethidium, acetaminophen ingestion appeared to visibly decrease the amount of
protein nitration (Figure 3.1). In an effort to further investigate the effect of chronic
acetaminophen ingestion on cardiac ROS, we also examined the presence of proteins
modified by lipid peroxidation using immunohistochemistry and an antibody that
recognizes the lipid peroxidation metabolite 4-hydroxy-2-nonenal (4-HNE) [241].
Compared to 6-months animals, the aged myocardium was characterized by a marked
increase in the amount of 4-HNE immunoreactivity (Figure 3.1). Relative to the aged
controls, acetaminophen treatment was found to visibly decrease 4-HNE levels (Figure
3.1). Taken together, these data suggest that chronic acetaminophen ingestion is
associated with diminished indices of cardiac ROS.
72
Acetaminophen decreases age-associated cardiomyocyte apoptosis
Excessive oxidative stress is a well characterized stimulus for cellular apoptosis.
To examine this relationship with aging and acetaminophen treatment we used TdT-
mediated dUTP nick end labeling (TUNEL) to determine the number of cardiomyocytes
that exhibited DNA fragmentation. Compared to adult animals, heart sections from the
aged control group showed significant increases in the number of TUNEL positive nuclei
(Figure 3.2).
To extend upon our TUNEL findings we next examined the regulation of Bax and
Bcl-2. Compared to 6-month animals, Bcl-2 protein levels were 40% lower (P < 0.05)
with aging (Figure 3.3). Acetaminophen treatment in aged animals increased Bcl-2
protein levels by 68% (P < 0.05). Conversely, the amount of Bax remains unchanged
with aging or treatment. It is thought that the ratio of Bax to Bcl-2 plays an important role
in regulating the progression to apoptosis with cell death favored as the balance shifts
toward Bax [103]. With aging, the ratio of Bax to Bcl2 was increased by 68% (P < 0.05).
Compared to 33-month control animals, chronic acetaminophen treatment decreased the
Bax to Bcl2 ratio by 35% (P < 0.05) (Figure 3.3).
Caspases are cysteine-dependent aspartate specific proteases that are activated
during cell death [104]. Compared to 6-month control animals, the amount of full length
(inactive) caspase-3 was 22% (P < 0.05) and 36% (P < 0.05) higher in 33-month control
and treated animals (Figure 3.4) (P < 0.05). Caspase-3 activation, as measured by
densitometric analysis of the cleaved 19 and 17 Kda caspase-3 fragments, was similar
between 6-month adult and 33-month old treated animals but 186% and 92% higher (P <
0.05) in the 33-month control hearts (Figure 3.4). Taken together, these data suggest that
73
chronic acetaminophen treatment decreases the incidence of age-related cardiomyocytes
apoptosis.
Acetaminophen attenuates age-associated alterations in cell survival signaling
The Akt/PKB is a serine/threonine protein kinase that functions as a critical
regulator of cell survival through its ability to prevent apoptosis [242]. Acetaminophen
increased Akt Thr308 and Ser473 phosphorylation (activation) by 29% (P<0.05) and
99% (P<0.05), respectively (Figure 3.5). To confirm these findings we also examined the
phosphorylation of the mammalian target of rapamycin (mTOR) on the basis of recent
data suggesting that the phosphorylation of this molecule mediates the activation of Akt
[243]. Compared to untreated control animals, acetaminophen increased mTOR (Ser
2448) phosphorylation by 79% (P<0.05) (Figure 3.6). Recent studies have suggested that
oxidative stress causes decreases in ERK1/2 phosphorylation [244] and that therapeutic
approaches to increase phosphorylation of ERK1/2 can confer cardioprotection [245].
Similar to these studies, six months of acetaminophen treatment significantly increased
phosphorylation of ERK1/2 by 32% compared to that observed in the untreated control
animals (P<0.05) (Figure 3.7). To further confirm this increase in ERK1/2 activity, we
next examined the phosphorylation status of the ERK1/2 substrate, p70S6K, at
Thr421/Ser424 [246]. As expected, acetaminophen treatment significantly increased
p70S6k phosphorylation (Thr421/Ser424) by ~110% compared to that observed in the
age-matched untreated control animals (P<0.05) (Figure 3.8). Like Akt/PKB, glycogen
synthase kinase 3-beta (GSK-3β) has been shown to hinder apoptosis as it can inhibit the
opening of the mitochondrial permeability transition pore (mPTP) when phosphorylated
74
[247]. Compared to untreated control animals, acetaminophen treatment significantly
increased the phosphorylation of GSK-3β (Ser 9) by 42% (P<0.05) (Figure 3.9).
75
Discussion
The age-related accumulation of ROS-induced biomolecules is thought to play a
major role in the development of cardiovascular disease [82]. For instance, both clinical
and experimental evidence have indicated that ROS are increased in chronic heart failure
[248]. Furthermore, antioxidants and free radical scavengers have been shown to
mitigate age-related and other ROS induced cardiac pathologies [249, 250]. In particular,
aspirin is an anti-oxidative compound with clearly demonstrated efficacy in the
prevention of heart disease [251]. Still, prophylactic aspirin therapy fails to fully prevent
the development of heart disease, increases the risk of bleeding problems, and is
associated with other safety concerns [251]. Thus, the prevention of heart disease in the
elderly could be improved significantly by the identification and characterization of
additional compounds that are safe, affordable, and effective for long-term use in
humans. In fact, characterization of the long-term effects of such compounds is crucial in
the development of combinatorial antioxidative treatments, which may eventually
represent the most effective treatment for multiple degenerative diseases [252]. Here, we
demonstrate that the chronic use of acetaminophen acts to diminish the incidence of
cardiomyocyte apoptosis in the aging F344XBN rat model.
Age-related increases in cardiac ROS levels have been implicated in the
pathological remodeling of the heart and heart failure [253]. Previous studies using the
F344XBN rat have demonstrated that aging in these animals is characterized by an
elevation in the amount of cardiac ROS, protein nitration and 4-hydroxy-2-nonenal (4-
HNE) [18]. The findings of the current study support these data while also demonstrating
76
that elevated indices of oxidative stress can be attenuated by chronic acetaminophen
treatment (Figure 3.1). To our knowledge, these findings have not been demonstrated
before.
How acetaminophen might decrease cardiac ROS is not well understood. Previous
reports suggest that acetaminophen may act as an antioxidant as it is a mono-phenol [23].
Possible sources of increased ROS in the aging myocardium have not been fully
elucidated but could include xanthine and NAD(P)H oxidoreductases, cyclooxygenase,
the mitochondrial electron transport chain and activated neutrophils, among many others
[254]. Although many of these sources could potentially produce ROS, it is thought that
increased NADH/NAD(P)H oxidase and / or alterations in mitochondrial function may
be the main cause of augmented ROS generation in the aged heart [255]. Whether age-
related alterations in these systems or others are responsible for the increases in the ROS
indices we see in the aging F344XBN heart is currently being investigated. Similarly,
whether acetaminophen affects these processes or others is not clear. Merrill and
colleagues have shown that acetaminophen significantly reduces ventricular dysfunction
caused by exogenously administered hydrogen peroxide [29] and that it attenuates the
burst of hydroxyl radicals released in the early minutes of reperfusion following global
myocardial ischemia [27]. Chronically administered acetaminophen has also been shown
to provide cardioprotection to the postischemic, reperfused rodent myocardium [28]. In a
recent study using Langendorff-perfused guinea pig hearts, Hadzimichalis and colleagues
demonstrated that administration of acetaminophen just before an ischemic attack can
result in attenuation of functional damage
via inhibition of MPTP opening and
cytochrome c release-induced apoptotic cell death following ischemia/reperfusion [199].
77
Acetaminophen has also been found to be effective in attenuating the deleterious effects
of peroxynitrate [197], myeloperoxidase [191], cyclooxygenase [189], and other
peroxides [193]. Whether acetaminophen targets these or other types of ROS and if this
action, if present, is responsible for the effects we observe in the aging F344XBN heart
will require further study.
Increases in cardiac apoptosis have been implicated in the pathological
remodeling of the heart and heart failure [20]. This increased apoptotic activity may be
mediated by the increased production of ROS [237, 252]. Indeed, oxidative stress seems
to function as a master regulator of apoptosis in cells [252]. In addition to apoptosis,
ROS levels may also act to expand the region of apoptotic activity surrounding damaged
cells during heart attack and stroke [256]. Given the potential linkages between ROS
levels and cardiomyocyte apoptosis we examined the effect of chronic acetaminophen
ingestion on the amount of TUNEL positive cells in heart sections of aged control and
acetaminophen treated animals. Consistent with our findings demonstrating that
acetaminophen can reduce indices of cardiac ROS, we also show that acetaminophen
may be efficacious in reducing the amount of age-related cardiac myocytes apoptosis
(Figure 3.2). In an effort to confirm these findings, we next investigated the ratio of Bax
to Bcl-2. Bax is a key pro-apoptotic regulator that can insert itself into the outer
mitochondrial membrane and promote the release of mitochondrial apoptogenic factors
[99]. Conversely, Bcl-2 is an anti-apoptotic factor that is thought to block the membrane
insertion of Bax by forming Bax/Bcl-2 heterodimers [100]. It is postulated that the ratio
of Bax to Bcl-2 plays an important role in regulating cellular apoptosis with cell death
being favored as the balance shifts toward Bax [103]. Consistent with our TUNEL data,
78
we found that chronic acetaminophen treatment reduces apoptotic Bax / Bcl-2 signaling
in the aged F344XBN rat heart (Figure 3.3).
It has been postulated that cytosolic cytochrome-c couples with apoptotic protease
activating factor-1 (Apaf-1) which leads to the activation of caspase-9. Activated
caspase-9 in turn has been shown to cleave caspase-3 resulting in caspase-3 activation
and proteolytic disassembly of the cell [104]. Similar to previous work [257], and
consistent with our TUNEL and Bax/Bcl-2 data, we also observed an increase in the level
of cleaved caspase-3 with aging (Figure 3.4). In addition, we also demonstrate that
acetaminophen treatment is associated with decreased caspase-3 activation (Figure 3.4).
Whether this decrease in caspase-3 activation by itself, or acting in concert with other
factors is responsible for the decreases in apoptosis we see with acetaminophen treatment
is currently unclear.
To investigate the possibility that molecules other than caspase-3 might also play
a role in regulating the acetaminophen-associated decreases in cardiac apoptosis
observed, we also examined the effects of acetaminophen on the regulation of protein
kinase B (Akt/PKB). The Akt/PKB is a serine/threonine protein kinase that functions as a
critical regulator of cell survival and proliferation [107, 258]. As expected from our
caspase-3 and Bax/Bcl-2 data, we found an increased expression of phosphorylated
(activated) Akt with acetaminophen treatment (Figure 3.5). As previous studies have
shown that mTOR may function to regulate Akt activity [259, 260], we also examined if
acetaminophen ingestion was associated with alterations in the phosphorylation (Ser
2448) of mTOR. Similar to our findings for Akt, we also observed a robust increase in
the amount of phosphorylated mTOR with chronic acetaminophen ingestion (Figure 3.6).
79
Why acetaminophen treatment may be associated with increases in Akt phosphorylation
is not clear as alterations in ROS have been shown to increase [261] or decrease [262,
263] Akt phosphorylation. Similarly, whether a direct linkage exists between changes in
Akt phosphorylation and the acetaminophen-associated changes in caspase-3 activation
we observe is unknown.
It has been suggested that increased phosphorylation of ERK1/2, is associated
with cell survival [245] and oxidative stress has been shown to be linked to diminished
ERK1/2 phosphorylation [244]. Similarly, other work has demonstrated that ERK1/2 can
protect against ischemia-reperfusion induced oxidative stress [264, 265] and that ERK1/2
is involved in regulating the cardioprotective effects of nitric oxide (NO) following
ischemia-reperfusion [266]. Like ERK1/2, activated (phosphorylated) GSK-3β has also
been shown to exhibit anti- apoptotic activity as it may be involved in regulating the
opening of the mitochondrial permeability transition pore [247]. Consistent with our
findings for Akt, here we demonstrate increased phosphorylation of ERK1/2 and GSK-3β
with acetaminophen treatment (Figures. 3.7, 3.9). Whether these changes in ERK1/2 and
GSK-3β are directly associated with the diminished cardiac myocyte apoptosis we
observe in the acetaminophen treated animals is currently unclear but warrants further
investigation.
In summary, we have demonstrated for the first time that chronic acetaminophen
use attenuates excess ROS and apoptotic Bax/Bcl2 signaling in aged cardiac cells,
thereby helping to prevent caspase-mediated apoptosis. These beneficial effects suggest
that chronic acetaminophen usage may protect the heart by promoting the development of
anti-oxidative capacity (Figure 3.10). Still, little is known about the mechanisms of
80
cardioprotection employed by chronic acetaminophen treatment. Increasing our
understanding of these mechanisms and how they may interact with other drugs and
disease processes could lead to new ways in improving our ability to manage heart
disease.
Table 1. Total body weight (BW), heart weight (HW) and heart weight to body weight
ratio (HW/BW %) of 6-, 33-month control and 33-month acetaminophen treated male
F344XBN rats. Results are expressed as gram (g) or HW/BW ratio (mean ± SD; n=6). (*)
indicates significant difference from adult (6-month) value (P<0.05). (†) indicates
significant difference from age matched 33-month control animals (P<0.05).
81
Figures
Figure 3. 1. Effect of aging and acetaminophen treatment on indices of cellular ROS. A.
Indices of oxidative (superoxide anion [O2•–], 4-hydroxy-2-nonenal [4-HNE]) and
nitrosative (protein nitrotyrosylation) stress in 6-month control, 33-month control, and
33-month treated hearts. Bar=100µm. B. Quantification of cardiac ROS as determined by
intensity of fluorescence. Results are expressed relative to the 6-month value. (*)
indicates significant difference from adult (6-month) value (P<0.05). (†) indicates
significant difference from age matched 33-month control animals (P<0.05). n = 4 hearts
per age group.
82
Figure 3. 2. Acetaminophen treatment decreases the incidence of TUNEL positive nuclei
in aged cardiac muscle. A. Double immunofluorescence labeling of dystrophin (Texas
Red), TUNEL (FITC) and nuclei (DAPI) in 6-month control, 33-month control, and 33-
month treated hearts. Bar = 100 μm. B. Number of TUNEL positive nuclei per mm2
in 6-
month control, 33-month control, and 33-month treated hearts. (*) indicates significant
difference from adult (6-month) value (P<0.05). (†) indicates significant difference from
age matched 33-month control animals (P<0.05). n = 4 hearts per age group.
83
Figure 3. 3. Acetaminophen treatment alters the ratio of Bax / Bcl-2 in aged cardiac
muscle. Protein isolates of hearts from 6-month contol, 33-month control and 33-month
treated hearts were analyzed by immunoblotting to determine Bax and Bcl2 protein
levels. Ponceau S staining of the nitrocellulose membrane along with densitometric
analysis of protein present was done to verify equivalent protein loading between the
lanes. (*) indicates significant difference from adult (6-month) value (P<0.05). (†)
indicates significant difference from age matched 33-month control animals (P<0.05). n
= 6 for all groups.
84
Figure 3. 4. Acetaminophen treatment decreases the age-associated activation of caspase-
3. Protein isolates of hearts from 6-month contol, 33-month control and 33-month treated
hearts were analyzed by immunoblotting to determine the amount of full length and
cleaved caspase-3. Ponceau S staining of the nitrocellulose membrane along with
densitometric analysis of protein present was done to verify equivalent protein loading
between the lanes (data not shown). (*) indicates significant difference from adult (6-
month) value (P<0.05). (†) indicates significant difference from age matched 33-month
control animals (P<0.05). n = 6 for all groups.
85
Figure 3. 5. Acetaminophen treatment increases Akt phosphorylation in aged cardiac
muscle. Protein isolates of hearts from 6-month contol, 33-month control and 33-month
treated hearts were analyzed by immunoblotting to determine the amount of total and
phosphorylated Akt (Thr 308, Ser 473). Ponceau S staining of the nitrocellulose
membrane along with densitometric analysis of protein present was done to verify
equivalent protein loading between the lanes (data not shown). (*) indicates significant
difference from adult (6-month) value (P<0.05). (†) indicates significant difference from
age matched 33-month control animals (P<0.05). n = 6 for all groups.
86
Figure 3. 6. Acetaminophen treatment increases mTOR phosphorylation in aged cardiac
muscle. Protein isolates of hearts from 6-month contol, 33-month control and 33-month
treated hearts were analyzed by immunoblotting to determine the amount of total and
phosphorylated mTOR (Ser 2448). Ponceau S staining of the nitrocellulose membrane
along with densitometric analysis of protein present was done to verify equivalent protein
loading between the lanes (data not shown). (*) indicates significant difference from
adult (6-month) value (P<0.05). (†) indicates significant difference from age matched 33-
month control animals (P<0.05). n = 6 for all groups.
87
Figure 3. 7. Acetaminophen treatment increases ERK1/2 phosphorylation in aged cardiac
muscle. Protein isolates of hearts from 6-month contol, 33-month control and 33-month
treated hearts were analyzed by immunoblotting to determine the amount of total and
phosphorylated ERK1/2. Ponceau S staining of the nitrocellulose membrane along with
densitometric analysis of protein present was done to verify equivalent protein loading
between the lanes (data not shown). (*) indicates significant difference from adult (6-
month) value (P<0.05). (†) indicates significant difference from age matched 33-month
control animals (P<0.05). n = 6 for all groups.
88
Figure 3. 8. Acetaminophen treatment increases p70S6K phosphorylation in aged cardiac
muscle. Protein isolates of hearts from 6-month contol, 33-month control and 33-month
treated hearts were analyzed by immunoblotting to determine the amount of total and
phosphorylated p70S6K (Thr421/Ser424). Ponceau S staining of the nitrocellulose
membrane along with densitometric analysis of protein present was done to verify
equivalent protein loading between the lanes (data not shown). (*) indicates significant
difference from adult (6-month) value (P<0.05). (†) indicates significant difference from
age matched 33-month control animals (P<0.05). n = 6 for all groups.
89
Figure 3. 9. Acetaminophen treatment increases GSK-3β phosphorylation in aged cardiac
muscle. Protein isolates of hearts from 6-month contol, 33-month control and 33-month
treated hearts were analyzed by immunoblotting to determine the amount of total and
phosphorylated GSK-3β. Ponceau S staining of the nitrocellulose membrane along with
densitometric analysis of protein present was done to verify equivalent protein loading
between the lanes (data not shown). (†) indicates significant difference from age matched
33-month control animals (P<0.05). n = 6 for all groups.
90
Figure 3. 10. Proposed scheme for the effects of chronic acetaminophen treatment in
attenuating aging-associated cardic ROS and apoptosis. Recent studies indicated that the
aging associated increases in oxidative stress play a key role in cardiomyocyte apoptosis.
Chronic acetaminophen treatment can attenuate excess ROS leading to diminished
cardiomyocyte apoptosis through changes in Bax/Bcl2 ratio, caspase activation and by
regulating Akt phosphorylation. Note: Solid red arrow indicates increase and solid blue
arrow indicates decrease.
91
PAPER 3
The following paper corresponds to the specific aim III
92
Anti-arrhythmic effect of chronic acetaminophen treatment in the aging F344XBN
rat involves diminished myocardial fibrosis and altered miRNAs regulation
Sunil K. Kakarla1,2
, Srininvas Thulluri1, Nandini D.P.K. Manne
1,3, Kevin M. Rice
1,3,4,5,6,
and Eric R. Blough1,2,3,4, 5,6
1 Center for Diagnostic Nanosystems, Marshall University, Huntington, WV
2 Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of
Medicine, Marshall University, Huntington, WV 3 Department of Biological Sciences, Marshall University, Huntington, WV
4 Department of Internal Medicine, Joan C. Edwards School of Medicine, Marshall
University, Huntington, WV 5
Department of Exercise Science, Sport and Recreation, College of Education and
Human Services, Marshall University, Huntington, WV 6 Department of Biology, West Virginia State University, Charleston, WV
Running title: Acetaminophen confers cardioprotection in old rats
Keywords: acetaminophen; aging; arrhythmias; fibrosis; Cx43; miRNA
93
Abstract
Background: There is a growing need for pharmacological agents to manage
cardiovascular disease in the rapidly increasing elderly population. Acetaminophen has
been shown to exhibit cardioprotective effects following ischemia-reperfusion injury by
acting as an antioxidant. Recent data suggests that chronic acetaminophen ingestion
significantly attenuates age-associated increases in cardiac ROS and apoptosis. Methods
and results: Here, we examine the effect of chronic acetaminophen treatment on the
incidence of cardiac arrhythmias, Cx43 expression, myocardial fibrosis and miRNA
expression in the aging Fischer344XBrown Norway (F344XBN) rat hearts. Aging male
F344XBN rats (27-mo; n=6) were treated with acetaminophen (30mg/kg/day p.o.) for six
months and underwent serial electrocardiography before and after the completion of the
study. Aging was associated with a higher incidence of premature atrial and ventricular
arrhythmias, greater myocardial fibrosis and diminished localization of the gap junction
protein Cx43, all of which were diminished with chronic acetaminophen ingestion. The
expression of miR-1, miR-133, miR-214, miR-30a and miR-30d was 66-, 64-, 54-, 73-
and 74-% lower, respectively, in 33-month control animals compared to that of 6-month
animals (P < 0.05). Acetaminophen treatment significantly increased the expression of
miR-1 and miR-214, while the other miRNA remain unchanged. Conclusion: These
results indicate that acetaminophen may prevent the incidence of age-associated
arrhythmias and that this alteration is associated with diminished fibrosis and changes in
cardiac miRNA expression.
94
Introduction
Cardiovascular disease remains the foremost cause of death and morbidity
worldwide and is especially prevalent in the elderly. Nearly half the deaths occurring
from cardiovascular causes take place suddenly due to fatal ventricular arrhythmias
[267]. Aging by itself represents a major risk factor for cardiovascular disease and is
associated with structural and functional alterations in the cardiovascular system
including increased LV wall thickness, increased LV mass, impaired LV ejection and a
higher incidence of arrhythmias [30, 32]. Echocardiographic studies have demonstrated
that aging in the Fischer 344/NNiaHSd X Brown Norway/BiNia (F344XBN) rat is
characterized by age-associated alterations in cardiac structure and function [16, 17],
while other work has demonstrated that cardiac aging in these animals is associated with
increases in the amount of cardiac reactive oxygen species (ROS) [18, 268] and myocyte
apoptosis [20]. It has also been established that myocardial aging is characterized by LV
LV fibrosis and a reduction in cardiomyocyte number, which together act to decrease
myocardial compliance and increase the risk of developing ventricular dysfunction and
arrhythmias [20, 85].
The factors that regulate age-associated cardiac remodeling have not been
elucidated. The “free radical theory of aging” proposes that aging is characterized by the
gradual accumulation of damage to biomolecules by reactive oxygen species (ROS) [31].
Why ROS levels may be altered with aging is not fully understood, but it is thought that
these changes may be due, at least in part, to altered mitochondrial electron transport
leakage [269], changes in the activity of xanthine oxidase [270],
variation in the
expression of nitric oxide synthase [270], or increases in the amount of NADPH oxidases
95
[271]. It has been hypothesized that elevations in tissue ROS levels with age can
influence several cellular processes including cardiac hypertrophy, the structure and
function of blood vessels, inflammation, apoptosis and alteration of the extracellular
matrix profile [61, 64]. Whether interventions aimed at decreasing age-related increases
in ROS may be beneficial for the treatment of age-associated cardiac arrhythmias or
dysfunction is not well understood [272].
The molecular mechanisms governing the cardiac remodeling are not well
understood. Recent data has suggested that miRNAs (miRNAs), small ~ 22-nucleotide
noncoding RNAs that inhibit transcription or translation by interacting with the 3′
untranslated region (3′UTR) of target mRNAs [156], may play a role in the regulation of
cardiac differentiation and disease [163]. In this context, several miRNAs, including
miR-1, 21, 29, 30, 195, 133, 206 and 208 appear to play an important role in the process
of cardiac remodeling, putatively by regulating changes in gene expression that
accompany pathological cardiac hypertrophy and contractile dysfunction [162, 164, 165,
170]. The factor(s) that regulate the expression of miRNA are not clear; however, recent
studies have suggested that alterations in cellular ROS levels may be involved [178].
Similarly, how aging may affect the expression of cardiac miRNA has not been
elucidated.
Acetaminophen is a potent antioxidant that has been used therapeutically for
several decades [22], and recently has been shown to exhibit cardioprotective properties
[21]. It is thought that acetaminophen can help protect cells and tissues from the
damaging effects of peroxynitrite, myeloperoxidase, cyclooxygenase, and other peroxides
96
[21]. Recent work has also suggested that acetaminophen exhibits potent cardioprotective
activity in preventing ROS-associated cardiac damage in the rat and dog heart [197].
Whether the chronic administration of acetaminophen is effective for the treatment of
age-associated cardiac remodeling is not well understood. Using the tissues from the
same animals employed in this study we have previously reported that chronic
acetaminophen ingestion significantly attenuates age-associated increases in cardiac ROS
and apoptosis [203]. Here we extend upon these studies and examined the effect of aging
and chronic low dose acetaminophen treatment on the expression of cardiac miRNA and
the development of cardiac arrhythmias. Our data suggest that aging in F344XBN rats is
associated with increased incidence of cardiac arrhythmias, lowered expression of gap
junction protein connexin-43 (Cx43), increased myocardial fibrosis and altered regulation
of miRNA. In addition, we also demonstrate that chronic acetaminophen ingestion is
efficacious in preventing the incidence of age-associated arrhythmias and that this
alteration is associated with diminished fibrosis and changes in cardiac miRNA
expression.
97
Materials and Methods
Animals
All procedures were performed in accordance with the Marshall University
Institutional Animal Care and Use Committee (IACUC) guidelines, using the criteria
outlined by the American Association of Laboratory Animal Care (AALAC). Fischer
344/NNiaHSD x Brown Norway/BiNia (F344XBN) rats aged 6 and 27 months, were
purchased from the National Institutes on Aging. Adult F344XBN rats at 27-months of
age were chosen for the study since they do not exhibit significant cardiovascular deficit
at this age and roughly coincide with humans in the 5th
decade of life [15]. Rats were
housed two per cage in an AAALAC approved vivarium with a 12 hour light-dark cycle
and temperature maintained at 22 ± 2°C. Animals were fed ad libitum and allowed to
acclimate for 2 weeks before the initiation of any treatment or procedures. All animals
were examined for precipitous weight loss, failure to thrive or unexpected gait alterations
and animals with apparent abnormalities or tumors were removed from the study.
Materials
Acetaminophen tablets or pure compound used in the study was provided by
McNeil Pharmaceuticals (Fort Washington, PA). Antibodies against Cx43 and rabbit IgG
antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Precast
10% SDS-PAGE gels were procured from Cambrex Biosciences (Baltimore, MD, USA),
and enhanced chemiluminescence (ECL) Western blot detection reagent was acquired
from Amersham Biosciences (Piscataway, NJ, USA). Restore Western blot stripping
98
buffer was obtained from Pierce (Rockford, IL, USA) and 3T3 cell lysates were from
Santa Cruz Biotechnology. All other chemicals were purchased from Sigma (St Louis,
MO, USA).
Acetaminophen treatment, electrocardiography and tissue collection
F344XBN rats (27-month; n = 6) were given acetaminophen (30 mg / kg body
weight / day) for 6 months in drinking water. Rats were treated until the age of 33-
months, which roughly coincides with humans in their seventh decade of life. Age-
matched F344×BN rats were maintained as controls. Heart rate and cardiac rhythm were
measured before the commencement of treatment and at the end of the treatment using
needle electrodes under ketamine-xylazine (4:1) cocktail (50 mg/kg, I/P) anesthesia.
Tissues were collected under anesthesia using a ketamine-xylazine cocktail (40:10
mg/kg i.p.) and supplemented as necessary for reflexive response. After midline
laparotomy, the heart was removed and placed in Krebs-Ringer bicarbonate buffer (KRB)
containing; 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 24.2 mM NaHCO3, and 10mM α-D-glucose, (pH 7.4) equilibrated with 5% CO2
/ 95% O2 and maintained at 37°C. Isolated hearts were quickly massaged to remove any
blood from the ventricles, cleaned of connective tissue, weighed, and immediately snap
frozen in liquid nitrogen. Liver and kidneys were excised immediately after removing
the heart and either snap-frozen under liquid nitrogen or stored in 5% buffered
formaldehyde.
99
Histology
Tissue specimens were serially sectioned (6 μm) using an IEC Minotome cryostat
and collected on poly-lysine coated slides. After fixing in acetone, (-20 °C for 2 min)
sections were stained with hematoxylin and eosin and mounted. Morphometric evaluation
was performed with the use of a computerized imaging analysis system (Olympus
MicroSuite™ Basic). Masson’s trichrome staining of serially sectioned cardiac tissue was
performed according to the manufacturer’s guidelines (Poly Scientific R & D Corp., Bay
Shore, NY).
Immunohistochemistry
Immunostaining for Cx43 was visualized by immunofluorescence as outlined by
the antibody manufacturer. Briefly, sections were washed three times with phosphate-
buffered saline (PBS) containing 0.5% Tween-20 (PBS-T), pH 7.5. After incubation for
30 min in a blocking solution (3% BSA), sections were incubated with specific antisera
diluted in PSB-T (antibody dilution of 1:100) for 1 h at 24°C in a humidified chamber.
After washing three times with PBS, sections were incubated with FITC anti-rabbit IgG
(1:200) for 30 min at 24°C in a humidified chamber. DAPI was also included in the
secondary antibody solution at a concentration of 1.5 μg/ml in order to visualize cell
nuclei. After a final PBS wash and mounting, specimens were visualized by
epifluorescence using an Olympus fluorescence microscope (Melville, NY, USA) fitted
with a 40x objective. Images were recorded digitally using a CCD camera and analyzed
using Olympus MicroSuite™ Basic from Olympus America (Melville, NY, USA).
100
Immunoblot analysis
Tissues were pulverized in liquid nitrogen using a mortar and pestle until a fine
powder was obtained and washed three times with ice-cold PBS as described previously
[273]. Samples were then suspended on ice in 100 μl of TPER (2 ml/g tissue weight;
Pierce, Rockford, IL, USA) containing protease (P8340, Sigma-Aldrich, Inc., St. Louis,
MO, USA) and phosphatase inhibitors (P5726, Sigma-Aldrich, Inc., St. Louis, MO,
USA). After lysis, homogenates were centrifuged 10 min at 13,000 x g and the
supernatant collected. Protein concentrations of homogenates were determined in
triplicate via the Bradford method (Pierce) using bovine serum albumin as a standard.
Samples were diluted to a concentration of 2.5 mg/ml in SDS-loading buffer and boiled
for 5 min. Aliquots (50 μg of total protein for each sample) were separated on a 10%
SDS-PAGE gel. Transfer of protein onto nitrocellulose membranes was performed using
standard conditions. Membranes were stained with Ponceau S and the amount of protein
quantified by densitometric analysis to confirm successful transfer of proteins and equal
loading of lanes [20, 203]. Semiquantitiative immunodetection of specific proteins was
performed using densitometry. Immunoblots were stripped with Restore Western blot
stripping buffer as described by the manufacturer to obtain direct comparisons between
expression and phosphorylation levels of different signaling molecules. After verifying
the absence of residual HRP activity by treating the membrane with the ECL reagent,
membranes were washed and reprobed. Randomization of antibody incubation was
utilized to minimize potential experimental error associated with membrane stripping.
101
miRNA analysis
Total RNA was harvested from cardiac tissues using TRIzol Reagent,
(Invitrogen). Total RNA was solubilized in RNase-free H2O and quantified in duplicate
by measuring the optical density (OD) at 260 nm. Purity of RNA was assured by
obtaining an OD260/OD280 ratio of ∼2.0. Two micrograms of RNA were reverse
transcribed (RT) using the QuantiMiR RT kit (System Bioscience) according to standard
methods (Systems Bioscience). Control RT reactions were done in which the RT enzyme
was omitted. The control RT reactions were PCR amplified to ensure that DNA did not
contaminate the RNA. Cyber green-based real-time qPCR was performed by using a
7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) and gene-specific
primers for the miRNA of interest designed according to the guidelines outlined in the
QuantiMiR RT kit (System Bioscience). Melt analysis was used after each PCR run to
ensure amplification of only a single product. All samples were normalized to an internal
control housekeeping gene (U1 small nuclear RNA (snRNA)) [274]. Relative fold
changes in miRNA within each group and basal differences between groups were
determined from the Ct values after normalization to their respective housekeeping genes
using the 2-∆ct
method [275].
Data Analysis
Results are presented as mean ± SEM. Data were analyzed by using the
SigmaStat 3.5 statistical program. A one-way analysis of variance was performed for
overall comparisons while the Student-Newman-Keuls post hoc test was used to
102
determine differences between groups. The level of significance accepted a priori was ≤
0.05.
Results
As reported previously [203], compared to 6-month animals, body weight was
significantly higher with age (6 C: 406 ± 15 vs. 33 C : 504 ± 23* vs. 33 T : 506 ± 13*;
(n=6; P < 0.05)). Chronic acetaminophen ingestion attenuated age-associated increases in
heart weight (6 C: 1.05 ± 0.04 vs. 33 C: 1.85 ± 0.06* vs. 33 T: 1.65 ± 0.05*†; (n=6; P <
0.05)) and heart / body weight ratio (6 C: 0.259 ± 0.01 vs. 33 C: 0.370 ± 0.02* vs. 33 T:
0.327 ± 0.01†; (n=6; P < 0.05)).
Aging is associated with increased incidence of cardiac arrhythmias
Confirming our previous observations [17], serial electrocardiography showed
evidence of increased incidence of arrhythmias with age (Figure 4.1). Chronic
acetaminophen treatment led to a marked decrease in the incidence of premature atrial
(PAC) and ventricular (PVC) contractions (Figures 4.1A, 4.1B).
Aging is associated with greater myocardial fibrosis
It is thought that the intrinsic properties of the myocardium change during aging
due to connective tissue alterations [276]. In agreement with this possibility, we found
increased perivascular fibrosis as well as subcellular alterations of the cardiomyocytes
and their junctions in the left ventricles of the aged control animals suggesting a
103
deterioration of heart function. Similarly, qualitative histological analysis on heart
sections of 33-month acetaminophen treated rats revealed the presence of fibrosis with
the incidence of these findings appearing to be less than that observed in the untreated
control animals (Figure 4.2).
Aging is associated with diminished connexin-43 levels
Compared to 6-month animals, Cx43 expression in the 33-month control animals
was 25% lower (P < 0.05) (Figure 4.3A). Acetaminophen treatment for 6-months
significantly increased Cx43 levels by 8% (P < 0.05) (Figure 4.3A). These results were
corroborated by immunohistochemical analysis of these hearts. Consistent with previous
findings [277], gap junctional Cx43 immunoreactivity was primarily observed at the
intercalated discs (Figure 4.3B). When compared to hearts obtained from 6-month rats,
Cx43 immunoreactivity at the intercalated disc regions of the membrane was markedly
lower in 33-mo control rat hearts, and cytosolic Cx43 immunoreactivity appeared to be
clustered inside the fibers of old hearts (Figure 4.3B).
Aging is associated with alterations in cardiac miRNA expression
In order to evaluate whether miRNAs play a significant role in the aging process,
a highly sensitive, semi-quantitative RT-PCR method was employed to analyze the
expression of miRNA thought to be involved in cardiac remodeling. Compared to 6-
month animals, the amount of miR-1, miR-133, miR-214, miR-30a and miR-30d was 66-
, 64-, 54-, 73- and 74-% lower, respectively in 33-month control animals (Figures 4.4-
104
4.6). The amount of miR-206, miR-21, miR-24, miR-195 and miR-29a was not altered
with aging (data not shown).
Acetaminophen treatment is associated with alterations in miRNA expression
Compared to 33-month control animals, acetaminophen treatment increased the
expression of miR-1 and miR-214 expression levels by 29-, and 53 %, respectively, (P <
0.05) (Figures 4.4 and 4.6). The amount of miR-133, miR-30a and miR-30d did not
change with treatment.
105
Discussion
Multiple experimental models have been used to assess the effects of aging on
ROS and CVD, including dogs, the Fisher 344 (F344) and Fischer 344/NNiaHSd X
Brown Norway/BiNia (F344XBN) aging rat models, and other species [278]. A recent
review of aging rat models concluded that the F344XBN rat strain seems to exhibit
“physiological aging” to a greater degree than others [13]. Indeed, previous studies have
demonstrated that aging in the F344XBN rats is characterized by higher cardiac ROS [18,
203] and apoptosis [20]. Other data have suggested that acetaminophen may function as a
potent antioxidant [27, 29]. Given the possible linkage between higher cardiac ROS and
cardiac dysfunction, we postulated that the chronic ingestion of acetaminophen would
attenuate detrimental changes in the aging F344XBN heart. The main findings of this
study were that chronic acetaminophen ingestion is capable of decreasing age-associated
increases in cardiac arrhythmias and that aging in the F344XBN heart is associated with
changes in miRNA expression.
Aging in the F344XBN heart is associated with greater incidence of cardiac
arrhythmias
Nearly half the deaths occurring from cardiovascular causes take place suddenly
due to fatal ventricular arrhythmias [267]. Similar to previous studies, we observed an
age-associated increase in the incidence of ventricular arrhythmias in the F344XBN rat
[16-18]. Of interest is our finding that 6-months of acetaminophen treatment appeared to
decrease the incidence of PAC and PVC (Figure 4.1). Recent data has implicated a link
106
between the loss of the gap junction protein connexin-43 (Cx43) and fatal arrhythmias.
For example, using a heart-specific Cx43 conditional knockout mouse model in which
cardiac Cx43 abundance decreases rapidly within the first few weeks of age, Danik and
colleagues demonstrated a decrease in cardiac conduction velocity and a dramatic
increase in susceptibility to inducible lethal ventricular tachyarrhythmias [279]. To
ascertain if alterations in Cx43 levels exist in the aging F344XBN heart we examined the
regulation of Cx43 using immunoblot analysis. Consistent with our electrocardiography
data, we observed that the expression of Cx43 is significantly lowered with aging and
importantly, that acetaminophen treatment is associated with increased protein levels of
Cx43 (Figure 4.3A). Immunohistochemical analysis revealed that the distribution of
Cx43 appears to be morphologically well preserved at the intercalated discs in the young
6-month animals. With increasing age, the amount of Cx43 immunoreactivity appeared to
decrease markedly at the intercalated disc regions of the 33-month control rat hearts
(Figure 4.3B).
Although these results, taken together, suggest that chronic acetaminophen
ingestion may be able to influence the expression of Cx43 it is unlikely that the
magnitude of the differences we see in the regulation of Cx43 can, by itself, explain the
decrease in age-associated arrhythmias we observed in the acetaminophen treated
animals. Indeed, previous data has suggested that Cx43 protein losses of up to ~40% are
insufficient by themselves to induce cardiac arrhythmias [279]. How acetaminophen may
decrease cardiac arrhythmias in the aging F344XBN heart is presently unclear. Given the
putative link between cardiac ROS levels and arrhythmias [248], it is likely that the anti-
107
arrhythmic effects of acetaminophen are related to its ability to act as an anti-oxidant [27,
29]. Using the same heart tissues from the present study, we have previously reported that
chronic acetaminophen ingestion significantly diminished age-associated increases in
cardiac ROS and apoptosis in the F344XBN rats [203]. Whether the changes we see in
Cx43 expression with acetaminophen treatment are directly linked to acetaminophen-
associated changes in cardiac ROS levels is unclear and beyond the scope of this study.
Nonetheless, these data are consistent with the notion that acetaminophen is capable of
increasing Cx43 expression and that long term acetaminophen ingestion may be
beneficial in reducing the incidence of age-related arrhythmias.
Using the F344XBN aging rat model, we have previously reported that aging is
associated with higher nitrosative and oxidative stress [18] ultimately leading to greater
cardiomyocyte apoptosis [20]. Other studies have also shown that myocardial aging is
characterized by LV fibrosis that has been linked to a progressive reduction in
cardiomyocyte number, increases in cardiac fibroblast (CF) proliferation, and the
deposition of collagen in the LV [85, 280]. Excessive fibrosis can cause decreases in
myocardial compliance and may be linked to the development of ventricular dysfunction
and arrhythmias [139]. Likewise, our findings also show significantly higher fibrosis in
33-month control animals compared to 6-month animals (Figure 4.2). Interestingly, six
months of chronic acetaminophen ingestion significantly diminished myocardial fibrosis
compared to that observed in age-matched control animals (Figure 4.2). These results are
in agreement with our previous report suggesting that chronic acetaminophen treatment
significantly diminishes age-related cardiomyocyte apoptosis in male F344XBN rats
[203]. Whether the decrease in cardiac fibrosis we see in acetaminophen treated aged rats
108
is linked to decreases in the amount of cardiomyocyte apoptosis or due to alterations in
signaling pathways that control cardiac fibrosis warrants further investigation. Similarly,
whether the decrease in cardiac fibrosis we observed with acetaminophen treatment is
directly responsible for the diminished cardiac arrhythmias we observe in the treated rats
is currently not clear.
Aging in the F344XBN heart is associated with diminished miR-1, miR-133, miR-
214, miR-30a and miR-30d expression
Previous work by our laboratory and others have demonstrated that aging in the
male F344XBN is associated with increases in heart mass, LV hypertrophy, increased
incidence of cardiac arrhythmias, decreased ventricular compliance and impairment in
systolic and diastolic function [16, 17] as well as increased expression of oxidative-
nitrosative stress [18] and cardiomyocyte apoptosis [20]. Using semi-quantitative real
time RT-PCR we demonstrate a lower amount of miR-1, miR-133, miR-214, miR-30a
and miR-30d with aging (Figures 4.4-4.6). The physiological significance of these
findings awaits further experimentation. Previous data has suggested that miR-1 and
miR-133 expression is upregulated, downregulated, and unchanged during cardiac
remodeling [164-166]. Why aging may decrease the expression of miR-1, miR-133,
miR-214, miR-30a and miR-30d levels is not fully understood. The reasons for divergent
results is unknown but may be related to differences in disease models, extent of disease,
or the myocardial regions sampled.
Recent reports have implicated miR-1 as participating in the etiology of
ventricular arrhythmias as it is thought that this miRNA post-transcriptionally represses
109
the expression of Cx43 [166]. Interestingly, the increase in arrhythmias we observe with
aging were associated with decreases in Cx43 (Figure 4.3), and decreases in miR-1
expression (Figure 4.4). This result is in opposition to the previous findings suggesting
that decreases in miR-1 expression will lead to increase in Cx43 expression. Indeed, the
role that miR-1 plays in regulating Cx43 in the aging heart is still unclear as treatment
associated increases in miR-1 expression (Figure 4.4) would lead one to predict that Cx43
levels would be decreased. Clearly this postulate was opposite to what we found as
acetaminophen treatment led to slight yet significant increases in Cx43 expression. In
addition to its role in regulating Cx43, it is also possible that miR-1 acts as an inhibitor of
cardiac hypertrophy. For example, the expression of miR-1 in culture inhibits serum-
induced hypertrophy and is associated with inhibited RasGAP, Cdk9 and fibronectin
expression [281]. Recent data has also shown that cardiac-specific over expression of
miR-1 in the embryonic heart inhibits cardiomyocyte proliferation and prevents
expansion of the ventricular myocardium [168]. As such, it is possible that the decrease
in miR-1 we see with aging and increase in the aged acetaminophen treated animals may
indicate a role for miR-1 to limit cardiac remodeling. The mechanism(s) underlying these
alterations is currently unclear but may be related to the antioxidant properties of
acetaminophen. Indeed, recent experiments have shown that the expression of several
miRNA species can be modulated by changes in cellular ROS [282]. For example, the
expression of miR-21 is increased following the exposure of cultured myocytes to
increased hydrogen peroxide while miR23b is down regulated in vascular smooth muscle
cells after exposure to hydrogen peroxide [283]. Further experiments perhaps using other
methods, pharmacological treatments or time points of observation will no doubt be
110
useful in increasing our understanding of how ROS levels may influence miRNA
expression.
Similar to many micro-RNAs, the role of miR-133 in cardiac remodeling has yet
to be established. Recent work has demonstrated that increased miR-133 results in the
suppression of protein synthesis and inhibition of hypertrophic growth in cultured
neonatal cardiac myocytes [164]. In this same study the authors also showed that
antisense RNA oligonucleotides targeting miR-133 resulted in cardiac hypertrophy and
reinduction of the fetal gene program [164]. Other studies have suggested that miR-133
may act as antiapoptotic factor by repressing the translation of caspase-9 [172]. Given
these postulates it is possible that the age-associated decrease in miR-133 we observe
may play a role in the ventricular hypertrophy seen in these animals with advanced age.
Alternatively, miR-133 has also been implicated in the pathogenesis of cardiac
arrhythmias as it has been shown that this molecule can regulate the ether-a-go-go related
gene (ERG), which encodes a key portion of the cardiac potassium channel [284].
Whether the changes in miR-133 we observe are related to factors controlling cardiac
hypertrophy or cardiac rhythm will require further investigation.
In addition to miR-1 and miR-133, recent studies have also implicated a role for
miR-30 in cardiac remodeling. Liu et al. showed that miR-133 knockout mice develop
severe fibrosis and heart failure [285]. Recently, Duisters et al. investigated the
regulation of connective tissue growth factor (CTGF), and showed that the expression of
miR-133 and miR-30 was inversely related to the amount of CTGF in rodent models of
heart disease and in human patients with pathological LV hypertrophy [176]. Similar to
their findings, the age-related increase in fibrosis we observe in F344XBN rat hearts are
111
associated with diminished expression of miR-133, miR-30a and miR-30d (Figures 4.4,
4.5). Conversely, the diminished myocardial fibrosis we see in acetaminophen treated
aged rats was not associated with any alterations in the expression of miR-133, miR-30a
and miR-30d.
The role of miR-214 in cardiac remodeling has not been elucidated. Recent study
suggests that miR-214 is upregulated during pathological hypertrophy; however,
transgenic mice over-expressing miR-214 exhibited no cardiac abnormalities [162]. Our
results indicate that miR-214 expression significantly decreases with age in aged control
rats and that the expression of this molecule remains unchanged with acetaminophen
ingestion (Figure 4.6). Understanding why the expression of miR-214 is altered
differently in aged hypertrophied hearts compared to other models of pathological
hypertrophy remains unclear and will require further investigation.
In conclusion, the results of this study show that chronic acetaminophen treatment
decreases the incidence of age-associated arrhythmias and that this alteration is
associated with increased Cx43, diminished myocardial fibrosis and alterations in cardiac
miRNA expression. Given that acetaminophen is well tolerated in humans and approved
by the FDA for over the counter sale, the possibility exists that this widely available drug
may be an attractive therapeutic option in the war against heart disease. Further
experiments using different animals and methods may be warranted to expand upon these
observations.
112
Funding
This study was supported by funding from McNeil Pharmaceuticals and the Department
of Energy Grant DE-PS02-09ER09-01 to ERB.
113
Figures
Figure 4. 1. Chronic acetaminophen treatment decreases incidence of cardiac
arrhythmias. (A) Percentage incidence of premature atrial (PAC) and ventricular (PVC)
contractions observed in the 33-mo control and 33-mo APAP-treated rats (n=6). (B)
Representative echocardiographs (Lead II) recorded from 6-mo control, 33-mo control
and 33-mo APAP-treated rats (n=6).
114
Figure 4. 2. Chronic acetaminophen attenuates myocardial fibrosis in aged rat hearts.
Representative ventricular myocardium sections from 6-mo control, 33-mo control and
33-mo treated rats treated with Masson’s Trichrome stain (A) and Hematoxylin & Eosin
stain (B) (n=4).
115
Figure 4. 3. Chronic acetaminophen preserves myocardial membrane Cx43. (A) Protein
isolates of hearts from 6-month control, 33-month control and 33-month treated hearts
were analyzed by immunoblotting to determine Cx43 protein levels. Ponceau S staining
of the nitrocellulose membrane along with densitometric analysis of protein present was
done to verify equivalent protein loading between the lanes. *Significant difference from
adult (6-month) value (P < 0.05). †Significant difference from age matched 33-month
control animals (P < 0.05). n = 6 for all groups (B) Representative immunohistochemical
images of hearts sections obtained from 6-mo control, 33-mo control and 33-mo APAP-
treated rat hearts using an antibody against Cx43.n=4 hearts per age group
116
Figure 4. 4. Aging decreases myocardial miR-1 and miR-133 expression. Quantitative
RT-PCR analyses of miRNA expression in 6-mo control, 33-mo control and 33-mo
treated rat hearts using SYBR green I. Relative expression of miR-1 (A) and miR-133 (B)
between groups was calculated after normalization to U1 miR expression in the same
samples. * Significantly different from 6-mo Control. † Significantly different from 33-
mo control. For all comparisons, P < 0.05; n = 4
117
Figure 4. 5. Aging decreases myocardial miR-30a and miR-30d expression. Quantitative
RT-PCR analyses of miRNA expression in 6-mo control, 33-mo control and 33-mo
treated rat hearts using SYBR green I. Relative expression of miR-30a (A) and miR-30d
(B) between groups was calculated after normalization to U1 miR expression in the same
samples. * Significantly different from 6-mo Control. † Significantly different from 33-
mo control. For all comparisons, P < 0.05; n = 4
118
Figure 4. 6. Chronic acetaminophen treatment alters myocardial miR-214 expression.
Quantitative RT-PCR analyses of miRNA expression in 6-mo control, 33-mo control and
33-mo treated rat hearts using SYBR green I. Relative expression of miR-214 between
groups was calculated after normalization to U1 miR expression in the same samples. *
Significantly different from 6-mo Control. † Significantly different from 33-mo control.
For all comparisons, P < 0.05; n = 4
119
CHAPTER IV
GENERAL DISCUSSION
The primary purpose of this study was to investigate the molecular mechanisms
underlying age-associated alterations in the structure and function of the aging F344XBN
rat heart. In addition, we also examined the efficacy of chronic acetaminophen ingestion
to prevent or reverse age-associated cardiac dysfunction. To accomplish these goals, we
determined the effects of aging and acetaminophen ingestion on heart size, mass,
myocyte cross sectional area, myocyte histology, indices of oxidative stress, the
incidence of cardiac apoptosis, protein expression and cardiac structure and function.
The aged (65+ years of age) now comprise the most rapidly growing segment of
the United States population and mounting evidence suggests that advancing age is
associated with increased risk of cardiovascular disease and a progressive decline in
cardiac function [3, 30]. It is well established that aging is associated with myocardial
structural changes including loss of myocytes, hypertrophy of the remaining
cardiomyocytes with progressive increase in myocyte cell volume per nucleus, reduction
in the number of conduction cells and wall thickening [1]. The exact molecular
mechanisms underlying the structural and functional alterations that lead to increased
susceptibility of the aging heart to fatal arrhythmias are not well understood. Age-related
alterations in cardiac structure and function have been mostly studied in human hearts at
the time of transplantation, while animal models have been found to be extremely useful
for the assessment of pathological processes and the development of new treatments [13].
A recent review of aging rat models concluded that the F344XBN rat strain seems to live
120
longer while also exhibiting “physiological aging” to a greater degree than other rat
models [13-15].
Age-related alterations in cardiac structure and function of F344XBN rats
Recent investigations using the Fischer F344XBN rat model have demonstrated
that aging in these animals is associated with an increased incidence of cardiac
arrhythmias as well as impairment of systolic and diastolic function [16, 17]. Hacker et
al. performed echocardiographic and hemodynamic analyses in male F344XBN rats from
adulthood to the very aged (n = 6 per 12-, 18-, 21-, 24-, 27-, 30-, 33-, 36-, and 39-mo-old
group) and revealed that aged rats had an increased LV mass-to-body weight ratio,
deterioration of systolic function, gradual increase in fibrosis and a decrease in
cardiomyocyte volume density [16]. In an another study, Walker et al. performed two-
dimensional ECHO measurements on male F344/BN rats aged 6-, 30-, and 36-month
and demonstrated that aging was associated with cardiac arrhythmias in 72% of 36-
month rats, 10% of 30-month rats and 0% in 6-month rats [17]. Previous work by our
laboratory has shown that aging in the F344XBN heart is characterized by increased
oxidative-nitrosative stress and that these findings were highly correlated with increases
in LV wall thickness [18]. Furthermore, this study has also demonstrated that age-related
increases in cardiac oxidative-nitrosative stress were significantly correlated with
changes in the expression and/or regulation of proteins involved in transcriptional (NF-
κB) activities, signaling (MAPKs along with Src), apoptotic (Bcl-2, Traf-2), and cellular
stress (HSPs) [18]. Other work has shown that aging in the F344XBN heart is associated
121
with increases in cardiac apoptosis and that this increase in cell death may be due, at least
in part, to elevations in cardiac ROS [20].
Findings from the present investigation appear to confirm that aging in F344XBN
rat heart is associated with increases in cardiac ROS, apoptosis and fibrosis ultimately
leading to an increased incidence of arrhythmias. In addition, our findings from this study
also demonstrate that aging in F344XBN rats is associated with significant increase in
heart size, heart weight, heart weight to body weight ratio, and cardiomyocyte fiber cross
sectional area. Specifically, we observed that heart size and body weight gradually
increased with age but that the heart to body weight ratio remain unchanged up to 30
months before increasing significantly at 36-months of age. In a similar fashion, the
cardiomyocyte fiber cross sectional areas tended to increase rapidly from 30 – 36 months
before reaching a level at 36 months that was nearly three times larger than that observed
in the 6-month adult animals. Furthermore, we also report that very aged hearts exhibit
the activation of multiple signaling mechanisms during the hypertrophic response. Our
findings suggest that age-related cardiac hypertrophy in F344XBN rats is predominantly
mediated via activation of the ERK1/2 and Akt signaling pathways. We observed
increased phosphorylation (activation) of ERK1/2, Akt at Ser 473 and Thr 308 in both the
33- and 36-month rat hearts. This activation of Akt was confirmed when we examined
the phosphorylation of Akt substrate, GSK3β. Likewise, PTEN, a negative modulator of
PI3K/Akt/mTOR pathway [225] that is inactivated upon phosphorylation, was found to
be significantly higher in the very aged 33- and 36-month animals. Like Akt, a growing
body of evidence suggests that mTOR may play a key role in regulating cell growth and
122
protein synthesis [129, 226]. Our findings also demonstrate a large increase in the amount
of mTOR phosphorylation in the very aged 36-month animals. It is thought that mTOR
may be involved in controlling a number of downstream signaling proteins that regulate
mRNA translation [133] such as the translational repressor proteins 4E-binding protein-1
(4E-BP1) and the ribosomal protein S6 kinase (rp6k) [123]. Consistent with our findings
of an increased mTOR phosphorylation, we observed that the phosphorylation of 4EBP1
and rpS6 was increased significantly in the very aged 36-month animals. Taken together,
our current and previous findings [17, 18, 20, 203] suggest that aging in the F344XBN rat
heart is associated with diminished cardiac function, elevations in oxidative stress,
increased cardiomyocyte apoptosis and cardiomyocyte hypertrophy with the latter
occurring probably as a result of increased activation of the ERK1/2 and Akt signaling
pathways.
Cardioprotective properties of acetaminophen in aged F344XBN rats
Acetaminophen exhibits antipyretic and analgesic activities and has been widely
used over the past several decades. Being a phenolic compound, acetaminophen has an
inherent ability to scavenge free radicals. A recent study has suggested that this
compound may be an effective antioxidant given its ability to protect against membrane
lipid oxidation through the scavenging of peroxyl radicals and peroxynitrite [192].
Merrill et al. have shown that acetaminophen protects the isolated guinea pig hearts from
the damaging effects of myocardial ischemia and reperfusion, that it preserves
myofibrillar ultrastructure, and is associated with the attenuated production of both
hydroxyl radicals and peroxynitrite [29]. In an another study, Merrill et al. investigated
123
the effect of acetaminophen on pentobarbital pretreated isolated guinea pig hearts that
underwent ischemia-reperfusion and reported that acetaminophen (0.35mmol/l)
significantly reduced the frequency of pentobarbital induced arrhythmias [27].
Collectively these findings suggest that under selective conditions (e.g., those causing
release of free radicals and other oxidants) acetaminophen can act as an antioxidant and
that this agent is able to prevent ventricular arrhythmias [27, 29]. Chronically
administered acetaminophen has also been shown to provide cardioprotection to the
postischemic, reperfused rodent myocardium [28]. Here for the first time, we have
investigated the efficacy of chronic acetaminophen ingestion to prevent or reverse the
oxidative-nitrosative stress mediated alterations in cardiac structure and function in aged
F344XBN rats.
Our findings suggest that acetaminophen is able to diminish age-related increases
in cardiac reactive oxygen species (ROS) and apoptosis in these animals. Compared to 6-
month control animals, indices of oxidative (superoxide anion [O2•–] and 4-hydroxy-2-
nonenal [4-HNE]) and nitrosative (protein nitrotyrosylation) stress were markedly
increased in 33-month old rat hearts. Long term treatment with acetaminophen
(30mg/kg/day p.o. for six months) exhibited diminished age-related increases in cardiac
ROS levels and TUNEL positive nuclei, and these changes were accompanied by
improvements in the Bax/Bcl2 ratio, diminished evidence of caspase-3 activation and
increased phosphorylation of protein kinase B (Akt), ERK1/2, p70S6K and GSK-3β.
How acetaminophen might decrease cardiac ROS is not well understood at this time and
warrants further investigation. Previous reports suggest that acetaminophen may act as an
antioxidant as it is a mono-phenol [23]. Improving our understanding of these
124
mechanisms and how they may interact with other drugs and disease processes could lead
to new ways in improving our ability to manage heart disease.
In addition, we have also examined the efficacy of chronic acetaminophen
treatment on the incidence of cardiac arrhythmias, Cx43 expression, myocardial fibrosis
and miRNA expression in the aging F344XBN rats. In agreement with our previous
findings [17], serial electrocardiography showed evidence of increased incidence of
arrhythmias with age. Importantly, chronic acetaminophen treatment led to a marked
decrease in the incidence of premature atrial (PAC) and ventricular (PVC) contractions.
Recent data has implicated a link between the loss of the gap junction protein Cx43 and
fatal arrhythmias. For example, using a heart-specific Cx43 conditional knockout mouse
model in which cardiac Cx43 abundance decreases rapidly within the first few weeks of
age, Danik and colleagues have demonstrated a decrease in cardiac conduction velocity
and a dramatic increase in susceptibility to inducible lethal ventricular tachyarrhythmias
[279]. Here, we observed that the expression of Cx43 is significantly lowered with aging
and importantly that acetaminophen treatment is associated with increased protein levels
of Cx43. Although these findings, taken together, suggest that chronic acetaminophen
ingestion may be able to influence the expression of Cx43, it is unlikely that the
magnitude of the differences we see in the regulation of Cx43 can, by itself, explain the
decrease in age-associated arrhythmias we observed in the acetaminophen treated
animals. Indeed, previous data have suggested that Cx43 protein losses of up to ~40% are
insufficient by themselves to induce cardiac arrhythmias [279]. How acetaminophen may
decrease cardiac arrhythmias in the aging F344XBN heart is presently unclear. Given the
putative link between cardiac ROS levels and arrhythmias [248], it is likely that the anti-
125
arrhythmic effects of acetaminophen are related to its ability to act as an anti-oxidant [27,
29]. Whether the changes we see in Cx43 expression with acetaminophen treatment are
directly linked to acetaminophen-associated changes in cardiac ROS levels is unclear and
beyond the scope of this study.
Previous studies have demonstrated that myocardial aging is characterized by LV
LV fibrosis that has been linked to a progressive reduction in cardiomyocyte number,
increases in cardiac fibroblast (CF) proliferation, and the deposition of collagen in the LV
[85, 280]. Excessive fibrosis can cause decreases in myocardial compliance and may be
linked to the development of ventricular dysfunction and arrhythmias [139]. Here, we
also observe significantly higher fibrosis in aged F344XBN rat hearts and, interestingly,
that six months of chronic acetaminophen ingestion significantly diminished myocardial
fibrosis, compared to that observed in age-matched control animals. These results are in
agreement with our previous findings suggesting that chronic acetaminophen treatment
significantly diminishes age-related cardiomyocyte apoptosis in male F344XBN rats.
Whether the decrease in cardiac fibrosis we see in acetaminophen treated aged rats is
linked to decreases in the amount of cardiomyocyte apoptosis or due to alterations in
signaling pathways that control cardiac fibrosis warrants further investigation. Similarly,
whether the decrease in cardiac fibrosis we observed with acetaminophen treatment is
directly responsible for the diminished cardiac arrhythmias we observe in the treated rats
is currently not clear. In summary, our findings suggest that chronic acetaminophen
treatment in F344XBN rats decreases age-related increases in cardiac ROS, the incidence
of age-associated arrhythmias, increased Cx43 protein expression, and diminished
evidence of myocardial fibrosis.
126
Regulation of miRNA with aging and acetaminophen treatment in F344XBN rats
MiRNAs (miRNAs) are small ~ 22-nucleotide noncoding RNAs that inhibit
transcription or translation by interacting with the 3′ untranslated region (3′UTR) of
target mRNAs [156]. Recent data has suggested that miRNA may play a role in the
regulation of cardiac differentiation and disease [163]. In this context, several miRNAs
including miR-1, 21, 29, 30, 195, 133, 206 and 208 have been described as playing an
important role in the process of cardiac remodeling, possibly by regulating changes in
gene expression that accompany pathological cardiac hypertrophy and contractile
dysfunction [162, 164, 165, 170]. The factors that regulate the expression of miRNA are
not clear although recent studies have suggested that alterations in cellular ROS levels
may be involved [178]. Similarly, how aging may affect the expression of cardiac
miRNA and the effect of chronic use of antioxidants have not been elucidated. Here, for
the first time, we have investigated the regulation of miRNA and the alterations with
chronic acetaminophen ingestion in aged F344XBN rat hearts. Our findings suggest that
aging in F344XBN rat hearts is associated with a lower amount of miR-1, miR-133, miR-
214, miR-30a and miR-30d. Acetaminophen treatment increased the expression of miR-1
and miR-214, while the amount of miR-133, miR-30a and miR-30d remain unchanged.
The physiological significance of these findings awaits further experimentation. Previous
data have suggested that miR-1 and miR-133 expression is upregulated, downregulated,
and unchanged during cardiac remodeling [164-166]. Why aging may decrease the
expression of miR-1, miR-133, miR-214, miR-30a and miR-30d levels is not fully
understood. The reasons for divergent results is unknown but may be related to
differences in disease models, extent of disease, or the myocardial regions sampled.
127
SUMMARY
1. Aging in F344XBN rats is associated with significant increases in heart size, heart
weight, heart weight to body weight ratio and cardiomyocyte fiber cross sectional
area.
2. Aging associated cardiac hypertrophy in F344XBN rats is predominantly regulated
via activation of the ERK1/2 and Akt signaling pathways.
3. Age-related cardiac hypertrophy in F344XBN rats is associated with increased
protein translational signaling.
4. Chronic acetaminophen ingestion diminished age-associated increases in indices of
oxidative (superoxide anion [O2•–] and 4-hydroxy-2-nonenal [4-HNE]) and
nitrosative (protein nitrotyrosylation) stress in F344XBN rat hearts.
5. Acetaminophen treated animals exhibited diminished age-related increases in cardiac
TUNEL positive nuclei, improvements in the Bax/Bcl2 ratio, and diminished
evidence of caspase-3 activation.
6. Acetaminophen increased phosphorylation of Akt, ERK1/2 and GSK-3β which play a
key role in cell survival signaling.
7. Aging in F344XBN rats is associated with increased incidence of cardiac
arrhythmias, lowered expression of gap junction protein connexin-43 (Cx43),
increased myocardial fibrosis and altered regulation of miRNA.
8. Chronic acetaminophen ingestion is efficacious in preventing the incidence of age-
associated arrhythmias and this alteration is associated with diminished fibrosis and
changes in cardiac miRNA expression.
128
FUTURE DIRECTIONS
The present study focused on investigating the molecular mechanisms underlying
age-related cardiac remodeling in F344XBN rats, with special emphasis on the efficacy
of chronic acetaminophen ingestion as a therapeutic approach. Future studies will be
planned to further extend on the findings of the current study with respect to the
corresponding specific aims as detailed below.
Specific Aim I:
Findings from the present study suggest that aging in male F344XBN rat heart is
associated with increased activation of ERK1/2 and Akt signaling pathways resulting in
increased protein synthesis ultimately leading to cardiac hypertrophy. Future studies will
treat the aged male F344XBN rats with antihypertrophic drugs to test whether a reduction
in cardiac hypertrophy is accompanied by the reduction in the activation of ERK1/2 and
Akt. Results from such studies will no doubt further the molecular signaling profile
underlying age-related cardiac hypertrophy. Whether aging in female F344XBN rat
hearts is associated with similar cardiac remodelling or not remain unexplored. Studies
using young, adult and aged ovariectomized or non-ovariectomized female F344XBN
rats will help to better understand the influence of age and gender on cardiac structure
and function in older age.
Specific Aims II and III
Findings from the present study suggest that chronic acetaminophen ingestion is
able to diminish age-related increases in cardiac oxidative-nitrosative stress, cardiac
129
apoptosis, myocardial fibrosis and the incidence of arrhythmias in male F344XBN rats.
Still, little is known about the mechanisms of cardioprotection employed by chronic
acetaminophen treatment. Previous reports suggest that acetaminophen may act as an
antioxidant as it is a mono-phenol [23]. Future studies will be directed at investigating the
effect of chronic acetaminophen treatment on the expression and activity of antioxidant
enzyme systems in aged male F344XBN rat hearts. Findings from such studies will
provide additional mechanistic data to delineate whether the cardioprotective properties
of acetaminophen are through free radical scavenging or by enhancement of inherent
antioxidant enzymes systems. Moreover, as the majority of studies examining the
cardioprotective properties of acetaminophen were conducted in male rats, the effect of
gender on such cardioprotection conferred by acetaminophen remain largely unknown.
130
REFERENCES
1. Karavidas, A., et al., Aging and the cardiovascular system. Hellenic J Cardiol, 2010. 51(5): p. 421-7.
2. Gerstenblith, G., et al., Echocardiographic assessment of a normal adult aging population. Circulation, 1977. 56(2): p. 273-8.
3. Lakatta, E.G., Age-associated cardiovascular changes in health: impact on cardiovascular disease in older persons. Heart Fail Rev, 2002. 7(1): p. 29-49.
4. Jacobzone S, O.H., Ageing and Health Care Costs. Internationale Politik und Gesellschaft Online (International Politics and Society) 1/2002.
5. Juhaszova, M., et al., Protection in the aged heart: preventing the heart-break of old age? Cardiovasc Res, 2005. 66(2): p. 233-44.
6. Tyagi, P. and G. Tayal, Ischemic preconditioning of myocardium. Acta Pharmacol Sin, 2002. 23(10): p. 865-70.
7. Boluyt, M.O., O.H. Bing, and E.G. Lakatta, The ageing spontaneously hypertensive rat as a model of the transition from stable compensated hypertrophy to heart failure. Eur Heart J, 1995. 16 Suppl N: p. 19-30.
8. d'Alessio, P., Aging and the endothelium. Exp Gerontol, 2004. 39(2): p. 165-71. 9. Delp, M.D., et al., Rat aortic vasoreactivity is altered by old age and hindlimb
unloading. J Appl Physiol, 1995. 78(6): p. 2079-86. 10. Josephson, I.R., et al., Alterations in properties of L-type Ca channels in aging rat
heart. J Mol Cell Cardiol, 2002. 34(3): p. 297-308. 11. Moreau, R., et al., Reversal by aminoguanidine of the age-related increase in
glycoxidation and lipoxidation in the cardiovascular system of Fischer 344 rats. Biochem Pharmacol, 2005. 69(1): p. 29-40.
12. Tschudi, M.R., et al., Effect of age on kinetics of nitric oxide release in rat aorta and pulmonary artery. J Clin Invest, 1996. 98(4): p. 899-905.
13. Lipman, R.D., Pathobiology of aging rodents: inbred and hybrid models. Exp Gerontol, 1997. 32(1-2): p. 215-28.
14. Lipman, R.D., G.E. Dallal, and R.T. Bronson, Effects of genotype and diet on age-related lesions in ad libitum fed and calorie-restricted F344, BN, and BNF3F1 rats. J Gerontol A Biol Sci Med Sci, 1999. 54(11): p. B478-91.
15. Turturro, A., et al., Growth curves and survival characteristics of the animals used in the Biomarkers of Aging Program. J Gerontol A Biol Sci Med Sci, 1999. 54(11): p. B492-501.
16. Hacker, T.A., et al., Age-related changes in cardiac structure and function in Fischer 344 x Brown Norway hybrid rats. Am J Physiol Heart Circ Physiol, 2006. 290(1): p. H304-11.
17. Walker, E.M., Jr., et al., Age-associated changes in hearts of male Fischer 344/Brown Norway F1 rats. Ann Clin Lab Sci, 2006. 36(4): p. 427-38.
131
18. Asano, S., et al., Aging influences multiple indices of oxidative stress in the heart of the Fischer 344/NNia x Brown Norway/BiNia rat. Redox Rep, 2007. 12(4): p. 167-80.
19. Tsutsui, H., S. Kinugawa, and S. Matsushima, Mitochondrial oxidative stress and dysfunction in myocardial remodelling. Cardiovasc Res, 2009. 81(3): p. 449-56.
20. Kakarla, S.K., et al., Possible molecular mechanisms underlying age-related cardiomyocyte apoptosis in the F344XBN rat heart. J Gerontol A Biol Sci Med Sci, 2010. 65(2): p. 147-55.
21. Spiler, N.M., T.H. Rork, and G.F. Merrill, An old drug with a new purpose: cardiovascular actions of acetaminophen (paracetamol). Curr Drug Targets Cardiovasc Haematol Disord, 2005. 5(5): p. 419-29.
22. Ameer, B. and D.J. Greenblatt, Acetaminophen. Ann Intern Med, 1977. 87(2): p. 202-9.
23. Dinis, T.C., V.M. Maderia, and L.M. Almeida, Action of phenolic derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch Biochem Biophys, 1994. 315(1): p. 161-9.
24. Rork, T.H., et al., Acetaminophen in the hypoxic and reoxygenated guinea pig myocardium. Exp Biol Med (Maywood), 2004. 229(11): p. 1154-61.
25. Merrill, G.F., Acetaminophen and low-flow myocardial ischemia: efficacy and antioxidant mechanisms. Am J Physiol Heart Circ Physiol, 2002. 282(4): p. H1341-9.
26. Golfetti, R., et al., Acetaminophen in the post-ischemia reperfused myocardium. Exp Biol Med (Maywood), 2002. 227(11): p. 1031-7.
27. Merrill, G.F. and E. Goldberg, Antioxidant properties of acetaminophen and cardioprotection. Basic Res Cardiol, 2001. 96(5): p. 423-30.
28. Golfetti, R., T. Rork, and G. Merrill, Chronically administered acetaminophen and the ischemia/reperfused myocardium. Exp Biol Med (Maywood), 2003. 228(6): p. 674-82.
29. Merrill, G., et al., Coronary and myocardial effects of acetaminophen: protection during ischemia-reperfusion. Am J Physiol Heart Circ Physiol, 2001. 280(6): p. H2631-8.
30. Lakatta, E.G., Aging and cardiovascular structure and function in healthy sedentary humans. Aging (Milano), 1998. 10(2): p. 162-4.
31. Harman, D., The free radical theory of aging. Antioxid Redox Signal, 2003. 5(5): p. 557-61.
32. Lakatta, E.G., Cardiovascular Aging: Perspectives From Humans to Rodents. Am J Geriatr Cardiol, 1998. 7(6): p. 32-45.
33. World Population Ageing, 2009; United Nations Department of Economic and Social Affairs/Population Division;ESA/P/WP/212 December 2009.
34. de Grey, A.D., et al., Antiaging technology and pseudoscience. Science, 2002. 296(5568): p. 656.
132
35. World Health Organization. World Health Report 2002, Annex Table 2 (deaths by cause, sex and mortality stratum in WHO Regions, estimates for 2001). Geneva, Switzerland: World Health Organization, 2002:186--91.
36. Kinsella K, V.V., U.S. Census Bureau. An Aging World: 2001. Washington, DC: U.S. Government Printing Office, 2001; series P95/01-1. 2001.
37. Donald G. Fowles, Saadia Greenberg; A Profile of Older Americans: 2009 ; Administration on Aging (AoA), U.S. .
38. Seidell, J.C. and T.L. Visscher, Body weight and weight change and their health implications for the elderly. Eur J Clin Nutr, 2000. 54 Suppl 3: p. S33-9.
39. Short, K.R. and K.S. Nair, Mechanisms of sarcopenia of aging. J Endocrinol Invest, 1999. 22(5 Suppl): p. 95-105.
40. Karakelides, H. and K.S. Nair, Sarcopenia of aging and its metabolic impact. Curr Top Dev Biol, 2005. 68: p. 123-48.
41. Fukagawa, N.K., L.G. Bandini, and J.B. Young, Effect of age on body composition and resting metabolic rate. Am J Physiol, 1990. 259(2 Pt 1): p. E233-8.
42. Harman, D., Aging: a theory based on free radical and radiation chemistry. J Gerontol, 1956. 11(3): p. 298-300.
43. Hulbert, A.J., et al., Life and death: metabolic rate, membrane composition, and life span of animals. Physiol Rev, 2007. 87(4): p. 1175-213.
44. Wahba, W.M., Influence of aging on lung function--clinical significance of changes from age twenty. Anesth Analg, 1983. 62(8): p. 764-76.
45. Leblanc, P., F. Ruff, and J. Milic-Emili, Effects of age and body position on "airway closure" in man. J Appl Physiol, 1970. 28(4): p. 448-51.
46. Martin, J.E. and M.T. Sheaff, Renal ageing. J Pathol, 2007. 211(2): p. 198-205. 47. Domenech, R.J. and P. Macho, [Cardiovascular aging]. Rev Med Chil, 2008.
136(12): p. 1582-8. 48. Cafagna, D. and E. Ponte, [Morphological and functional aspects of the
cardiovascular system related to aging: does "aging heart" exist?]. Minerva Med, 1997. 88(12): p. 491-500.
49. Ferrari, A.U., A. Radaelli, and M. Centola, Invited review: aging and the cardiovascular system. J Appl Physiol, 2003. 95(6): p. 2591-7.
50. Frey, N. and E.N. Olson, Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol, 2003. 65: p. 45-79.
51. Downes, T.R., et al., Mechanism of altered pattern of left ventricular filling with aging in subjects without cardiac disease. Am J Cardiol, 1989. 64(8): p. 523-7.
52. Bikkina, M., M.G. Larson, and D. Levy, Prognostic implications of asymptomatic ventricular arrhythmias: the Framingham Heart Study. Ann Intern Med, 1992. 117(12): p. 990-6.
53. Haider, A.W., et al., Increased left ventricular mass and hypertrophy are associated with increased risk for sudden death. J Am Coll Cardiol, 1998. 32(5): p. 1454-9.
133
54. Morales, M.A., et al., Age dependency of myocardial structure: a quantitative two-dimensional echocardiographic study in a normal population. Echocardiography, 2000. 17(3): p. 201-8.
55. Zaragoza, C., et al., Animal models of cardiovascular diseases. J Biomed Biotechnol, 2011. 2011: p. 497841.
56. Anversa, P., et al., Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart. Circ Res, 1990. 67(4): p. 871-85.
57. Anversa, P. and J.M. Capasso, Cellular basis of aging in the mammalian heart. Scanning Microsc, 1991. 5(4): p. 1065-73; discussion 1073-4.
58. Boluyt, M.O., et al., Echocardiographic assessment of age-associated changes in systolic and diastolic function of the female F344 rat heart. J Appl Physiol, 2004. 96(2): p. 822-8.
59. Sohal, R.S., L.A. Arnold, and B.H. Sohal, Age-related changes in antioxidant enzymes and prooxidant generation in tissues of the rat with special reference to parameters in two insect species. Free Radic Biol Med, 1990. 9(6): p. 495-500.
60. Runge, M.S., K. Molnar, and N.R. Madamanchi, "Old" hearts and arteries: the role of oxidative stress. Trans Am Clin Climatol Assoc, 2010. 121: p. 52-8; discussion 59-60.
61. Anilkumar, N., A. Sirker, and A.M. Shah, Redox sensitive signaling pathways in cardiac remodeling, hypertrophy and failure. Front Biosci, 2009. 14: p. 3168-87.
62. Kinugawa, S., et al., Treatment with dimethylthiourea prevents left ventricular remodeling and failure after experimental myocardial infarction in mice: role of oxidative stress. Circ Res, 2000. 87(5): p. 392-8.
63. Nozik-Grayck, E. and K.R. Stenmark, Role of reactive oxygen species in chronic hypoxia-induced pulmonary hypertension and vascular remodeling. Adv Exp Med Biol, 2007. 618: p. 101-12.
64. Siwik, D.A. and W.S. Colucci, Regulation of matrix metalloproteinases by cytokines and reactive oxygen/nitrogen species in the myocardium. Heart Fail Rev, 2004. 9(1): p. 43-51.
65. de la Asuncion, J.G., et al., Mitochondrial glutathione oxidation correlates with age-associated oxidative damage to mitochondrial DNA. FASEB J, 1996. 10(2): p. 333-8.
66. Shigenaga, M.K., T.M. Hagen, and B.N. Ames, Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci U S A, 1994. 91(23): p. 10771-8.
67. Bejma, J., P. Ramires, and L.L. Ji, Free radical generation and oxidative stress with ageing and exercise: differential effects in the myocardium and liver. Acta Physiol Scand, 2000. 169(4): p. 343-51.
68. Kim, J.D., R.J. McCarter, and B.P. Yu, Influence of age, exercise, and dietary restriction on oxidative stress in rats. Aging (Milano), 1996. 8(2): p. 123-9.
69. Nohl, H., V. Breuninger, and D. Hegner, Influence of mitochondrial radical formation on energy-linked respiration. Eur J Biochem, 1978. 90(2): p. 385-90.
134
70. Sohal, R.S. and B.H. Sohal, Hydrogen peroxide release by mitochondria increases during aging. Mech Ageing Dev, 1991. 57(2): p. 187-202.
71. Yu, B.P., Cellular defenses against damage from reactive oxygen species. Physiol Rev, 1994. 74(1): p. 139-62.
72. Kevin, L.G., E. Novalija, and D.F. Stowe, Reactive oxygen species as mediators of cardiac injury and protection: the relevance to anesthesia practice. Anesth Analg, 2005. 101(5): p. 1275-87.
73. Nabeebaccus, A., M. Zhang, and A.M. Shah, NADPH oxidases and cardiac remodelling. Heart Fail Rev, 2011. 16(1): p. 5-12.
74. Cave, A., et al., NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology. Philos Trans R Soc Lond B Biol Sci, 2005. 360(1464): p. 2327-34.
75. Nakamura, K., et al., Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-alpha and angiotensin II. Circulation, 1998. 98(8): p. 794-9.
76. Delbosc, S., et al., Simvastatin prevents angiotensin II-induced cardiac alteration and oxidative stress. Hypertension, 2002. 40(2): p. 142-7.
77. Sabri, A., H.H. Hughie, and P.A. Lucchesi, Regulation of hypertrophic and apoptotic signaling pathways by reactive oxygen species in cardiac myocytes. Antioxid Redox Signal, 2003. 5(6): p. 731-40.
78. Ghosh, M.C., et al., Regulation of calcineurin by oxidative stress. Methods Enzymol, 2003. 366: p. 289-304.
79. Izumiya, Y., et al., Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensin II-induced cardiac hypertrophy and remodeling. Circ Res, 2003. 93(9): p. 874-83.
80. Kwon, S.H., et al., H(2)O(2) regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways. J Mol Cell Cardiol, 2003. 35(6): p. 615-21.
81. von Harsdorf, R., P.F. Li, and R. Dietz, Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation, 1999. 99(22): p. 2934-41.
82. Flora, S.J., Role of free radicals and antioxidants in health and disease. Cell Mol Biol (Noisy-le-grand), 2007. 53(1): p. 1-2.
83. Nishida, K. and K. Otsu, Cell death in heart failure. Circ J, 2008. 72 Suppl A: p. A17-21.
84. Centurione, L., et al., Age-related death-survival balance in myocardium: an immunohistochemical and biochemical study. Mech Ageing Dev, 2002. 123(4): p. 341-50.
85. Olivetti, G., et al., Cardiomyopathy of the aging human heart. Myocyte loss and reactive cellular hypertrophy. Circ Res, 1991. 68(6): p. 1560-8.
86. Melissari, M., et al., [The aging of the heart: weight and structural changes in the left ventricle with age]. G Ital Cardiol, 1991. 21(2): p. 119-30.
87. Anversa, P., et al., Myocyte cell loss and myocyte hypertrophy in the aging rat heart. J Am Coll Cardiol, 1986. 8(6): p. 1441-8.
135
88. Kajstura, J., et al., Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats. Am J Physiol, 1996. 271(3 Pt 2): p. H1215-28.
89. Wang, X., et al., The cellular response to oxidative stress: influences of mitogen-activated protein kinase signalling pathways on cell survival. Biochem J, 1998. 333 ( Pt 2): p. 291-300.
90. Xia, Z., et al., Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science, 1995. 270(5240): p. 1326-31.
91. Sugden, P.H. and A. Clerk, "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res, 1998. 83(4): p. 345-52.
92. Mizukami, Y., et al., Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation. Biochim Biophys Acta, 2001. 1540(3): p. 213-20.
93. Clerk, A., et al., Stimulation of "stress-regulated" mitogen-activated protein kinases (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses. J Biol Chem, 1998. 273(13): p. 7228-34.
94. Yue, T.L., et al., Inhibition of extracellular signal-regulated kinase enhances Ischemia/Reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res, 2000. 86(6): p. 692-9.
95. Kumar, D., et al., Apoptosis in adriamycin cardiomyopathy and its modulation by probucol. Antioxid Redox Signal, 2001. 3(1): p. 135-45.
96. Jacobson, M.D., et al., Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature, 1993. 361(6410): p. 365-9.
97. Cook, S.A., P.H. Sugden, and A. Clerk, Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential. Circ Res, 1999. 85(10): p. 940-9.
98. Wolter, K.G., et al., Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol, 1997. 139(5): p. 1281-92.
99. Zha, H., et al., Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J Biol Chem, 1996. 271(13): p. 7440-4.
100. Burlacu, A., Regulation of apoptosis by Bcl-2 family proteins. J Cell Mol Med, 2003. 7(3): p. 249-57.
101. Danial, N.N. and S.J. Korsmeyer, Cell death: critical control points. Cell, 2004. 116(2): p. 205-19.
102. Sharpe, J.C., D. Arnoult, and R.J. Youle, Control of mitochondrial permeability by Bcl-2 family members. Biochim Biophys Acta, 2004. 1644(2-3): p. 107-13.
136
103. Oltvai, Z.N., C.L. Milliman, and S.J. Korsmeyer, Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 1993. 74(4): p. 609-19.
104. Hengartner, M.O., The biochemistry of apoptosis. Nature, 2000. 407(6805): p. 770-6.
105. Phaneuf, S. and C. Leeuwenburgh, Cytochrome c release from mitochondria in the aging heart: a possible mechanism for apoptosis with age. Am J Physiol Regul Integr Comp Physiol, 2002. 282(2): p. R423-30.
106. Song, H.P., et al., The phosphatidylinositol 3-kinase-Akt pathway protects cardiomyocytes from ischaemic and hypoxic apoptosis via mitochondrial function. Clin Exp Pharmacol Physiol, 2010. 37(5-6): p. 598-604.
107. Song, G., G. Ouyang, and S. Bao, The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med, 2005. 9(1): p. 59-71.
108. Daneshvar, D., et al., Diastolic dysfunction: improved understanding using emerging imaging techniques. Am Heart J, 2010. 160(3): p. 394-404.
109. Levy, D., et al., Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med, 1990. 322(22): p. 1561-6.
110. Badano, L., et al., [Age-induced changes in the cardiovascular system in normal subjects]. G Ital Cardiol, 1992. 22(9): p. 1023-34.
111. Kitzman, D.W., Diastolic heart failure in the elderly. Heart Fail Rev, 2002. 7(1): p. 17-27.
112. Barry, S.P., S.M. Davidson, and P.A. Townsend, Molecular regulation of cardiac hypertrophy. Int J Biochem Cell Biol, 2008. 40(10): p. 2023-39.
113. Schaub, M.C., et al., Various hypertrophic stimuli induce distinct phenotypes in cardiomyocytes. J Mol Med, 1997. 75(11-12): p. 901-20.
114. Hunter, J.J. and K.R. Chien, Signaling pathways for cardiac hypertrophy and failure. N Engl J Med, 1999. 341(17): p. 1276-83.
115. Stanley, W.C. and M.P. Chandler, Energy metabolism in the normal and failing heart: potential for therapeutic interventions. Heart Fail Rev, 2002. 7(2): p. 115-30.
116. Sugden, P.H., Signalling pathways in cardiac myocyte hypertrophy. Ann Med, 2001. 33(9): p. 611-22.
117. Nicol, R.L., et al., Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy. Embo J, 2001. 20(11): p. 2757-67.
118. Ikeda, Y., et al., Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis. J Biol Chem, 2005. 280(33): p. 29661-6.
119. Sanna, B., et al., Direct and indirect interactions between calcineurin-NFAT and MEK1-extracellular signal-regulated kinase 1/2 signaling pathways regulate cardiac gene expression and cellular growth. Mol Cell Biol, 2005. 25(3): p. 865-78.
137
120. Molkentin, J.D., Calcineurin-NFAT signaling regulates the cardiac hypertrophic response in coordination with the MAPKs. Cardiovasc Res, 2004. 63(3): p. 467-75.
121. McMullen, J.R. and G.L. Jennings, Differences between pathological and physiological cardiac hypertrophy: novel therapeutic strategies to treat heart failure. Clin Exp Pharmacol Physiol, 2007. 34(4): p. 255-62.
122. Heineke, J. and J.D. Molkentin, Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol, 2006. 7(8): p. 589-600.
123. Proud, C.G., Ras, PI3-kinase and mTOR signaling in cardiac hypertrophy. Cardiovasc Res, 2004. 63(3): p. 403-13.
124. Oh, H., et al., Activation of phosphatidylinositol 3-kinase through glycoprotein 130 induces protein kinase B and p70 S6 kinase phosphorylation in cardiac myocytes. J Biol Chem, 1998. 273(16): p. 9703-10.
125. Sugden, P.H. and A. Clerk, Cellular mechanisms of cardiac hypertrophy. J Mol Med, 1998. 76(11): p. 725-46.
126. Aikawa, R., et al., Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest, 1997. 100(7): p. 1813-21.
127. Sabri, A., et al., Hydrogen peroxide activates mitogen-activated protein kinases and Na+-H+ exchange in neonatal rat cardiac myocytes. Circ Res, 1998. 82(10): p. 1053-62.
128. Ziolo, M.T., H. Katoh, and D.M. Bers, Expression of inducible nitric oxide synthase depresses beta-adrenergic-stimulated calcium release from the sarcoplasmic reticulum in intact ventricular myocytes. Circulation, 2001. 104(24): p. 2961-6.
129. Proud, C.G., mTORC1 signalling and mRNA translation. Biochem Soc Trans, 2009. 37(Pt 1): p. 227-31.
130. Dorn, G.W., 2nd and T. Force, Protein kinase cascades in the regulation of cardiac hypertrophy. J Clin Invest, 2005. 115(3): p. 527-37.
131. McMullen, J.R., et al., Inhibition of mTOR signaling with rapamycin regresses established cardiac hypertrophy induced by pressure overload. Circulation, 2004. 109(24): p. 3050-5.
132. Bodine, S.C., et al., Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol, 2001. 3(11): p. 1014-9.
133. Gingras, A.C., B. Raught, and N. Sonenberg, Control of translation by the target of rapamycin proteins. Prog Mol Subcell Biol, 2001. 27: p. 143-74.
134. Lakatta, E.G. and D. Levy, Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part II: the aging heart in health: links to heart disease. Circulation, 2003. 107(2): p. 346-54.
135. Kitzman, D.W., et al., Importance of heart failure with preserved systolic function in patients > or = 65 years of age. CHS Research Group. Cardiovascular Health Study. Am J Cardiol, 2001. 87(4): p. 413-9.
138
136. Capasso, J.M., et al., Severe myocardial dysfunction induced by ventricular remodeling in aging rat hearts. Am J Physiol, 1990. 259(4 Pt 2): p. H1086-96.
137. Gielen, S., et al., Aging and heart failure--similar syndromes of exercise intolerance? Implications for exercise-based interventions. Heart Fail Monit, 2005. 4(4): p. 130-6.
138. Stewart, S., et al., Heart failure and the aging population: an increasing burden in the 21st century? Heart, 2003. 89(1): p. 49-53.
139. Creemers, E.E. and Y.M. Pinto, Molecular mechanisms that control interstitial fibrosis in the pressure-overloaded heart. Cardiovasc Res, 2011. 89(2): p. 265-72.
140. Chen, W. and N.G. Frangogiannis, The role of inflammatory and fibrogenic pathways in heart failure associated with aging. Heart Fail Rev, 2010. 15(5): p. 415-22.
141. Centurione, L., et al., Correlations between protein kinase C zeta signaling and morphological modifications during rat heart development and aging. Mech Ageing Dev, 2003. 124(8-9): p. 957-66.
142. Eghbali, M., et al., Collagen accumulation in heart ventricles as a function of growth and aging. Cardiovasc Res, 1989. 23(8): p. 723-9.
143. Orlandi, A., et al., Role of ageing and coronary atherosclerosis in the development of cardiac fibrosis in the rabbit. Cardiovasc Res, 2004. 64(3): p. 544-52.
144. Lin, J., et al., Age-related cardiac muscle sarcopenia: Combining experimental and mathematical modeling to identify mechanisms. Exp Gerontol, 2008. 43(4): p. 296-306.
145. Sawada, M. and J.C. Carlson, Changes in superoxide radical and lipid peroxide formation in the brain, heart and liver during the lifetime of the rat. Mech Ageing Dev, 1987. 41(1-2): p. 125-37.
146. Siwik, D.A., P.J. Pagano, and W.S. Colucci, Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. Am J Physiol Cell Physiol, 2001. 280(1): p. C53-60.
147. Cheng, T.H., et al., Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts. J Am Coll Cardiol, 2003. 42(10): p. 1845-54.
148. Frangogiannis, N.G., et al., Critical role of monocyte chemoattractant protein-1/CC chemokine ligand 2 in the pathogenesis of ischemic cardiomyopathy. Circulation, 2007. 115(5): p. 584-92.
149. Dewald, O., et al., CCL2/Monocyte Chemoattractant Protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res, 2005. 96(8): p. 881-9.
150. Brooks, W.W. and C.H. Conrad, Myocardial fibrosis in transforming growth factor beta(1)heterozygous mice. J Mol Cell Cardiol, 2000. 32(2): p. 187-95.
151. Barcellos-Hoff, M.H. and T.A. Dix, Redox-mediated activation of latent transforming growth factor-beta 1. Mol Endocrinol, 1996. 10(9): p. 1077-83.
139
152. Park, S.K., et al., Hydrogen peroxide is a novel inducer of connective tissue growth factor. Biochem Biophys Res Commun, 2001. 284(4): p. 966-71.
153. Lee, A.A., et al., Angiotensin II stimulates the autocrine production of transforming growth factor-beta 1 in adult rat cardiac fibroblasts. J Mol Cell Cardiol, 1995. 27(10): p. 2347-57.
154. Campbell, S.E. and L.C. Katwa, Angiotensin II stimulated expression of transforming growth factor-beta1 in cardiac fibroblasts and myofibroblasts. J Mol Cell Cardiol, 1997. 29(7): p. 1947-58.
155. Rosenkranz, S., TGF-beta1 and angiotensin networking in cardiac remodeling. Cardiovasc Res, 2004. 63(3): p. 423-32.
156. Ambros, V., microRNAs: tiny regulators with great potential. Cell, 2001. 107(7): p. 823-6.
157. Lee, R.C., R.L. Feinbaum, and V. Ambros, The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 1993. 75(5): p. 843-54.
158. Esquela-Kerscher, A. and F.J. Slack, Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer, 2006. 6(4): p. 259-69.
159. Costinean, S., et al., Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci U S A, 2006. 103(18): p. 7024-9.
160. Hammond, S.M., MicroRNAs as oncogenes. Curr Opin Genet Dev, 2006. 16(1): p. 4-9.
161. Ambros, V., MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell, 2003. 113(6): p. 673-6.
162. van Rooij, E., et al., A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure. Proc Natl Acad Sci U S A, 2006. 103(48): p. 18255-60.
163. Latronico, M.V., D. Catalucci, and G. Condorelli, MicroRNA and cardiac pathologies. Physiol Genomics, 2008. 34(3): p. 239-42.
164. Care, A., et al., MicroRNA-133 controls cardiac hypertrophy. Nat Med, 2007. 13(5): p. 613-8.
165. van Rooij, E., et al., Control of stress-dependent cardiac growth and gene expression by a microRNA. Science, 2007. 316(5824): p. 575-9.
166. Yang, B., et al., The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2. Nat Med, 2007. 13(4): p. 486-91.
167. Zhao, Y., et al., Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell, 2007. 129(2): p. 303-17.
168. Zhao, Y., E. Samal, and D. Srivastava, Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature, 2005. 436(7048): p. 214-20.
140
169. Ikeda, S., et al., Altered microRNA expression in human heart disease. Physiol Genomics, 2007. 31(3): p. 367-73.
170. Tatsuguchi, M., et al., Expression of microRNAs is dynamically regulated during cardiomyocyte hypertrophy. J Mol Cell Cardiol, 2007. 42(6): p. 1137-41.
171. Shan, Z.X., et al., Upregulated expression of miR-1/miR-206 in a rat model of myocardial infarction. Biochem Biophys Res Commun, 2009. 381(4): p. 597-601.
172. Xu, C., et al., The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes. J Cell Sci, 2007. 120(Pt 17): p. 3045-52.
173. Xiao, J., et al., MicroRNA miR-133 represses HERG K+ channel expression contributing to QT prolongation in diabetic hearts. J Biol Chem, 2007. 282(17): p. 12363-7.
174. Thum, T., et al., MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature, 2008. 456(7224): p. 980-4.
175. Cheng, Y., et al., MicroRNAs are aberrantly expressed in hypertrophic heart: do they play a role in cardiac hypertrophy? Am J Pathol, 2007. 170(6): p. 1831-40.
176. Duisters, R.F., et al., miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remodeling. Circ Res, 2009. 104(2): p. 170-8, 6p following 178.
177. Lukiw, W.J. and A.I. Pogue, Induction of specific micro RNA (miRNA) species by ROS-generating metal sulfates in primary human brain cells. J Inorg Biochem, 2007. 101(9): p. 1265-9.
178. Shilo, S., et al., Evidence for the involvement of miRNA in redox regulated angiogenic response of human microvascular endothelial cells. Arterioscler Thromb Vasc Biol, 2008. 28(3): p. 471-7.
179. Prescott, L.F., Paracetamol: past, present, and future. Am J Ther, 2000. 7(2): p. 143-7.
180. Spooner, J.B. and J.G. Harvey, The history and usage of paracetamol. J Int Med Res, 1976. 4(4 Suppl): p. 1-6.
181. Anderson, B.J., Paracetamol (Acetaminophen): mechanisms of action. Paediatr Anaesth, 2008. 18(10): p. 915-21.
182. Botting, R.M., Mechanism of action of acetaminophen: is there a cyclooxygenase 3? Clin Infect Dis, 2000. 31 Suppl 5: p. S202-10.
183. Dahlin, D.C., et al., N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen. Proc Natl Acad Sci U S A, 1984. 81(5): p. 1327-31.
184. James, L.P., P.R. Mayeux, and J.A. Hinson, Acetaminophen-induced hepatotoxicity. Drug Metab Dispos, 2003. 31(12): p. 1499-506.
185. Bertolini, A., et al., Paracetamol: new vistas of an old drug. CNS Drug Rev, 2006. 12(3-4): p. 250-75.
186. Stirnimann, G., K. Kessebohm, and B. Lauterburg, Liver injury caused by drugs: an update. Swiss Med Wkly, 2010. 140: p. w13080.
141
187. Hinson, J.A., D.W. Roberts, and L.P. James, Mechanisms of acetaminophen-induced liver necrosis. Handb Exp Pharmacol, 2010(196): p. 369-405.
188. Tang, W., Drug metabolite profiling and elucidation of drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol, 2007. 3(3): p. 407-20.
189. Boutaud, O., et al., Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H(2) synthases. Proc Natl Acad Sci U S A, 2002. 99(10): p. 7130-5.
190. Brennan, M.L., et al., Prognostic value of myeloperoxidase in patients with chest pain. N Engl J Med, 2003. 349(17): p. 1595-604.
191. Chou, T.M. and P. Greenspan, Effect of acetaminophen on the myeloperoxidase-hydrogen peroxide-nitrite mediated oxidation of LDL. Biochim Biophys Acta, 2002. 1581(1-2): p. 57-63.
192. Maharaj, D.S., et al., Acetaminophen and aspirin inhibit superoxide anion generation and lipid peroxidation, and protect against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats. Neurochem Int, 2004. 44(5): p. 355-60.
193. Nakamoto, K., et al., Protective effect of acetaminophen against acute gastric mucosal lesions induced by ischemia-reperfusion in the rat. Pharmacology, 1997. 54(4): p. 203-10.
194. Cheung, P.Y., et al., Matrix metalloproteinase-2 contributes to ischemia-reperfusion injury in the heart. Circulation, 2000. 101(15): p. 1833-9.
195. Wang, W., G. Sawicki, and R. Schulz, Peroxynitrite-induced myocardial injury is mediated through matrix metalloproteinase-2. Cardiovasc Res, 2002. 53(1): p. 165-74.
196. Rork, T.H., et al., Acetaminophen attenuates peroxynitrite-activated matrix metalloproteinase-2-mediated troponin I cleavage in the isolated guinea pig myocardium. J Mol Cell Cardiol, 2006. 40(4): p. 553-61.
197. Merrill, G.F., et al., Acetaminophen and myocardial infarction in dogs. Am J Physiol Heart Circ Physiol, 2004. 287(5): p. H1913-20.
198. Merrill, G.F., et al., Antiarrhythmic properties of acetaminophen in the dog. Exp Biol Med (Maywood), 2007. 232(9): p. 1245-52.
199. Hadzimichalis, N.M., et al., Acetaminophen-mediated cardioprotection via inhibition of the mitochondrial permeability transition pore-induced apoptotic pathway. Am J Physiol Heart Circ Physiol, 2007. 293(6): p. H3348-55.
200. Jaques-Robinson, K.M., et al., Acetaminophen is cardioprotective against H2O2-induced injury in vivo. Exp Biol Med (Maywood), 2008. 233(10): p. 1315-22.
201. Lakatta, E.G. and S.J. Sollott, The "heartbreak" of older age. Mol Interv, 2002. 2(7): p. 431-46.
202. Lipman, R.D., et al., Pathologic characterization of brown Norway, brown Norway x Fischer 344, and Fischer 344 x brown Norway rats with relation to age. J Gerontol A Biol Sci Med Sci, 1996. 51(1): p. B54-9.
142
203. Kakarla, S.K., et al., Chronic acetaminophen attenuates age-associated increases in cardiac ROS and apoptosis in the Fischer Brown Norway rat. Basic Res Cardiol, 2010. 105(4): p. 535-44.
204. Hannan, R.D., et al., Cardiac hypertrophy: a matter of translation. Clin Exp Pharmacol Physiol, 2003. 30(8): p. 517-27.
205. Aoki, Y., et al., The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders. Hum Mutat, 2008. 29(8): p. 992-1006.
206. Ueyama, T., et al., Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy. J Mol Cell Cardiol, 2000. 32(6): p. 947-60.
207. Yamazaki, T., et al., Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes. J Biol Chem, 1993. 268(16): p. 12069-76.
208. Bogoyevitch, M.A., et al., Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem, 1994. 269(2): p. 1110-9.
209. Bueno, O.F. and J.D. Molkentin, Involvement of extracellular signal-regulated kinases 1/2 in cardiac hypertrophy and cell death. Circ Res, 2002. 91(9): p. 776-81.
210. Yu, W., et al., Insulin signaling: a possible pathogenesis of cardiac hypertrophy. Cardiovasc Ther, 2010. 28(2): p. 101-5.
211. Towbin, H., T. Staehelin, and J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A, 1979. 76(9): p. 4350-4.
212. Gupte, A.A., G.L. Bomhoff, and P.C. Geiger, Age-related differences in skeletal muscle insulin signaling: the role of stress kinases and heat shock proteins. J Appl Physiol, 2008. 105(3): p. 839-48.
213. Marzetti, E., et al., Age-related activation of mitochondrial caspase-independent apoptotic signaling in rat gastrocnemius muscle. Mech Ageing Dev, 2008. 129(9): p. 542-9.
214. Wheeler, M.T., S. Zarnegar, and E.M. McNally, Zeta-sarcoglycan, a novel component of the sarcoglycan complex, is reduced in muscular dystrophy. Hum Mol Genet, 2002. 11(18): p. 2147-54.
215. Inoki, K., et al., Signaling by target of rapamycin proteins in cell growth control. Microbiol Mol Biol Rev, 2005. 69(1): p. 79-100.
216. Gingras, A.C., et al., Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev, 2001. 15(21): p. 2852-64.
217. Ruvinsky, I., et al., Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev, 2005. 19(18): p. 2199-211.
143
218. Bogoyevitch, M.A., C.J. Marshall, and P.H. Sugden, Hypertrophic agonists stimulate the activities of the protein kinases c-Raf and A-Raf in cultured ventricular myocytes. J Biol Chem, 1995. 270(44): p. 26303-10.
219. Clerk, A., et al., Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts. J Biol Chem, 1994. 269(52): p. 32848-57.
220. Schluter, K.D., et al., Role of phosphatidylinositol 3-kinase activation in the hypertrophic growth of adult ventricular cardiomyocytes. Cardiovasc Res, 1998. 40(1): p. 174-81.
221. Shioi, T., et al., Akt/protein kinase B promotes organ growth in transgenic mice. Mol Cell Biol, 2002. 22(8): p. 2799-809.
222. Matsui, T., et al., Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J Biol Chem, 2002. 277(25): p. 22896-901.
223. Cantley, L.C., The phosphoinositide 3-kinase pathway. Science, 2002. 296(5573): p. 1655-7.
224. Antos, C.L., et al., Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A, 2002. 99(2): p. 907-12.
225. Oudit, G.Y. and J.M. Penninger, Cardiac regulation by phosphoinositide 3-kinases and PTEN. Cardiovasc Res, 2009. 82(2): p. 250-60.
226. Wang, X. and C.G. Proud, The mTOR pathway in the control of protein synthesis. Physiology (Bethesda), 2006. 21: p. 362-9.
227. Manning, B.D., et al., Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell, 2002. 10(1): p. 151-62.
228. Wang, L., I. Gout, and C.G. Proud, Cross-talk between the ERK and p70 S6 kinase (S6K) signaling pathways. MEK-dependent activation of S6K2 in cardiomyocytes. J Biol Chem, 2001. 276(35): p. 32670-7.
229. Wang, L. and C.G. Proud, Ras/Erk signaling is essential for activation of protein synthesis by Gq protein-coupled receptor agonists in adult cardiomyocytes. Circ Res, 2002. 91(9): p. 821-9.
230. Nojima, H., et al., The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J Biol Chem, 2003. 278(18): p. 15461-4.
231. Franklin, S.S., Cardiovascular risks related to increased diastolic, systolic and pulse pressure. An epidemiologist's point of view. Pathol Biol (Paris), 1999. 47(6): p. 594-603.
232. Lakatta, E., Aging Effects on the Vasculature in Health: Risk Factors for Cardiovascular Disease. Am J Geriatr Cardiol, 1994. 3(6): p. 11-17.
233. Lauer, T., et al., Age-dependent endothelial dysfunction is associated with failure to increase plasma nitrite in response to exercise. Basic Res Cardiol, 2008. 103(3): p. 291-7.
144
234. Laurent, S., Arterial wall hypertrophy and stiffness in essential hypertensive patients. Hypertension, 1995. 26(2): p. 355-62.
235. Hare, J.M., Oxidative stress and apoptosis in heart failure progression. Circ Res, 2001. 89(3): p. 198-200.
236. Wencker, D., et al., A mechanistic role for cardiac myocyte apoptosis in heart failure. J Clin Invest, 2003. 111(10): p. 1497-504.
237. Lee, H.C. and Y.H. Wei, Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging. Exp Biol Med (Maywood), 2007. 232(5): p. 592-606.
238. Wu, M., et al., Acetaminophen prevents aging-associated hyperglycemia in aged rats: effect of aging-associated hyperactivation of p38-MAPK and ERK1/2. Diabetes Metab Res Rev, 2009. 25(3): p. 279-86.
239. Zhao, H., et al., Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med, 2003. 34(11): p. 1359-68.
240. Carter, W.O., P.K. Narayanan, and J.P. Robinson, Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol, 1994. 55(2): p. 253-8.
241. Comporti, M., Lipid peroxidation and biogenic aldehydes: from the identification of 4-hydroxynonenal to further achievements in biopathology. Free Radic Res, 1998. 28(6): p. 623-35.
242. Los, M., et al., Switching Akt: from survival signaling to deadly response. Bioessays, 2009. 31(5): p. 492-5.
243. Rosner, M., et al., Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle. Hum Mol Genet, 2009. 18(17): p. 3298-310.
244. Dhingra, S., et al., p38 and ERK1/2 MAPKs mediate the interplay of TNF-alpha and IL-10 in regulating oxidative stress and cardiac myocyte apoptosis. Am J Physiol Heart Circ Physiol, 2007. 293(6): p. H3524-31.
245. Das, A., et al., ERK phosphorylation mediates sildenafil-induced myocardial protection against ischemia-reperfusion injury in mice. Am J Physiol Heart Circ Physiol, 2009. 296(5): p. H1236-43.
246. Casas-Terradellas, E., et al., ERK and p38 pathways regulate amino acid signalling. Biochim Biophys Acta, 2008. 1783(12): p. 2241-54.
247. Wagner, C., et al., Ischemic post-conditioning reduces infarct size of the in vivo rat heart: role of PI3-K, mTOR, GSK-3beta, and apoptosis. Mol Cell Biochem, 2010.
248. Ungvari, Z., et al., Role of oxidative-nitrosative stress and downstream pathways in various forms of cardiomyopathy and heart failure. Curr Vasc Pharmacol, 2005. 3(3): p. 221-9.
145
249. Dost, T., M.V. Cohen, and J.M. Downey, Redox signaling triggers protection during the reperfusion rather than the ischemic phase of preconditioning. Basic Res Cardiol, 2008. 103(4): p. 378-84.
250. Besse, S., et al., Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts. J Physiol Pharmacol, 2006. 57(4): p. 541-52.
251. Berger, J.S., et al., Aspirin for the primary prevention of cardiovascular events in women and men: a sex-specific meta-analysis of randomized controlled trials. Jama, 2006. 295(3): p. 306-13.
252. Ray, S.D., et al., Oxidative stress is the master operator of drug and chemically-induced programmed and unprogrammed cell death: Implications of natural antioxidants in vivo. Biofactors, 2004. 21(1-4): p. 223-32.
253. Boengler, K., R. Schulz, and G. Heusch, Loss of cardioprotection with ageing. Cardiovasc Res, 2009. 83(2): p. 247-61.
254. Griendling, K.K., D. Sorescu, and M. Ushio-Fukai, NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res, 2000. 86(5): p. 494-501.
255. Adler, A., et al., NAD(P)H oxidase-generated superoxide anion accounts for reduced control of myocardial O2 consumption by NO in old Fischer 344 rats. Am J Physiol Heart Circ Physiol, 2003. 285(3): p. H1015-22.
256. Pletjushkina, O.Y., et al., Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal. Biochemistry (Mosc), 2006. 71(1): p. 60-7.
257. Kwak, H.B., W. Song, and J.M. Lawler, Exercise training attenuates age-induced elevation in Bax/Bcl-2 ratio, apoptosis, and remodeling in the rat heart. Faseb J, 2006. 20(6): p. 791-3.
258. Fischer, P. and D. Hilfiker-Kleiner, Survival pathways in hypertrophy and heart failure: the gp130-STAT axis. Basic Res Cardiol, 2007. 102(5): p. 393-411.
259. Balasubramanian, S., et al., mTOR in growth and protection of hypertrophying myocardium. Cardiovasc Hematol Agents Med Chem, 2009. 7(1): p. 52-63.
260. Rosner, M., et al., Functional interaction of mTOR complexes in regulating mammalian cell size and cell cycle. Hum Mol Genet, 2009.
261. Qin, S. and P.B. Chock, Implication of phosphatidylinositol 3-kinase membrane recruitment in hydrogen peroxide-induced activation of PI3K and Akt. Biochemistry, 2003. 42(10): p. 2995-3003.
262. Konishi, H., et al., Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27. FEBS Lett, 1997. 410(2-3): p. 493-8.
263. Liu, L., et al., Heat shock protein 27 regulates oxidative stress-induced apoptosis in cardiomyocytes: mechanisms via reactive oxygen species generation and Akt activation. Chin Med J (Engl), 2007. 120(24): p. 2271-7.
264. Shimizu, N., et al., Activation of mitogen-activated protein kinases and activator protein-1 in myocardial infarction in rats. Cardiovasc Res, 1998. 38(1): p. 116-24.
146
265. Xu, Z., X. Ji, and P.G. Boysen, Exogenous nitric oxide generates ROS and induces cardioprotection: involvement of PKG, mitochondrial KATP channels, and ERK. Am J Physiol Heart Circ Physiol, 2004. 286(4): p. H1433-40.
266. Li, D.Y., et al., Role of ERK1/2 in the anti-apoptotic and cardioprotective effects of nitric oxide after myocardial ischemia and reperfusion. Apoptosis, 2006. 11(6): p. 923-30.
267. Huikuri, H.V., A. Castellanos, and R.J. Myerburg, Sudden death due to cardiac arrhythmias. N Engl J Med, 2001. 345(20): p. 1473-82.
268. Rice, K.M., et al., Fluprostenol-induced MAPK signaling is independent of aging in Fischer 344/NNiaHSd x Brown Norway/BiNia rat aorta. Ann Clin Lab Sci, 2010. 40(1): p. 26-31.
269. Ide, T., et al., Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium. Circ Res, 1999. 85(4): p. 357-63.
270. Saavedra, W.F., et al., Imbalance between xanthine oxidase and nitric oxide synthase signaling pathways underlies mechanoenergetic uncoupling in the failing heart. Circ Res, 2002. 90(3): p. 297-304.
271. Li, J.M., et al., Activation of NADPH oxidase during progression of cardiac hypertrophy to failure. Hypertension, 2002. 40(4): p. 477-84.
272. Landmesser, U., K.C. Wollert, and H. Drexler, Potential novel pharmacological therapies for myocardial remodelling. Cardiovasc Res, 2009. 81(3): p. 519-27.
273. Rice, K.M., et al., Effects of aging on pressure-induced MAPK activation in the rat aorta. Pflugers Arch, 2005. 450(3): p. 192-9.
274. Marasa, B.S., et al., MicroRNA profiling in human diploid fibroblasts uncovers miR-519 role in replicative senescence. Aging (Albany NY), 2010. 2(6): p. 333-43.
275. Livak, K.J. and T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001. 25(4): p. 402-8.
276. Sangaralingham, S.J., et al., The aging heart, myocardial fibrosis, and its relationship to circulating C-type natriuretic Peptide. Hypertension, 2011. 57(2): p. 201-7.
277. Sasano, C., et al., Internalization and dephosphorylation of connexin43 in hypertrophied right ventricles of rats with pulmonary hypertension. Circ J, 2007. 71(3): p. 382-9.
278. Melov, S., Animal models of oxidative stress, aging, and therapeutic antioxidant interventions. Int J Biochem Cell Biol, 2002. 34(11): p. 1395-400.
279. Danik, S.B., et al., Modulation of cardiac gap junction expression and arrhythmic susceptibility. Circ Res, 2004. 95(10): p. 1035-41.
280. Weber, K.T., C.G. Brilla, and J.S. Janicki, Myocardial fibrosis: functional significance and regulatory factors. Cardiovasc Res, 1993. 27(3): p. 341-8.
281. Sayed, D., et al., MicroRNAs play an essential role in the development of cardiac hypertrophy. Circ Res, 2007. 100(3): p. 416-24.
147
282. Lin, Y., et al., Involvement of MicroRNAs in hydrogen peroxide-mediated gene regulation and cellular injury response in vascular smooth muscle cells. J Biol Chem, 2009. 284(12): p. 7903-13.
283. Cheng, Y., et al., MicroRNA-21 protects against the H(2)O(2)-induced injury on cardiac myocytes via its target gene PDCD4. J Mol Cell Cardiol, 2009. 47(1): p. 5-14.
284. Xiao, J., et al., MicroRNA miR-133 represses HERG K+ channel expression contributing to QT prolongation in diabetic hearts. J Biol Chem, 2007. 282(17): p. 12363-7.
285. Liu, N., et al., microRNA-133a regulates cardiomyocyte proliferation and suppresses smooth muscle gene expression in the heart. Genes Dev, 2008. 22(23): p. 3242-54.
148
CURRICULUM VITAE
SUNIL K. KAKARLA
6633 Country club drive, Huntington, WV 25705
Phone: 919-656-6414. E-mail: [email protected]
OBJECTIVE
To work as an assistant professor in a renowned College of Veterinary Medicine and to
be placed in the elite group of researchers where I can explore the field of science to
quench my thirst for knowledge and grow along with the institute.
EDUCATION
July 2007-Present Ph. D., Biomedical Science (Doctoral candidate) at John C Edward
School of Medicine, Marshall University, Huntington, WV
Dissertation: “AGE-ASSOCIATED ALTERATIONS IN THE
F344XBN RAT HEART AND THE EFFICACY OF
CHRONIC ACETAMINOPHEN INGESTION AS A
THERAPEUTIC APPROACH”
Aug 2005-Jun 2007 MS in Biological Sciences (emphasis in Molecular Physiology) at
Marshall University, Huntington, WV
Thesis: “AGE-RELATED CHANGES IN CARDIAC
DYSTROPHIN GLYCOPROTEIN COMPLEX STRUCTURE
AND FUNCTION IN THE F344XBN RATS”
Nov 1994-Oct 1999 DVM at College of Veterinary Science, ANGRAU, Tirupati, A.P,
INDIA
LICENSURE NC state veterinary license
CERTIFICATIONS Completed Education Commission for Foreign Veterinary
Graduates (ECFVG) certification
PROFESSIONAL EXPERIENCE
RESEARCH EXPERIENCE
149
Aug 2005-Present Research Assistant, Laboratory of Molecular physiology,
Marshall University, Huntington, WV.
July 2002-July 2005 Veterinary Assistant Surgeon, Animal Husbandry
Department, INDIA.
Nov 1999-June 2002 Poultry Pathologist, Vijayanagaram Hatcheries Pvt Ltd,
VSP, A.P, INDIA.
PUBLICATIONS
PUBLISHED
Wang Y, Wu M, Al-Rousan R, Liu H, Fannin J, Paturi S, Arvapalli R, Katta A, Kakarla
SK, Rice K, Triest WE, Blough ER. Iron-induced cardiac damage: role of apoptosis and
deferasirox intervention. J Pharmacol Exp Ther. 2011 Jan;336(1):56-63
Katta A, Kundla S, Kakarla SK, Wu M, Fannin J, Paturi S, Liu H, Addagarla HS,
Blough ER. Impaired overload-induced hypertrophy is associated with diminished mTOR
signaling in insulin resistant skeletal muscle of the obese Zucker rat. Am J Physiol Regul
Integr Comp Physiol. 2010 Dec;299(6):R1666-75.
Wu M, Liu H, Fannin J, Katta A, Wang Y, Arvapalli RK, Paturi S, Kakarla SK, Rice
KM, Blough ER. Acetaminophen Improves Protein Translational Signaling in Aged
Skeletal Muscle. Rejuvenation Res. 2010 Oct;13(5):571-9
Kakarla SK, Fannin JC, Keshavarzian S, Katta A, Paturi S, Nalabotu SK, Wu M, Rice
KM, Manzoor K, Walker EM Jr, Blough ER; Chronic acetaminophen attenuates age-
associated increases in cardiac ROS and apoptosis in the Fischer Brown Norway Hybrid
Rat. Basic Res Cardiol. 2010 Jul; 105(4):535-44
Kolli MB, Day BS, Takatsuki H, Nalabotu SK, Rice KM, Kohama K, Gadde MK,
Kakarla SK, Katta A, Blough ER; Application of PAMAM dendrimers for use in
bionanomotor systems. Langmuir. 2010 May 4; 26(9):6079-82
Arvapalli RK, Paturi S, Laurino JP, Katta A, Kakarla SK, Gadde MK, Wu M, Rice KM,
Walker EM, Wehner P, Blough ER. Deferasirox decreases age-associated iron
accumulation in the aging F344XBN rat heart. Cardiovasc Toxicol. 2010 Jun; 10(2):108-
16
150
Paturi S, Gutta AK, Katta A, Kakarla SK, Arvapalli RK, Gadde MK, Nalabotu SK, Rice
KM, Wu M, Blough E; Effects of aging and gender on regulators of muscle adaptation in
the aging F344/BN rat model. Mech Ageing Dev. 2010 Mar; 131(3):202-9
Rice KM, Walker EM, Kakarla SK, Paturi S, Wu M, Narula S, Blough ER; Aging Does
Not Alters Fluprostenol-Induced MAPK Signaling in the Fischer 344/NNiaHSd X Brown
Norway /BiNia Rat Aorta. Ann Clin Lab Sci. 2010 Winter; 40 (1):26-31
Kakarla SK, Rice KM, Katta A, Paturi S, Wu M, Kolli M, Keshavarzian S, Manzoor K,
Wehner PS, Blough ER; Possible molecular mechanisms underlying age-related
cardiomyocyte apoptosis in the F344XBN Rat Heart. J Gerontol A Biol Sci Med Sci.
2010 Feb; 65(2):147-55.
Kakarla SK, Rice KM, Mupparaju SP, Paturi S, Katta A, Wu M, Harris RT, Blough ER;
Shear stress activates Akt during vascular smooth muscle cell reorientation. Biotechnol
Appl Biochem. 2010 Feb 18; 55(2):85-90.
Al-Rousan RM, Paturi S, Laurino JP, Kakarla SK, Gutta AK, Walker EM, Blough ER.
2009. Deferasirox Removes Cardiac Iron and Attenuates Oxidative Stress in the Iron-
Overloaded Gerbil. Am J Hematol. 2009 Sep; 84(9):565-70.
Paturi S, Gutta AK, Kakarla SK, Katta A, Arnold EC, Wu M, Rice KM, Blough ER;
Impaired overload-induced hypertrophy in obese zucker rat slow-twitch skeletal muscle.
J Appl Physiol. 2010 Jan; 108(1):7-13. Epub 2009 Sep 24.
Wu M, Katta A, Gadde MK, Liu H, Kakarla SK, Fannin J, Paturi S, Arvapalli RK, Rice
KM, Wang Y, Blough ER; Aging-associated dysfunction of Akt / protein kinase B: S-
nitrosylation and acetaminophen intervention. PLoS One. 2009 Jul 29; 4(7):e6430.
Wu M, Desai DH, Kakarla SK, Katta A, Paturi S, Gutta AK, Rice KM, Walker EM Jr,
Blough ER. Acetaminophen prevents aging-associated hyperglycemia in aged rats: effect
of aging-associated hyperactivation of p38-MAPK and ERK1/2. Diabetes Metab Res
Rev. 2009 Mar; 25(3):279-86.
Katta A, Kakarla SK, Wu M, Meduru S, Desai DH, Rice KM, Blough ER. Lean and
obese Zucker rats exhibit different patterns of p70s6 kinase regulation in the tibialis
anterior muscle in response to high-force muscle contraction. Muscle Nerve. 2009 Apr;
39(4):503-11.
151
Kakarla SK, Katta A, Wu M, Paturi S, Gadde MK, Arvapalli R, Kolli M, Rice KM,
Blough ER; Altered regulation of contraction-induced Akt / mTOR / p70S6k pathway
signaling in skeletal muscle of Syndrome-X Obese Zucker Rat Model. Exp Diabetes Res.
2009; 2009:384683.
Katta A, Preston DL, Kakarla SK, Asano S, Meduru S, Mupparaju SP, Yokochi E, Rice
KM, Desai DH, Blough ER. Diabetes alters contraction-induced mitogen activated
protein kinase activation in the rat soleus and plantaris. Exp Diabetes Res. 2008;
2008:738101.
Asano S, Rice KM, Kakarla SK, Katta A, Desai DH, Walker EM, Wehner P, Blough
ER. Aging influences multiple indices of oxidative stress in the heart of the Fischer
344/NNia x Brown Norway/BiNia rat. Redox Rep. 2007; 12(4):167-80.
Rice KM, Desai DH, Kakarla SK, Katta A, Preston DL, Wehner P, Blough ER. Diabetes
alters vascular mechanotransduction: pressure-induced regulation of mitogen activated
protein kinases in the rat inferior vena cava. Cardiovasc Diabetol. 2006 Sep 8; 5:18.
MANUSCRIPTS IN REVIEW
Kakarla SK, Thulluri S, Manne N.D.P.K, Rice KM, Blough ER; Anti-arrhythmic effect
of chronic acetaminophen treatment in the aging F344XBN rat involves diminished
myocardial fibrosis and altered miRNAs regulation.
Kakarla SK, Sudarsanam K, Kolli MB, Katta A, Nalabotu SK, Rice KM, Blough ER;
Mechanisms of age-related cardiac hypertrophy in the F344XBN rat model.
Anjaiah K, kakarla SK, Wu M, Nalabotu SK, Rice KM, Blough ER; Attenuation in
overload-induced skeletal muscle hypertrophy in obese insulin- resistant Zucker rat is
associated with hyperphosphorylation of AMP-activated protein kinase and dsRNA-
dependent protein kinase (PKR).
Nalabotu S, Takatsuki H, Kolli MB, Frost L, Yoshiyama S, Gadde M, Kakarla SK,
Kohama K, and Blough ER; Control of myosin enzyme activity by the reversible
alteration of protein structure.
ABSTRACTS
Presented several abstracts and posters at scientific meetings.
152
CLINICAL EXPERIENCE
Sep, 2010-Present Volunteer at Help for Animals Clinic, Huntington, WV
Oct, 2009-Mar, 2010 Volunteer at Proctorville Animal Clinic, Proctorville, OH
Oct, 2009- Jan, 2010 Volunteer at Equine Medical Center, Chesapeake, OH
July, 2003-Jun, 2005 Lead Veterinarian at Govt. Small animal clinic, Vizag, A.P,
India
July, 2002-Jun, 2003 Shelter Veterinarian, SPCA, Vizag, A.P, India
Jan, 2000-Jun, 2002 Veterinary Assistant Surgeon, Veterinary Polyclinic, A.P,
INDIA.
July, 1999-Dec, 1999 Intern, Veterinary Polyclinic, Hyderabad, A.P, INDIA.
Duties performed as a Veterinarian
Responsible for primary care including vaccination, prevention, diagnosis
and treatment of sick animals, and advising owners about nutrition and
management practices in an urban hospital setting which routinely dealt
with a mix of small (70%) and large (30%) animals.
Performed elective surgeries like spay, neuter, declawing, tail docking etc.
Performed reproductive soundness exams in dairy cattle, artificial
insemination, pregnancy diagnosis, handled dystocia cases and caesarian
section in needy situations.
Conducted farmer awareness camps about deworming, vaccination and
better management practices for livestock.
Trained technicians about animal handling, workplace safety, work
protocols, equipment handling and client interaction
Expertise:
Can perform dog spay in 15-20 minutes
Can perform dog neuter in 7-8 minutes
Can perform cat spay in 7-8 minutes
Can perform cat neuter in 5 minutes
Experienced in handling stainless steel suture material
Duties performed as volunteer in USA
Assisted veterinarians handle small animal medicine cases, learnt to
communicate with clients and prescribe appropriate medications.
153
Assisted with physical exams, obtaining and interpreting radiographs and
performing ultrasound exams.
Learned to analyze laboratory test results, interpret radiographic and
Ultrasound findings. Ability to operate both X-ray and Ultrasound
machines. Proficient in handling IDEXX and Vet Scan Laboratory
Systems.
Performed Urine and Fecal analysis.
Performed several spay and neuters, habituated to aseptic precautions,
anesthesia delivery (Induction to Recovery), vaccination and deworming
protocols.
Gained knowledge of record keeping and training of technicians.
HONORS AND AWARDS
Best veterinarian of the district (2002), Visakhapatnam, A.P, India
PROFESSIONAL MEMBERSHIPS
Member of AVMA
Member of Indian Veterinary Council
Eligibility to practice veterinary medicine in India.
Member of Andhra Pradesh Veterinary Association
License to practice veterinary medicine in the state of Andhra Pradesh, India.
RESEARCH SKILLS
Immunohistochemistry
Flourescent and Confocal Microscopy
Different staining procedures
Sample preparation from Laboratory animals
Protein Quantitation using Bradford assay
SDS gel electrophoresis
Immunoprecipitation and Westerns
Quantitation using Densitometry
DNA, RNA and miRNA isolation
DNA and Protein gel electrophoresis
PCR, RT-PCR
Cell culture
154
Small animal surgery
Handling of Laboratory animals
Cardiac Ischemia-Reperfusion Experiments in Rats and Mice
ELISA
COMPUTER SKILLS
Excellent experience with MS Office. (MS Word, Excel, PowerPoint,
Outlook and Access)
Excellent experience with statistical software – SigmaStat
Excellent experience with imaging software – Alpha Ease, Image J
Excellent experience with Adobe software (Acrobat, Photoshop), Endnote.
REFERENCES
Dr. Eric R. Blough, Ph.D.,
Assosciate Professor
Department of Biology
Director, Laboratory of Molecular physiology
Marshall University, Huntington, WV
E-mail: [email protected],
Phone: (304) 696-2708
(Master’s and PhD adviser)
Dr. Nalini Santanam, Ph.D.,
Professor and Cluster Coordinator
Dept of Physiology, Pharmacology and Toxicology
Joan C Edward School of Medicine
Marshall University, Huntington, WV
Phone: (304) 696-7321
E-mail: [email protected],
Dr. Todd L. Green, Ph.D.,
Associate Professor and
Director of Graduate studies, Biomedical Sciences Graduate programme
Dept of Pharmacology, Physiology and Toxicology
Phone: (304) 696-3531 Fax: (304) 696-7272
E-Mail: [email protected]