Deferasirox decreases age-associated iron accumulation in the

101 Pages·2013·4.26 MB·English

by  Ravi K. Arvapalli

Marshall University
Marshall Digital Scholar
Theses, Dissertations and Capstones
1-1-2009
Deferasirox decreases age-associated iron
accumulation in the aging F344BN rat heart
Ravi K. Arvapalli
Marshall University, [email protected]
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Arvapalli, Ravi K., "Deferasirox decreases age-associated iron accumulation in the aging F344BN rat heart" (2009).Theses,
Dissertations and Capstones.Paper 445.
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TITLE

Deferasirox decreases age-associated iron accumulation in
the aging F344BN rat heart

By
Ravi Kumar Arvapalli
A thesis submitted to the
Graduate faculty of the Department of Biology
at
Marshall University
In partial fulfillment of the requirements for the degree
of
Master of Science

Committee:

_____________________________ Dr. Eric R Blough, Thesis adviser

_____________________________ Dr. David Mallory, Committee member

_____________________________ Dr. Bin Wang, Committee member

_____________________________ Dr. Miaozong Wu, Committee member

Date: 07-06-2009 Summer 2009
Marshall University
Huntington, West Virginia
ABSTRACT

It is thought that aging in rats and humans is associated with increases in iron
accumulation and these increases in iron may be associated with increased cellular
apoptosis. Here we examine the relationship between cardiac iron levels and
cardiomyocyte apoptosis in aged F344BN rats which were treated with an oral iron
chelator (Deferasirox; 100mg/kg body. weight/day) for 6 months. Compared with 6-
month controls, the levels of cardiac iron, cardiac apoptosis, FLC and DMT-1 were
higher in 33-month hearts. Deferasirox treatment for six months decreased cardiac iron
and this was associated with decreases in the number of apoptotic cardiac myocytes.
Age-associated increases in cardiac apoptosis were coupled with alterations in the
amount of Bcl-2 and Bax. Deferasirox treatment increased Bcl-2 expression and
decreased Bad and activated caspase-12 expression. Taken together, these data
suggest deferasirox may be effective in diminishing age-associated iron accumulation
and cardiac apoptosis in the aging F344BN rat model.
1

ACKNOWLEDGEMENTS

I deeply express my gratitude to Dr. Eric Blough, my advisor, mentor and
committee chair for warmly welcoming me into his research group at Marshall
University. I would not be where I am today, had it not been for his kindness, generosity,
support and assistance. Thank you Eric for providing me with an ideal work
environment; for setting high standards in my professional and personal education; for
your never ending support, zeal, generosity and accessibility; for inspiring me,
supporting me and always having faith in me. Working under your direction has been an
honor and privilege and I am very much grateful for that. I would like to thank Dr. David
Mallory, Dr. Bin Wang and Dr. Miaozong Wu my committee members for their precious
remarks and suggestions on my thesis. I would like to thank all my friends and
colleagues in our research group for the superb discussions about research and
science; for creating a warm and inspiring atmosphere in the laboratory and for being so
helpful at all times. Working in such a research group was always fun. Thanks to Kevin,
Sunil, Anjaiah, Anil, Satya, Madhukar, Murali, Hari and Siva. Also I would like to convey
my sincere thanks to my parents, family members and friends back home in India for
their encouragement, love and never ending support.
2

ABBREVIATIONS

ALB Albumin
ALP Alkaline phosphatase
ALT Alanine aminotransferase
AMY Amylase
ANOVA One-way analysis of variance on ranks
AST Aspartate aminotransferase
BUN Blood urea nitrogen
BSA Bovine serum albumin
CRE Creatinine
CVD Cardiovascular disease
DH O Distilled Water
2
DMT-1 Divalent metal Transporter-1
DFO Deferoxamine
ECL Enhanced chemiluminiscence
ECM Extracellular Matrix
EDTA Ethylene diamine tetra acetic Acid
F344BN Fischer 344/NNiaHSd X Brown Norway/BiNia
GLOB Globulin
ICP-AES Inductively coupled plasma-atomic emission
spectrometry
IOD Integrated Optical Densities
IRP-1 Iron regulatory protein-1
3

KRB Krebs-ringers buffer solution
NIA National Institute of Aging
PBS Phosphate buffered saline
PBST Phosphate buffered saline with 0.5% tween-20
PHOS Phosphorous
ROS Reactive oxygen species
SEM Standard Error of Mean
SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis
TBS Tris buffered saline
TBST Tris buffered saline with 0.5% tween-20
TBIL Total bilirubin
TP Total protein
TfR-1 Transferrin receptor-1
TUNEL Terminal deoxynucleotidyl transferase dUTP nick end
labeling
WW Wet weight
4

LIST OF TABLES

Table 1 ......................................................................................................................................57
Table 2 ......................................................................................................................................58
Table 3 ......................................................................................................................................59
5

LIST OF FIGURES

Figure 1. Deferasirox treatment prevents age-associated increases in cardiac iron ..................62
Figure 2. Aged hearts exhibit increases in the amount of ferritin light chain and divalent metal
transporter-1 .............................................................................................................................63
Figure 3. Deferasirox treatment attenuates age-associated increases in cardiac apoptosis ......64
Figure 4. Bax / Bcl2 content is increased with age ....................................................................65
Figure 5. Bad protein levels are higher in aged rats and decreased by deferasirox treatment ...66
Figure 6. Deferasirox treatment decreases the amount of activated caspase-12 .......................67
6

TABLE OF CONTENTS

TITLE ........................................................................................................................................................... iii
ABSTRACT ................................................................................................................................................ iv
ACKNOWLEDGEMENTS ......................................................................................................................... 1
ABBREVIATIONS ...................................................................................................................................... 2
List of Tables ............................................................................................................................................... 4
List of figures ............................................................................................................................................... 5
CHAPTER 1 ................................................................................................................................................ 8
Introduction .............................................................................................................................................. 8
Purpose of the Study ........................................................................................................................... 11
Specific Aims and Hypothesis ............................................................................................................ 12
CHAPTER 2 .............................................................................................................................................. 13
REVIEW OF LITERATURE ................................................................................................................ 13
Effects of aging on cardiovascular structure and function ...................................................13
Reactive oxygen species ...................................................................................................14
Apoptosis ...........................................................................................................................15
Iron ....................................................................................................................................16
Age-associated iron accumulation......................................................................................17
Iron Chelation ....................................................................................................................18
Deferasirox ........................................................................................................................19
CHAPTER 3 .............................................................................................................................................. 22
Abstract .................................................................................................................................................. 23
Introduction ............................................................................................................................................ 24
Materials and Methods ........................................................................................................................ 26
Animals ..............................................................................................................................26
Materials ............................................................................................................................26
Iron chelation .....................................................................................................................27
Laboratory evaluation of iron-induced cardiac, liver, and renal damage .............................27
Heart collection ..................................................................................................................28
Determination of organ metal levels ...................................................................................28
TUNEL staining ..................................................................................................................29
7

Immunoblot analysis ..........................................................................................................30
Data analysis .....................................................................................................................31
Results ................................................................................................................................................... 33
Deferasirox decreases age-associated increases in cardiac iron .......................................33
Aging increases the expression of iron regulatory proteins ................................................33
Age-associated changes in indices of liver and kidney function .........................................34
Deferasirox treatment decreases indices of age-associated cardiomyocyte apoptosis .......34
Discussion ............................................................................................................................................. 35
Acknowledgements .............................................................................................................................. 41
CHAPTER 4 .............................................................................................................................................. 42
Conclusions ........................................................................................................................................... 42
Future Directions .................................................................................................................................. 43
REFERENCES ......................................................................................................................................... 44
APPENDICES ........................................................................................................................................... 51
Appendix A ............................................................................................................................................ 56
AppendixB ............................................................................................................................................. 68