Pharmacological Activation of Pro-survival
Pathways as a Strategy for Improving Donor
Heart Preservation
Jair Chau Kwan
Transplant Programme, Victor Chang Cardiac Research Institute
Department of Clinical Pharmacology & Toxicology and
Heart Lung Transplant Unit, St. Vincent’s Hospital Sydney
And
Faculty of Medicine, The University of New South Wales,
Sydney, Australia
This thesis is submitted in fulfilment of the requirements for the
degree of Doctor of Philosophy
2009 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet
Surname or Family name: Kwan
First name: Jair Chau Other name/s: N.A.
Abbreviation for degree as given in the University calendar: PhD
School: Clinical School of Medicine, St. Vincent’s Hospital Faculty: Medicine
Title: Pharmacological activation of pro-survival pathways as a strategy for improving donor heart preservation
Abstract 350 words maximum: (PLEASE TYPE)
Despite the development and use of specialised cardiac preservation solutions, the quality of the donor heart may still be compromised by its obligatory exposure to periods of ischaemia (both cold and warm) followed by reperfusion upon reintroduction of the recipient circulation. This is reflected in Transplant Registry data showing increased primary allograft failure as a function of increasing ischaemic time.
The research described in this thesis is designed to further the understanding of the mechanisms by which the donor heart may be adapted to these prolonged periods of ischaemia and reperfusion by the activation of endogenous pro-survival signalling pathways by the addition of pharmacological agents to Celsior, a clinical preservation solution.
Studies were conducted in an isolated working rat heart model of donor heart preservation. The first study investigated the cardioprotective effects of a novel inhibitor of poly(ADP-ribose) polymerase 1, INO-1153. Maximum protective effect (after a 6 hour storage period) was observed when the PARP inhibitor was administered prior to cardiac arrest and storage and when the agent was added to the Celsior cardioplegic / storage solution. This protective affect was associated with activation of the Akt signalling pathway and could be prevented by inhibition of Akt phosphorylation and activation. The second study examined functional protection and pro-survival signalling pathway activation in hearts arrested and stored for 6 hours in Celsior supplemented with glyceryl trinitrate (an exogenous source of nitric oxide) and Cariporide (an inhibitor of sodium hydrogen exchange). Here, cardiac protection was accompanied by activation of the ERK 1/2 pro-survival pathway as well as a decrease in apoptosis. The third study examined the cardioprotective effect of supplementation of Celsior with all three agents after an extended (10 hour) period of hypothermic storage. Significant recovery of function was only observed in the triply supplemented hearts, being accompanied by activation of both the Akt and ERK pathways.
These studies demonstrate for the first time the feasibility of recruitment of endogenous pro-survival pathways as an approach to increasing the post-storage function of the donor heart. Importantly, for the logistics of clinical transplantation, these pathways can be recruited by addition of appropriate pharmacological agents to the arresting and storage solution.
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(such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only).
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ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’
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- ii - ACKNOWLEDGEMENTS
Firstly, my sincere thanks to Dr. Mark Hicks for the guidance, advice, continual inspiration and excellence on the path to research.
With Dr. Hicks as my ever inspiring supervisor, mentor and friend, I am humbled by his outstanding knowledge, infectious enthusiasm and academic ability which are formidable and yet he carries them with ease and charm. I thank him for his selfless and unrelenting support to share his wisdom as well as his tireless and meticulous efforts on the publications, published abstracts, seminar talks and valuable comments on the draft versions of this thesis arising from this body of work.
My sincere thanks to my co-supervisor, Professor Peter Macdonald, for welcoming me into the laboratory, introducing me to the clinical arena of cardiology and giving me the opportunity to work on this critical area of cardiac transplantation. His constant encouragements, discussions and graciousness to share his wealth of knowledge have enabled me to benefit immensely in the production of this thesis. I am very grateful for his guidance and contributions towards my findings established in this body of work.
My special thanks also to Dr. Ling Gao. Throughout my research undertaken for this Ph.D. thesis her tireless help and guidance has been a key determinant to my success in mastering the animal model and has
- iii - generated many insights into further understanding and improvement in organ preservation and transplantation.
My special thanks to fellow colleagues Dr. Alfred Hing, Dr. Alasdair
Watson, Dr. Andrew Jabbour and Jireh Tsun all of whom I have enjoyed stimulated discussions and are fun to work with.
I would like to acknowledge and thank Professor Jacob George, Professor
Geoffrey Farrell, and Dr. Roslyn London for their invaluable guidance, support and sharing the many aspects of clinical gastroenterology and hepatology at Westmead Hospital in Sydney which provided the catalyst for me to undertake a path in medical research.
My thanks and gratitude to the Victor Chang Cardiac Research Institute,
National Heart Foundation, Gastroenterology Society of Australia for their funding and support of this project.
To my father Cheng Fai, mother Set Lan, and sisters Cailyn and Cai Nan, to whom this work is dedicated. My deepest thanks and appreciation for their endless love, support, encouragements and sacrifices they have made over these years. Finally, to my wife May Ling. Her endless love, care, encouragement, warmth, support and believing in me is what I cherish everyday. I especially dedicate the scientific endeavour in this thesis to her.
- iv - ABSTRACT
Despite the development and use of specialised cardiac preservation solutions, the quality of the donor heart may still be compromised by its obligatory exposure to periods of ischaemia (both cold and warm) followed by reperfusion upon re-introduction of the recipient circulation. The research described in this thesis is designed to further the understanding of the mechanisms by which the donor heart may be adapted to these prolonged periods of ischaemia and reperfusion by the activation of endogenous pro-survival signalling pathways through addition of pharmacological agents to Celsior, a clinical preservation solution.
Studies were conducted in an isolated working rat heart model of donor heart preservation. Hearts were rapidly excised and arrested and mounted on a perfusion circuit then stabilised in working mode. Baseline indices of cardiac function - aortic flow (AF), coronary flow (CF), cardiac output (CO) and heart rate (HR), were measured immediately before arrest and hypothermic storage for 6 or 10 hours. After storage, hearts were remounted on the perfusion apparatus, stabilised in working mode for 30 minutes and the above indices of cardiac function were remeasured.
Recovery of cardiac function was expressed as a percentage of the pre- storage baseline function. Hearts were then sampled and stored for western blotting and immunohistochemistry.
- v - The first study investigated the cardioprotective effects of a novel inhibitor of poly(ADP-ribose) polymerase 1, INO-1153. Exposure of hearts to INO-
1153 either before or at cardioplegia and storage or at reperfusion improved all measured indices of post-storage cardiac function after 6 hours of hypothermic storage compared to hearts stored in the absence of
INO-1153. When INO-1153 was present in the preservation solution, post- storage recovery of CF was 79% vs 40%; AF was 43% vs 15%; CO was
55% vs 23% and HR was 100% vs 50% of pre-storage baseline levels compared with the recovery of control hearts arrested and stored in unsupplemented celsior storage solution (P<0.05, treated vs unsupplemented control). This functional recovery was associated with activation of the Akt signalling pathway (a 4.3 fold increase in Akt phosphorylation) and a 2.4 fold increase in Erk1/2 phosphorylation (both
P<0.05 vs control). Functional recovery and increases in Akt and Erk phosphorylation could be prevented by pre-treatment of hearts with wortmannin, an inhibitor of the Akt pathway, before exposure to INO-1153.
The second study examined post-storage recovery of cardiac function and pro-survival signalling pathway activation in hearts arrested and stored for 6 hours in Celsior supplemented with glyceryl trinitrate (an exogenous source of nitric oxide) and Cariporide (an inhibitor of sodium hydrogen exchange). Hearts arrested and stored in celsior supplemented with GTN and cariporide for 6 hours recovered 80% vs 30% of baseline
CF, 58% vs 16% of AF, 70% vs 20% CO and 95% vs 47% of HR
- vi - compared to hearts arrested and stored in unsupplemented celsior
(P<0.05). Functional recovery was accompanied by a 7 fold increase in
Erk1/2 phosphorylation (P<0.001 vs control); a 2 fold increase in Bcl-2 phosphorylation (P<0.001 vs control); no increase in Akt phosphorylation and a 3 fold decrease in pro-apoptotic cleaved caspase 3 (P<0.001 vs control) compared to hearts arrested and stored in unsupplemented celsior. Pre-treatment of hearts with PD98059, an inhibitor of Erk1/2 pathway, produced a non significant decrease in all indices of cardiac function.
The third study examined the cardioprotective effect of supplementation of
Celsior with all three agents after an extended (10 hour) period of hypothermic storage. Hearts exposed to triply supplemented Celsior during arrest and storage recovered 67% vs 15% of baseline CF (P<0.01 vs any single supplement), 42%vs 1% of AF (P<0.02 vs any single supplement), 49% vs 4%of CO (P<0.05 vs any single supplement) and
86% vs 17% of HR (P<0.02 vs any single supplement). Functional recovery in the triple supplemented group was associated with a 6.5 fold increase in Erk1/2 phosphorylation (P<0.01 vs control), a 6 fold increase in
Akt phosphorylation (P<0.01 vs control), a 5.3 fold increase in phosphorylation of downstream target GSK3β (P<0.01 vs control) and a
5.4 fold increase in phosphorylation of the cytoskeletal element, ERM
(P<0.01 vs control).
- vii - In summary, these studies demonstrate for the first time the feasibility of recruitment of endogenous pro-survival pathways as an approach to increasing the post-storage function of the donor heart. Importantly, for the logistics of clinical transplantation, these pathways can be recruited by addition of appropriate pharmacological agents to the arresting and storage solution.
- viii - PUBLICATIONS ARISING FROM THIS THESIS
Published manuscripts
Kwan JC, Gao L, Macdonald PS, Hicks M. Protective effect of glyceryl trinitrate and cariporide in a model of donor heart preservation: Role of extracellular signal-regulated kinase activation. (Submitted)
Kwan JC, Gao L, Macdonald PS, Hicks M. Improved Long term Heart
Preservation by Combined Activation of Erk1/2 and Akt kinases.
(Submitted)
Gao L, Kwan JC, Macdonald PS, Yang L, Preiss T, Hicks M. Improved poststorage cardiac function by poly (ADP-ribose) polymerase inhibition: role of phosphatidylinositol 3-kinase Akt pathway. Transplantation 2007;
84: 380-6.
Published abstracts
Kwan JC, Gao, L, Madconald P, Hicks M. Protective effect of glyceryl trinitrate and cariporide in a model of donor heart preservation: Activation of Erk1/2 pathway. Transplantation 2008; 86; 2S, 1150
Gao L, Kwan JC, Jabbour A, Macdonald P, Hicks M. Polypharmaceutical survival kinase activation provides significant cardiac protection after prolonged hypothermic storage. Transplantation 2008; 86; 2S, 496
- ix -
Kwan JC, Gao L, Macdonald PS, Hicks M. Decrease in apoptosis associated with cardioprotective strategies in a model of donor heart preservation. J Mol Cell Cardiol 2007; 42; No. 6, S81
Kwan JC, Gao L, Macdonald PS, Hicks M. Cardioprotective effect of poly(ADP-ribose) polymerase inhibition: Role of PI3K/Akt.
J Mol Cell Cardiol 2007; 42; No. 6, S206-S207
- x - TABLE OF CONTENTS PAGE
Originality statement ii
Acknowledgements iii
Abstract v
Publications arising from this thesis vii
Table of contents ix
List of tables xvi
List of figures xvii
List of abbreviations xxii
CHAPTER 1: Introduction
1.1 Overview 2
1.2 History of heart transplantation 4
1.2.1 Pre-clinical research era 4
1.2.2 Early clinical experience era 8
1.2.3 The modern era (post 1984) 10
1.3 Ischaemia reperfusion injury 12
1.3.1 Background and definitions 12
1.3.2 Metabolic changes during ischaemia 13
1.3.2.1 Energy metabolism in non ischaemic heart 13
1.3.2.2 Energy metabolism in ischaemic heart 15
- xi - PAGE
1.3.3 Changes to intracellular ion and water 17
homeostasis during ischaemia
1.3.4 Reperfusion injury 20
1.3.4.1 Changes to intracellular ion and water 21
homeostasis during reperfusion
1.3.4.2 Free radical formation 22
1.3.4.3 Formation of superoxide 23
1.3.4.4 Recent research into the role of free 27
radicals and oxidants – Protective effects
1.3.4.5 Role of calcium and mitochondria in cell 28
injury and death
1.3.4.6 Reperfusion associated changes to the 35
endothelium and microcirculation
1.4 Preservation of the donor heart 38
1.4.1 The need for donor heart preservation 38
1.4.2 Major elements of heart preservation 39
1.4.2.1 Immediate mechanical arrest of the heart 39
1.4.2.2 Hypothermia 40
1.4.2.3 Minimisation of ischaemia reperfusion injury 42
1.4.3 Development of current cardioplegic / storage 47
solutions
1.4.4 Need to improve and extend cardiac preservation 59
- xii - PAGE
1.5 Novel pharmacological and physiological approaches 61
to donor heart protection
1.3.4 Nitric oxide donor 61
1.3.5 Inhibition of sodium hydrogen exchange 66
1.3.6 Inhibition of poly(ADP-ribose) polymerase 70
1.3.7 Ischaemic pre and post conditioning 74
1.3.8 Pro-survival signalling pathways as a common 82
mechanism to ischaemic pre and post conditioning
1.5.5.1 Proximal elements 82
1.5.5.2 Downstream targets of RISK pathway 87
1.5.5.3 Endogenous “Anti-RISK” elements 90
1.5.5.4 Pharmacological modulation of pro-survival 92
pathways
1.6 Experimental model chosen for current study 97
1.7 Thesis hypotheses and aims 100
CHAPTER 2: General Materials and Methods
2.1 Materials 104
2.2 Ethical conduct of experimental studies 108
2.3 Animals 109
- xiii - PAGE
2.4 Surgical procedures 109
2.4.1 Surgical instruments 109
2.4.2 Pre-medication and anaesthesia 110
2.4.3 Surgical removal of heart 111
2.4.4 Attaching the heart to perfusion circuit 113
2.4.5 Cardiac functional parameters measured in a 117
typical donor heart preservation study
2.5 Cardioplegia and storage 119
2.6 Collection and preparation of heart for molecular studies 121
and histology
2.7 Preparation of tissue lysates from cardiac tissue 121
2.8 SDS-PAGE and immuno (western) blotting conditions 122
2.9 Processing of tissue for immunohistochemical and 125
immunofluorescence analysis
2.10 Statistical analysis 127
CHAPTER 3: Improved Post-storage Cardiac Function by Poly(ADP- ribose) Polymerase Inhibition - Role of Phosphatidylinositol 3-kinase
Akt Pathway
3.1 Introduction 129
3.2 Materials and methods 132
3.3 Results 137
- xiv - PAGE
3.3.1 Effect of pharmacological agents on baseline 137
measurements of cardiac function
3.3.2 Effect of INO-1153 treatment during heart 138
preservation improves post-storage cardiac function
3.3.3 Effect of INO-1153 on cardiac Poly(ADP-ribose) 142
deposition
3.3.4 Effect of Wortmannin pretreatment against improved 144
post-storage cardiac function by INO-1153 treatment
3.3.5 Effects of Wortmannin and INO-1153 treatment on 147
Akt activation after post-storage
3.3.6 Effects of Wortmannin and INO-1153 treatment on 149
Erk1/2 activation after post-storage
3.3.7 Effects of Wortmannin and INO-1153 treatment on 151
phospholamban phosphorylation after post-storage
3.3.8 Effects of Wortmannin and INO-1153 treatment on 153
eNOS activation after post-storage
3.4 Discussion 155
CHAPTER 4: Improved Post-storage Cardiac Function by the
Presence of Glyceryl Trinitrate and Cariporide in Celsior Solution at
Arrest and during Hypothermic Storage – Role of Erk1/2 Pathway
4.1 Introduction 167
4.2 Materials and methods 170
- xv - PAGE
4.3 Results 176
4.3.1 Prestorage baseline measurements of cardiac 176
function
4.3.2 Effect of cardioplegia/storage solution composition 177
on post-reperfusion recovery of cardiac function
4.3.3 Effect of cardioplegia/storage solution composition 180
on post-reperfusion phosphorylation status of Erk1/2
and Akt
4.3.4 Effect of cardioplegia/storage solution composition 184
on post-reperfusion phosphorylation status of Bcl-2
4.3.5 Effect of cardioplegia/storage solution composition 186
on apoptotic marker post-reperfusion
4.3.6 Effect of MEK / ERK inhibitor, PD98059 on 190
post-storage recovery of hearts arrested and stored
in Celsior supplemented with GTN and Cariporide
4.3.7 Effect of MEK / ERK inhibitor, PD98059 on 192
post-storage phosphorylation status of Erk1/2 and Akt
4.4 Discussion 194
CHAPTER 5: Enhanced Cardiac Recovery Post-Reperfusion after a
Prolonged Period of Hypothermic Storage by Pharmacological
Recruitment of Pro-survival Kinases Akt and Erk1/2
5.1 Introduction 203
- xvi - PAGE
5.2 Materials and methods 207
5.3 Results 213
5.3.1 Prestorage baseline measurements of Cardiac 213
Function
5.3.2 Effect of cardioplegia / storage solution composition 214
on post-reperfusion recovery of cardiac function
5.3.3 Effect on cardioplegia / storage solution composition 222
on post-reperfusion phosphorylation status of Erk1/2
and Akt activation
5.3.4 Effect on cardioplegia / storage solution composition 226
on post-reperfusion phosphorylation status of GSK3β
5.3.5 Effect on cardioplegia / storage solution composition 228
on post-reperfusion phosphorylation status of ERM
5.3.6 Effect on cardioplegia / storage solution composition 228
on post-reperfusion phosphorylation status of BAD
and Bcl-2
5.4 Discussion 231
CHAPTER 6: General Discussion 243
BIBLIOGRAPHY 255
- xvii - LIST OF TABLES PAGE
Table 1.1 Summary of features between Necrosis and 32
Apoptosis
Table 1.2 Composition of some commercial preservation 49
solutions
Table 2.1 Anaesthesia and Pre-medications 104
Table 2.2 Perfusion buffer components 104
Table 2.3 Pharmacological supplements 106
Table 2.4 Chemicals and agents used in the preparation of 107
lysis buffer for Western Blotting
Table 2.5 Reagents and equipment used in protein 108
preparation, electrophoresis and transfer
Table 2.6 Components for casting of SDS-polyacrylamide 123
separation and stacking gels
Table 2.7 Conditions of electrophoresis and transfer process 123
Table 2.8 List of antibodies used throughout study 124
Table 3.1 Pre-storage baseline cardiac measurements and 138
Values
Table 4.1 Pre-storage baseline cardiac measurements and 178
values
Table 5.1 Pre-storage baseline cardiac measurements and 213
values
Table 5.2 Numerical values for indices of functional recovery 221
from Figures 5.3 - 5.6
- xviii - LIST OF FIGURES PAGE
Figure 1.1 Drawing of the triangulation technique 5
Figure 1.2 Overview of myocardial energy substrate 14
metabolism
Figure 1.3 Overview of ion homeostasis 19
Figure 1.4 Oxygen free radicals and tissue injury 24
Figure 1.5 Overview of mPTP opening in apoptosis and 36
necrosis
Figure 1.6 Comparision of adult heart transplants survival by 60
era
Figure 1.7 Representive scheme of the PARP pathway 71
Figure 1.8 Schematic representation of the mechanisms of 85
major pro-survival kinase pathways associated
with pre- and post-conditioning
Figure 2.1 Surgical instruments 110
Figure 2.2 Rat anti-coagulated by an injection of heparin 110
Figure 2.3 Surgical exposure of the heart 111
Figure 2.4 Excision of the heart – lung block 111
Figure 2.5 Hypothermic arrest of the heart 112
Figure 2.6 Trimming the aorta 112
Figure 2.7 Cannulation of the aorta 113
Figure 2.8 Venting the pulmonary artery 114
Figure 2.9 Ligation (a) and removal of lungs (b) 114
Figure 2.10 Rotation of heart for cannulation 115
- xix - PAGE
Figure 2.11 Clipping and securing the left atrial cannnula 115
Figure 2.12 Completed picture of a working heart preparation 116
Figure 2.13 Transonic flow probe (a) and flow meter (b) 117
Figure 2.14 MacLab data acquisition system 118
Figure 2.15 Arrested heart mounted onto the perfusion cannulae 120
stored in a beaker
Figure 3.1 Experimental protocol and treatment groups 135
Figure 3.2 Coronary flow and Heart rate represented as a 140
percentage of baseline values
Figure 3.3 Aortic flow and Cardiac output represented as a 141
percentage of baseline values
Figure 3.4 Representative immunohistochemical sections 143
from control and INO-1153 treated hearts
Figure 3.5 Representative trace recordings of aortic flow and 145
heart rate
Figure 3.6 Effect of PI3-k inhibition using Wortmannin 146
Figure 3.7 Representative immunoblots and histogram 148
showing Akt phosphorylation, Akt and β–actin
levels and ratio
Figure 3.8 Representative immunoblots and histogram 150
showing Erk1/2 phosphorylation and total Erk1/2
levels and ratio
- xx - PAGE
Figure 3.9 Representative immunoblots and histogram 152
showing phospholamban phosphorylation and
total phospholamban levels and ratio
Figure 3.10 Representative immunoblots and histogram 154
showing eNos phosphorylation and total eNOS
levels and ratio
Figure 4.1 Study protocol and treatment groups 173
Figure 4.2 Representative trace recordings of aortic flow and 178
heart rate
Figure 4.3 Summary comparision of various treatment groups 179
Figure 4.4 Representative immunoblots and histogram showing 182
Erk1/2 phosphorylation, total Erk1/2 and β-actin
levels and ratio
Figure 4.5 Representative immunoblots and histogram showing 183
Akt phosphorylation, total Akt and β-actin levels
and ratio
Figure 4.6 Representative immunoblots and histogram showing 185
Bcl-2 phosphorylation, total Bcl-2 and β-actin levels
and ratio
Figure 4.7 Representative immunoblots and histogram showing 188
cleaved caspase 3, pro-caspase 3 and β-actin
levels and ratio
- xxi - PAGE
Figure 4.8 Representative cleaved caspase 3 189
immunofluorescence images and histogram
showing quantification
Figure 4.9 Effect of PD98059 on recovery of post-storage 191
function
Figure 4.10 Effect of PD98059 on the extent of Akt and 193
Erk1/2 phosphorylation
Figure 5.1 Timeline and experimental groups 210
Figure 5.2 Representative trace recordings of pressure and 216
aortic flow
Figure 5.3 Aortic flow as a percentage of baseline values 217
Figure 5.4 Coronary flow as a percentage of baseline values 218
Figure 5.5 Cardiac output as a percentage of baseline values 219
Figure 5.6 Heart rate as a percentage of baseline values 220
Figure 5.7 Representative immunoblots and histogram 223
showing Erk1/2 phosphorylation, total Erk1/2 and
β-actin levels and ratio
Figure 5.8 Representative immunoblots and histogram 225
showing Akt phosphorylation, total Akt and β-actin
levels and ratio
Figure 5.9 Representative immunoblots and histogram 227
showing GSK3β phosphorylation, total GSK3β and
β-actin levels and ratio
- xxii - PAGE
Figure 5.10 Representative immunoblots and histogram 229
showing ERM phosphorylation, total ERM and
β-actin levels and ratio
Figure 5.11 Representative immunoblots showing expressions 230
of BAD and Bcl-2 phosphorylation
- xxiii - LIST OF ABBREVIATIONS
µM micromolar
AIF apoptosis inducing factor
Akt protein kinase B
ANOVA analysis of variance
ANT adenine nucleotide transporter
ATP adenosine triphosphate
Bcl-2 B-cell leukemia 2
Bcl-XL B-cell leukemia extra long
BH4 tetrahydrobiopterin
Ca2+ calcium ion
CABG coronary artery bypass graft
cGMP cytoplasmic guanosine monophosphate
Da daltons
DAB diaminobenzadine
DAPI 4', 6-diamidino-2-phenylindole
DMSO dimethylsulphoxide
DNA deoxyribonucleic acid
ECSOD extracellular superoxide dismutase
eNOS endothelial nitric oxide synthasae
Erk extra cellular signal regulated kinase
ERM ezrin-radixin-moesin
ESR erythrocyte sedimentation rate
FADH flavin adenine dinucleotide hydrogenase
GDP guanosine diphosphate
- xxiv - GMP guanosine monophosphate
Grb2 growth factor receptor-bound protein 2
GSH glutathione-SH
GSK-3β glycogen+C109 synthase kinase 3 beta
GTN glyceryl trinitrate
GTP guanosine triphosphate
H+ hydrogen peroxide
H2O2 hydrogen peroxide
Hg mercury
HO* hydroxyl radical
HTK histadine-trytophan-ketoglutarate
IPC ischaemic preconditioning
IR ischaemia reperfusion
IRI ischaemia reperfusion injury
IU international units
JNK c-Jun N-terminal kinase
K+ potassium ion kg kilogram
LAD left anterior descending (coronary artery)
MAPK mitogen activated protein kinase mdm2 murine double minute clone 2
MEK mitogen-activated protein kinase kinase mg milligram
MI myocardial infarction mitoKATP mitochondria potassium ATP
- xxv - mm millimetre mPTP mitochondria permeability transition pore
Na+ sodium ion
NAD nicotinamide adenine dinucleotide
NADH nicotinamide adenine dinucleotide dehydrogenase
NHE sodium hydrogen exchange nM nanometre
NO nitric oxide
NOS nitric oxide synthase
• O2- superoxide anion oC degrees celsius
ONOO- peroxynitrite p70S6K p70 S6 kinase
PARP poly(ADP-ribose) polymerase
PCI percutaneous coronary intervention
PDK1 phosphoinositide-dependent kinase 1
PI3-k phosphatidylinositol 3-kinase
PIP3 phosphatidylinositol 3,4,5-triphosphate
PKA protein kinase A
PKC protein kinase C
PLSD protected least significant difference
PTEN phosphatase and tensin homolog
RISK reperfusion injury salvage kinase
ROS reactive oxygen species siRNA small interfering ribonucleic acid
- xxvi - SD standard deviation
SE standard error
SERCA sarcoplasmic reticulum Ca2+ ATPase
SOD superoxide dismutase
ST2 St. Thomas solution 2
TCA tricarboxylic acid
TNF-α tumour necrosis factor-alpha
UW university of wisconsin
VDAC voltage dependent anion channel
VEGF vascular endothelial growth factor
- xxvii - CHAPTER 1
Introduction
1 1.1 OVERVIEW
Heart transplantation is now firmly established as the most effective therapy for end-stage heart disease with approximately 90% of heart transplant recipients in Australia and New Zealand returning to levels of activity that would be considered normal for people of their age (2007
Report of the Australia and New Zealand Cardiothoracic Organ Transplant
Registry). In spite of its effectiveness, heart transplantation remains a relatively rare procedure because of the scarcity of suitable donors.
The quality of the donor heart is determined by a variety of factors including: i) donor age and pre-existing disease; ii) pathophysiological changes associated with brain death; iii) donor management prior to organ procurement; iv) the duration and conditions of hypothermic storage; v) the circumstances of reperfusion. Even with the development of specialised cardioplegic and storage solutions to minimise the effects of the obligatory period of cold ischemia and warm reperfusion, there is still a significant incidence of primary graft failure or delayed function immediately post transplant, especially when the donor heart is derived from a marginal donor. The discard rate for referred donor hearts from marginal donors may be as high as 60% - an unacceptably high rate for such a scarce resource (Rosengard et al., 2002).
Given this disconnect between supply and demand, there is intense interest in further pharmacological and physiological approaches to
2 minimize the damage accrued by the donor heart during the transplant process. The present thesis explores the feasability of harnessing some of the recent advances in the elucidation of mechanisms of protection against ischemia reperfusion damage (pharmacological recruitment and activation of protective signal transduction pathways) in the setting of a model of donor heart preservation.
3 1.2 HISTORY OF HEART TRANSPLANTATION
The development and history of heart transplantation spans over 100 years to the present with important lessons and contributions from many individuals all over the world including France, the United States of
America, South Africa, Britain and Russia. It is largely because of the commitments of these individuals and various groups through the early and late 20th Century that the potential of heart transplantation has been realised. This section outlines a brief history of heart transplantation. It is divided into 3 sections; i) the pre-clinical research era, ii) early clinical experience and iii) the modern (post-cyclosporine) era; briefly illustrating the the technical developments and advances through each era.
1.2.1 Pre-clinical research era
The careful studies of the Frenchman, Alexis Carrel in the late 19th and early 20th centuries provided the foundations of transplantation and organ preservation on a number of levels. A key element in the transplantation of any organ is the successful joining of the donor and recipient blood vessels. In a series of studies begun in Lyon in 1902 and continued in the
USA, Carrel developed a triangulation technique for vascular anastomosis
(Carrel, 1902) (Figure 1.1).
4
Figure 1.1 Drawing of the triangulation technique. Positioning of the 3 stay sutures (arrowed) enables 3 lines of fine running sutures to be placed between them. Adapted from (Dutkowski et al., 2008).
In order to investigate the usefulness of this anastamotic technique, Carrel and Charles Guthrie performed a series of transplants including the first heterotopic heart transplant in the neck of a dog (Carrel et al., 1905). The distal end of the divided recipient carotid artery supplied blood flow to the donor heart with blood flowing through left atrium, ventricle and aorta into the distal end of the divided jugular vein. The proximal sides of the carotid and jugular supplied right heart circulation.
This study provided an important proof of principle for future transplantation research: i.e. the heart survived (albeit for a short period) the removal from the “donor” and the sew-in period into the “recipient”.
The transplanted coronary circulation was successfully perfused and the heart regained contractile activity, but because of its positioning, cardiac output was not possible.
5 Carrel was also interested in strategies of organ and tissue preservation.
His 1912 paper entitled “The preservation of tissues and its application in surgery” summarising a series of studies undertaken between 1906-12 anticipated the development of modern tissue and organ transplantation in all its forms (Carrel, 1912). For this body of work Carrel was awarded the
Nobel Prize in Physiology and Medicine in 1912, his citation reading “in recognition of his work on vascular suture and the transplantation of blood vessels and organs.”
In 1933, Mann and colleagues simplified the anasomoses to facilitate coronary perfusion in 2 heterotopic models - auto-transplantation and homo-transplantation (Mann et al., 1933). Results of auto-transplantation studies showed that the viability of the organ after re-implantation in the same subject was superior to those implanted into another subject of the same species (homo-transplantation). They also anticipated the problem of rejection by commenting that the reasons for graft failure were
“biological” rather than “technical”.
Between 1946 and 1955, Vladimir Demikhov in Russia performed many heart and lung transplants in animals (Demikhov, 1962). Amongst them, the most far-reaching experiment was a canine heart that was successfully transplanted into the orthotopic position by attaching the aorta, pulmonary artery and venae cavae of the donor heart to the corresponding recipient blood vessels while the pulmonary veins of the donor heart were attached
6 to the recipient’s left atrium. These experiments reported allograft survival times of 11.5 – 15 hours and showed for the first time that a cardiac allograft could function in a recipient animal (Cooper, 1968). Unfortunately, this significant body of work was unknown at the time in the West as his studies, originally published in Russian, were not published in English until
1962.
In 1951, Marcus and associates from Chicago devised a heterotopic technique involving 3 dogs to support the donor heart when it was disconnected from the donor circulation and it was the first method of donor heart preservation (Marcus et al., 1951). The development of supportive strategies for the recipient led to the first use of hypothermia in
1953 by Neptune and colleagues (Neptune et al., 1953) from Philadelphia during their attempt at orthotopic transplantation. Their approach was similar to that of Vladimir Demikhov which was described a decade earlier.
They were successful in showing that the cardiac allograft could support the circulatory load of the recipient, also demonstrated by Demikhov without the assistance of hypothermia.
Results of orthotopic transplantation gradually improved as a result of further technological advances. Use of a pump oxygenerator for further recipient support was employed in 1957 for heart-lung transplantation by
W.R. Webb (Webb et al., 1957). The team published the technique for heart only in 1959 and documented the cardiac allograft survival up to 7.5
7 hours (Webb et al., 1959). The donor heart was stored in Tyrode’s solution supplemented with 10% serum at approximately 4°C.
1960 was the year Lower and Shumway demonstrated the use of orthotopic transplantation of a number of canine hearts with the use of a rotating disk oxygenator with successful post transplant survival between 6 and 21 days (Lower et al., 1960). During 1961 Shumway and colleagues refined the method for canine heart transplantation and survival times ranged from 8 hours to 8 days. They concluded that immunologic injury due to graft rejection or absence of immunosuppression was the barrier left between long and short term post transplant survival (Lower et al.,
1961).
1.2.2 Early clinical experience era
The first heart transplant was performed in human by James Hardy in
1964 at University of Mississippi Medical Centre in Jackson – Mississippi, when he transplanted a chimpanzee heart into a patient, who died soon after as the chimpanzee heart could not handle the large venous return of the human being (Hardy et al., 1964). In December 1967, Christian
Barnard performed the first human to human heart transplant using the
Shumway method (Lower et al., 1960) on a 54 year old patient suffering from end-stage ischaemic cardiomyopathy at Groote Schuur Hospital,
Cape Town South Africa (Barnard, 1967). The recipient recovered and lived for 18 days after the operation before dying of pneumonia despite
8 being on immunosuppressive therapy. Dr. Barnard and his team performed a second transplant operation in January 1968 and this patient lived for almost 20 months after the procedure but died of coronary atherosclerosis (Brink et al., 2005).
The same year, Dr. Harry Windsor and a team at St Vincent’s Hospital in
Sydney performed the first heart transplant in Australia (Windsor, 1969).
The 58 year old recipient died on the 45th day post transplant as a consequence of aortic rupture secondary to infection. After a second unsuccessful transplant, the programme was voluntarily halted. Norman
Shumway carried out the first “successful” human heart transplant in the
USA also in 1968 (Stinson et al., 1968). Although the procedure was technically successful, the recipient’s postoperative course was complicated and he died as a result of severe bacterial infection 15 days after transplant.
Cooley and colleagues documented their experiences of 12 human heart transplants by using blood group compatibility, lymphocyte crossmatch studies and a grading system to improve the success of the transplant outcome (Cooley et al., 1968). They reported post transplant survival times of up to 4.5 months in 7 human heart recipients.
As Shumway and his team compiled and reviewed their experience in heart transplantation and post transplant survival of the recipients, it
9 became clear that the area of cardiac allograft rejection was not well resolved as there was the problem of chronic rejection of the allograft that ultimately claimed the lives of post transplant recipients (Clark et al.,
1973). In 1973 Shumway’s group introduced percutaneous transvenous endomyocardial biopsy (Caves et al., 1973) to allow them to follow histologic events that occurred in the heart to determine the degree of rejection so that they could tailor the levels of immunosuppressive medication required by the recipient. By 1974 their new technique led to higher survival rates and the number of human heart transplants performed was increased considerably (Graham et al., 1974).
During the late 1970s J.F. Borel (Borel, 1976) was the first to report the immunosuppressive effects of cyclosporine A and Calne and his associates (Calne et al., 1978) discovered the powerful immunosuppressive effect of cyclosporine A using porcine orthotopic cardiac allografts. He proposed it could be an attractive candidiate for use in clinical immunosuppression. During the early 1980s, Shumway and his team introduced the use of Cyclosporine as an immunosuppressive drug
(McGregor et al., 1986) and found that the recipient survival rates improved markedly and thereafter made heart transplantation safer.
1.2.3 The modern era (post 1984)
The introduction of cyclosporine A ultimately led to the tremendous success of cardiac transplantation and received worldwide interest through
10 the late 1980s. In Australia, the heart transplantation programme at St
Vincent’s Hospital recommenced in 1984 under the direction of Dr. Victor
Chang, with the first recipient surviving until 1991 (Chang, 1984). The same year, a national cardiopulmonary centre was established in
Auckland, New Zealand, along with another 3 units throughout Australia.
The survival rates up to end of 2007 are reported to be 87% at 1 year,
61% at 10 years and 38% at 20 years post-transplant, based on more than
1900 heart transplants performed (2007 Report of the Australia and New
Zealand Cardiothoracic Organ Transplant Registry). The 2007 report also reported approximately 80 heart transplants are performed each year in contrast to the almost 30,000 patients that die each year as a result of heart failure (Krum et al., 2006).
Given this scarcity of suitable heart donors in Australia, there is an intense need to develop alternative therapies such as permanent mechanical circulatory assistance, cell and gene based therapies. However, broad introduction of these latter approaches to replace clinical heart transplantation may still be a long way off. In the mean time, improvements to pre-existing pharmacological and physiological approaches to minimize the damage to the donor heart caused by ischaemia reperfusion during the transplant process maybe more easily adapted for clinical use. This is the focus of the present thesis.
11 1.3 ISCHAEMIA REPERFUSION INJURY
1.3.1 Background and definitions
Rudolf Virchow, the 19th century German pathologist coined the term
“ischaemia” in an attempt to characterise the results of “hinderance of blood supply” to tissue (Virchow, 1858). In the mid 1990’s, Hearse proposed a “consensus” definition of myocardial ischaemia as a state where ischaemic conditions lead to an inability of the supply of oxygen and nutrients to meet the metabolic demand of the tissue (Hearse, 1994).
Rapid and timely reperfusion of ischaemic myocardium is essential for the resupply of oxygen and nutrients and the removal of various cellular waste products that facilitates salvage of ischaemic tissue. However, reperfusion has also been accompanied by a range of physiological and biochemical alterations (termed “reperfusion injury”) (Braunwald et al., 1985).
Reperfusion injury may manifest as myocardial stunning, arrhythmias, microvascular and endothelial injury that can lead to myocardial cell death
(also termed lethal reperfusion injury) (Glazier, 2005, Moens et al., 2005).
A number of the key mechanisms thought to be responsible for reperfusion injury will be discussed in subsequent sections.
Reperfusion injury to the donor heart in the context of clinical cardiac transplantation may be more complex than that observed during acute myocardial infarction or ischaemic heart disease. Some of the differences include i) donor brain death, ii) total global ischaemia with no possibility of
12 myocardial support from collateral circulation, iii) global ischaemia accompanied by hypothermia during the period of cardiac storage, iv) management of the recipient on cardiopulmonary bypass and v) a period of warm ischaemia during the reimplantation of the donor heart into the recipient before reperfusion (Hicks et al., 2006). Toledo-Pereyra and colleagues (Toledo-Pereyra, 1987) recognised the logistic difficulties in assessing the incremental cardiac damage during each phase of the process, especially in clinical conditions. He suggested that a working definition for reperfusion injury in organ transplantation should encompass
“all injury to the organ during donor maintenance, warm ischemia, preservation, transplantation and reperfusion”.
1.3.2 Metabolic changes during ischaemia
1.3.2.1 Energy metabolism in non-ischaemic heart
The energy required for the contractile function of the heart and ion homeostasis is provided by hydrolysis of the high energy phosphate bond of ATP produced in the mitochondria. Around 66% of the ATP consumed by the heart is involved in provision of cardiac pump function and around
33% provides energy for basal metabolic activity including maintenance of intra-cellular ion gradients and pumps and biosynthetic functions (Suga,
1990). Under normal (non-ischaemic) conditions, fatty acids are the primary substrates for energy production in the heart. However, other substrates such as lactate and glucose may also contribute to the energy
13 (A) Normoxic Cardiac Myocyte Free Fatty Acids Glucose 60 – 90%
CPT-1
PDH Acetyl Glycolysis Pyruvate Krebs CoA Cycle 10 – 40% 2 ATP 2 H+ ETC 2% Lactate O2 ATP (98%)
(B) Ischaemic Cardiac Myocyte Free Fatty Acids Glucose
PDH Glycolysis Pyruvate
+ 2 ATP 2 H Lactate
Figure 1.2: Overview of myocardial energy substrate metabolism in non- ischemic (A) and ischaemic myocardial cells (B). See section 1.3.2 for details.
ATP: adenosine triphosphate; CPT-1: carnitine palmitoyltransferase; PDH: pyruvate dehydrogenase. Adapted from (Stanley et al., 2002).
14 production in the heart (Figure 1.2A). In the isolated working heart model, where energy substrates can be closely controlled, it has been shown that fatty acids inhibit oxidation of glucose to a greater extent than glucose inhibits fatty acid oxidation, although optimal cardiac function was observed when both substrates were oxidised simultaneously
(Taegtmeyer et al., 1980).
Energy production in myocardial cells under non-ischaemic conditions is summarized in Figure 1.2 (A). The bulk of energy formation, (60-90%), is derived from fatty acids (Stanley et al., 2002). The fatty acids oleic acid and palmitic acid are the major lipid substrates for energy production.
Fatty acids enter the cardiac myocyte via a membrane bound translocase or a fatty acid binding protein. Once inside the cell they are rapidly esterified to fatty acyl-CoA by fatty acyl-CoA synthetase. These products can further esterified to triglycerides or converted to a fatty acylcarnitine derivative that can be transported into the mitochondria. In the mitochondrial matrix, the fatty acids undergo ß-oxidation leading to production of acetyl-CoA. Acetyl-CoA then enters the Krebs cycle to produce NADH and FADH2 which subsequently generates ATP via oxidative phosphorylation (Stanley et al., 2005).
1.3.2.2 Energy metabolism in ischaemic heart
When the donor aorta is clamped preparatory to cardiac procurement, the heart is subjected to complete global ischaemia until it is reperfused with
15 the recipient’s circulation after implantation. Cardiomyocytes must now maintain ATP levels by the less efficient glycolytic ATP production as energy production shifts from aerobic to anaerobic route.
The glycolytic pathway converts glycogen or glucose to pyruvate in the cytosol by a series of enzymatic reactions not dependent on the presence of oxygen. In aerobic ATP production where pyruvate enters the mitochondrion and is fed into the Krebs cycle providing reducing power to drive the electron transport chain. In the absence of oxygen, pyruvate is converted to lactate in the cytosol (Figure 1.2 B).
During severe ischaemia, sustained accumulation of lactate and intracellular hydrogen ions will result in myocardial acidosis (King et al.,
1998) and may eventually inhibit glycolysis and any residual ATP formation damaging the cardiac cells irreversibly (Rovetto et al., 1975).
During this period, breakdown products of ATP accumulate, especially hypoxanthine and xanthine which are important potential substrates for free radical and oxidant production (discussed in Section 1.3.4.2).
During ischaemia, fatty acids, previously the major substrates for oxidative phosphorylation in normoxic myocytes are now accumulated in the cytosol of cardiac myocytes along with their acyl (CoA and carnitine) metabolites
(Liedtke, 1981). Because of their amphipathic properties (ie a charged polar head group and long hydrophobic tails) it was postulated that these
16 fatty acids could insert themselves into the lipid phase of biological membranes producing significant changes to the physical properties of the membranes (Katz et al., 1981).
Other subtle metabolic changes occurring during ischaemia may mitigate some of the effects of suboptimal energy levels. Firstly, the mitochondria change from being ATP producers to ATP consumers. The mitochondrial
F1F0-ATPase instead of being a key component of ATP synthesis now hydrolyses ATP in order to maintain the inner mitochondrial membrane potential which may preserve mitochondrial integrity and ensure proper mitochondrial function at reperfusion (St-Pierre et al., 2000).
Cell fractionation studies indicate that rather than being homogeneously distributed through the cytosol, glycolytic enzymes are associated with the cell membrane and the endoplasmic reticulum (Pierce et al., 1985). Such glycolytic ATP production has been shown to drive calcium uptake into the endoplasmic reticulum (Entman et al., 1977) and maintain basal plasma membrane Na+ / K+ ATPase activity (Jeremy et al., 1993).
1.3.3 Changes to intracellular ion and water homeostasis during ischaemia
The maintenance of tightly controlled ion homeostasis is crucial for all facets of cardiac function. Of particular importance for the development of ischaemia-reperfusion related deficits are the Na, K ATPase, the sodium
17 calcium (Na+/Ca2+) exchanger and the sodium hydrogen (Na+/H+) exchanger (Figure 1.3). The Na, K ATPase uses energy from hydrolysis of mitochondrial ATP to pump 3 sodium ions out of the cell and 2 potassium ions into the cell to establish the normal membrane potential for propagation of cardiac action potentials (Bers et al., 2003). This Na+ gradient established by the normally functioning Na, K ATPase also creates the driving force for extrusion of calcium by the sodium calcium exchanger (Schmidt et al., 1998). Under non-ischaemic conditions, the intra-myocyte pH is around 7.2, rendering the sodium hydrogen exchanger quiescent (Flaherty et al., 1982).
Application of the aortic cross-clamp to the donor heart produces an abrupt change from efficient mitochondrial ATP production during aerobic metabolism to relatively inefficient glycolytic ATP production. The fall in
ATP production and rise in ADP and inorganic phosphate results in a marked decrease in Na, K ATPase activity leading to a rapid increase in intracellular sodium (H.R. Cross et al., 1995). This leads to rapid depolarisation of the cardiac myocyte cell membrane and facilitates influx of negative chloride ions and efflux of positive potassium ions. Such a nett gain in intracellular solute now draws water into the cells, culminating in cell swelling (Leaf, 1973, Leaf et al., 1954).
The exact mechanism of sodium influx during ischaemia is contentious with activation of the sodium hydrogen exchanger by hydrogen ions from
18 (A): Non-Ischaemic Conditions + 3Na Na+ K+ ATPase
Na/Ca 3Na+ Exch N/H Exch 2K+
2+ Ca Na+ K+ ATP
(B): Ischaemic Conditions + + Na K Cl-, H O ATPase 2 Na+ Na/Ca Exch Ca2+ N/H Exch 3Na+ H+ Glycolysis
+ + Na ATP K
Figure 1.3 Overview of ion homeostasis. (A) Under non-ischaemic conditions activity of Na+/K+ ATPase is maintained and establishes the normal intracellular membrane potential. Sodium hydrogen exchanger (N/H Exch) is quiescent and
Na/Ca exchanger maintains normal Ca levels. (B) During ischaemia, mitochondrial ATP synthesis is impaired with inhibition of the Na+/K+ ATPase.
Increased glycolysis leads to decreased intracellular pH and activation of N/H
Exch. Rise in cytosolic Na+ levels leads to influx of water (cell swelling) and reverse mode Na/Ca exchange resulting in influx of Ca.
19 glycolysis and persistent non-activating sodium channels both suggested as major players (Lazdunski et al., 1985, Murphy et al., 1999). The activation of the sodium hydrogen exchanger during global ischaemia is likely to be self limiting as extrusion of intracellular hydrogen ions into the extracellular space will rapidly equalise the trans-membrane pH gradient which drives the exchange process.
Whatever the route of ischaemia-associated influx of intracellular sodium, this process will lead to some amount of calcium influx via the sodium calcium exchanger operating in “reverse mode” (ie exporting sodium out of the cell and importing calcium into the cell) (Barcenas-Ruiz et al., 1987).
However, the extent of calcium entry via this pathway may be limited as the sodium calcium exchanger is inhibited by the low pH conditions associated with ischaemia (Lazdunski et al., 1985).
1.3.4 Reperfusion injury
The process of reintroduction of oxygen to an ischaemic area of the myocardium (reperfusion) can paradoxically lead to further tissue injury despite the necessity for restoration oxygenated blood flow to ensure its survival. This phenomenon later known as the “oxygen paradox” was first observed by Hearse and colleagues (Hearse et al., 1973). The degree of reperfusion injury to the myocardium is directly dependant on the duration and severity of ischaemia sustained. Some of the known reperfusion associated pathologies are myocardial stunning (Ambrosio et al., 2001),
20 arrhythmias, irreversible cell damage or necrosis, as well as injury to the microvasculature and endothelium (Granger, 1999, Moens et al., 2005,
Park et al., 1999). The major mediators of cellular events involved in reperfusion injury are thought to be calcium overload and free radical damage (Verma et al., 2002).
1.3.4.1 Changes to intracellular ion and water homeostasis during
reperfusion
Reperfusion of previously ischaemic myocardium provides a source of oxygen and nutrients. In addition, waste products are rapidly removed from the extra-cellular space. However, the ability of the ischaemic tissue to resume ATP synthesis at baseline (pre-ischaemic) levels may be severely compromised even for some hours (DeBoer et al., 1980). As mentioned previously, ATP levels drop and cytosolic levels of nucleoside precursors (eg adenosine, hypoxanthine and inosine) rise (Benson et al.,
1961). During ischaemia, these products may diffuse through the cell membrane and are washed out upon reperfusion. The resynthesis of
(myocardial) these ATP precursors occurs via salvage pathways and de novo synthesis of ATP will be by no means immediate (Maguire et al.,
1972).
The reperfusate will remove the accumulation of extracellular hydrogen ions, which will now result in reactivation of the sodium hydrogen exchanger to relieve intracellular acidosis (Frolich et al., 1997). The
21 resultant increase in intracellular sodium is now removed by the sodium calcium exchanger working in the “reverse mode” with resultant increase in the levels of intracellular calcium (Schafer et al., 2001). Loss of the usually tight control of cardiac cellular calcium homeostasis can lead to a number of cytotoxic consequences (Dong et al., 2006) and is discussed in greater detail below.
Earlier studies by Karmazyn and co-workers (Karmazyn et al., 1993) have demonstrated that pharmacological inhibition of the sodium-hydrogen exchanger is a potent protective strategy against myocardial ischaemia reperfusion injury and proposed the use of such pharmacological agents as an adjunct therapy in reperfusion protocols. The use of this approach to protect the donor heart forms a major part of this thesis.
1.3.4.2 Free radical formation
Restoration of oxygenated blood flow to previously ischaemic myocardial tissue leads to a rapid and significant production of oxygen free radicals which have been detected directly by electron spin resonance spectroscopy (Bolli et al., 1989, Zweier et al., 1987).
A free radical is defined as any species capable of independent existence that contains one or more unpaired electrons, (an unpaired electron being one that occupies an atomic or molecular orbital by itself). Ground (triplet) state molecular oxygen contains 2 unpaired electrons in its π*2p orbitals.
22 Molecular oxygen may undergo a series of single electron reductions to
- form (in order) the superoxide anion (O2• ), hydrogen peroxide (H2O2), the hydroxyl radical (HO•), and water (Halliwell, 1989a, Halliwell, 1989b).
Oxygen free radicals can be generated from a range of enzymic and non- enzymic reactions in vivo in a number of organelles and cell types. Of particular relevance to ischaemia and reperfusion are those generated by mitochondria, the vascular endothelium and neutrophils. A scheme summarising the important biological routes of formation and decomposition of oxygen centred free radicals is shown in Figure 1.4.
1.3.4.3 Formation of Superoxide
Mitochondria: Under non-ischaemic conditions, oxygen is the terminal electron acceptor for 2 pairs of electrons after passage down the mitochondrial electron transport chain, with the formation of water.
However there are several sites where single electron transfer to oxygen may occur (eg complex I and complex III), with the formation of superoxide
(Turrens, 2003). Such single electron transfer normally represents a small fraction of the total electron transfer along the mitochondrial electron transfer chain and is controlled by intra-mitochondrial anti-oxidant enzymes such as Mn-superoxide dismutase and GSH peroxidase (Ferrari et al., 1993). The highly reduced intra-mitochondrial redox state produced during ischaemia is likely to favour single electron donation to residual oxygen (Hess et al., 1984). Single electron transfer at the level of
23 ubiquinone has been strongly implicated in this process (Becker et al.,
1999).
Normal mitochondrial electron transport
• Oxidases Catalase, glutathione peroxidase •e- leak from mitochondrial electron trans • Superoxide • Reducing chain dismutase conditions • Uncoupled • Spontaneous after ischemia NOS dismutation •Incr in Fe2+ O O -•HO HO• H O 2 2 2 2 Fenton 2 Reaction ? e- e- + 2H+
NO•
Damage to lipids, ONOO- proteins & DNA
Tissue injury, Cardiac dysfunction
Figure 1.4 Oxygen free radicals and tissue injury. Scheme summarising the important routes of formation and decomposition of oxygen free radicals during ischaemia and reperfusion, leading to intracellular damage followed by tissue injury and cardiac dysfunction.
24 Oxidases: Monoamine oxidase, located in the outer mitochondrial membrane is an important site of hydrogen peroxide formation that may diffuse into the cytosol or the inner mitochondrial space (Cadenas et al.,
2000). This may be a potentially significant but unrealised source of oxidant damage during the donor management phase of the transplant process due to the rapid release of endogenous catecholamines immediately after donor brain death and the common clinical practice of maintaining donor blood pressure with one or more exogenous catecholamines (Hicks et al., 2006).
The xanthine oxidoreductase enzyme was identified as a potential source of superoxide during ischaemia and reperfusion (McCord, 1985). This enzyme consists of two types: i) the “D” type dominates under non- ischaemic conditions and uses NAD+ as an electron acceptor for its reaction that catalyses the conversion of hypoxanthine and xanthine to uric acid; ii) during ischaemia, limited proteolysis of the “D” form by a calcium-dependant protease creates the “O” form of the enzyme with a subtle change in substrate specificity in that it now uses oxygen as an electron acceptor. At the same time, ATP is continually dephosphorylated and catabolized to hypoxanthine, the substrate for the so-called xanthine oxidase. Thus, when oxygen is reintroduced at reperfusion, superoxide can now be formed via the following reaction:
- + xanthine + H2O + 2O2 Æ uric acid + 2 O2 • + 2H
25 NADPH oxidases have been identified in phagocytic cells (Babior, 1999) and more recently in the vasculature (Griendling et al., 2000). NADPH oxidase from phagocytes produces superoxide in response to invading pathogens (Babior, 1999), whilst the vascular form is activated by a range of vasoactive agents (including angiotensin II and thrombin) and is thought to play a role in numerous signalling processes (Griendling et al., 2000).
However, neither is thought to play a role in ischaemia-reperfusion derived superoxide production (Cave et al., 2006, Hoffmeyer et al., 2000).
Nitric Oxide Synthase (NOS): Another potential route for cytosolic and mitochondrial superoxide formation is via “uncoupled” nitric oxide synthase. Under normal physiological conditions the nitric oxide synthase dimer catalyses the conversion of L-arginine to L-citrulline and nitric oxide
(NO•) using several cofactors, including tetrahydrobiopterin (BH4), but if
BH4 is depleted, electrons are transferred to oxygen rather than the natural substrate, arginine, with the formation of superoxide (Stuehr et al., 2001).
Recently, irreversible BH4 depletion has been observed during periods of up to 60 min myocardial ischaemia with a corresponding increase in NOS- derived superoxide (Dumitrescu et al., 2007).
Down-stream of Superoxide: Superoxide may be converted to hydrogen peroxide by spontaneous dismutation or more usually with the aid of mitochondrial Mn – superoxide dismutase. Rapid removal of hydrogen peroxide is then catalysed by glutathione peroxidase (Flohe, 1982). Under
26 appropriate conditions, an alternative fate for superoxide may be the rapid reaction with nitric oxide to form the highly oxidising peroxynitrite species,
ONOO- (Huie et al., 1993). These conditions may well be met in the post- ischaemic reperfused heart. NOS-independent nitric oxide formation has been demonstrated by reduction of tissue nitrite in the acid conditions of ischaemia (Zweier et al., 1995). Peroxynitrite formation in the heart after reperfusion was demonstrated unambiguously by ESR techniques and was directly linked to observed contractile deficits and poor coronary flows post-reperfusion (P. Wang et al., 1996). Although conditions immediately after ischaemia may also favour Fenton chemistry with resultant formation of the highly oxidising hydroxyl radical (HO•), its biological relevance as a damaging species has been questioned, with ONOO- possessing equivalent reactivity and toxicity profiles (Koppenol, 1998, Koppenol,
2001).
1.3.4.4 Recent Research into the role of free radicals & oxidants –
Protective effects
The role of reactive oxygen species and free radicals has evolved over time. The cytotoxic potential of these agents was highlighted by early studies which postulated that the common mechanism between toxicity of hyperbaric oxygen and the damaging effects of ionising radiation was the formation of oxygen-centred free radicals (Gerschman et al., 1954). In support of this concept, many experimental studies have demonstrated that cardiac ischaemia reperfusion injury could be minimised by enzymatic
27 and non-enzymatic anti-oxidants (Kukreja et al., 1997). However, despite numerous highly powered trials, the usefulness of antioxidant therapy in preventing morbidity and mortality associated with cardiovascular disease in the clinical arena has not been demonstrated (Kris-Etherton et al.,
2004). These findings are in line with recent evidence that endogenously produced free radicals may also play a pivotal role in protective signalling pathways within the cell (discussed in subsequent sections). This idea has its genesis in a series of studies that identified ischaemic preconditioning
(a series of short episodes of ischaemia and reperfusion before a long period of index ischaemia) delayed subsequent lethal cell injury (Murry et al., 1986). Subsequent studies revealed that: i) Oxygen radicals released during the preconditioning process were responsible for cardioprotection
(Baines et al., 1997) and ii) a short burst of oxidant formation with exogenous xanthine oxidase before index ischaemia produced a protective effect equivalent to these short episodes of ischaemia and reperfusion and this protective effect could be inhibited by prior exposure of the heart to anti-oxidants (Tritto et al., 1997).
1.3.4.5 Role of calcium and mitochondria in cell injury and death
The influx of extracellular calcium into the intracellular environment was found to be a common pathway for a number of cytotoxic insults (Schanne et al., 1979). Cellular calcium homeostasis in non-ischaemic cells is thus under rigid control of multiple pumps, transporters and binding proteins
(Berridge et al., 2003, Carafoli et al., 2001). Whilst extracellular calcium
28 levels are normally maintained in the millimolar range (Schanne et al.,
1979), cytosolic levels of Ca2+ are in the nanomolar range (20 – 50 nM, depending on the measurement technique) (Dipolo et al., 1976). The endoplasmic reticulum represents the largest intra-cellular calcium store, with resting levels at around 500 µM (Szabadkai et al., 2008). Basal levels of calcium in the mitochondrial matrix are around 100 nM, (Silverman,
1993). The increases in intracellular calcium as a result of ischaemia and reperfusion may have deleterious effects at many levels including inappropriate activation of degradative enzymes and changes to mitochondrial function which may ultimately lead to the cell and tissue death.
Many phospholipases which calalyse hydrolysis of phospholipids are activated by calcium. Products arising from phospholipase activity include free fatty acids such as arachidonate and lysophospholipids which may have a detergent effect on biological membranes. Others such as diacylglycerol and inositol 1,4,5-trisphosphate produced by phospholipase
C activity are powerful signalling agents (Burke et al., 2008). For example, myocardial ischaemia reperfusion injury in mouse infarct model was significantly reduced in a strain where the sPLA(2)-X gene was knocked out (Fujioka et al., 2008). Also, inhibition of phospholipase C in an isolated heart (Langendorff) model significantly improved post ischaemic cardiac recovery (Asemu et al., 2004). Recently, in a transplantation context, increased serum phospholipase A2 activity in a non-heart-beating model
29 of liver transplantation was associated with an increase in ischaemia reperfusion injury (Monbaliu et al., 2009).
Calpains are a family of cysteine proteases activated by micromolar and millimolar concentrations of calcium (Goll et al., 2003). They are considered to be distributed in the cytosol, however, may be associated with organellar membranes (Molinari et al., 1997). Recent studies also demonstrated the existence of a mitochondrial form of calpain (Badugu et al., 2008).
A likely mechanism for Ca – induced calpain cell injury was recently proposed (Syntichaki et al., 2002). A rise in intracellular calcium by any means initiates cytosolic calpain activation. Activated calpain then may localise to lysosomal membranes leading to lysosomal rupture and release of cathepsin, another powerful protease as well as an array of lysosomal hydrolases (Yamashima, 2000). Calpain may in turn initiate caspase activation and inactivation of key metabolic pathways in the cytosol and mitochondria resulting in cell death (Ferri et al., 2001, Norberg et al.,
2008). Calpain activation and mitochondrial dysfunction has been shown to be an important component of myocardial ischaemia reperfusion injury
(Chen et al., 2002). Other targets for activated calpain in myocardial ischaemia reperfusion include the sarcoplasmic reticulum and the sodium potassium – ATPase (Inserte et al., 2005, Singh et al., 2004).
30 During reperfusion, the increase in mitochondrial membrane potential and accumulated Ca2+ during ischaemia can activate the mitochondrial permeability transition pore (mPTP) when extracellular pH is being restored to normal. This phenomenon can result in irreversible damage to the mitochondria, cessation of ATP production, contractile dysfunction and cell death by necrosis or apoptosis (Crompton, 1999, Lemasters et al.,
1998). The consequence of ionic alterations during ischaemia and reperfusion appears to be correlated with the rise in cytosolic and/or mitochondrial Ca2+ (Steenbergen et al., 1990), especially the increase of
Ca2+ at the start of reperfusion.
Changes in cellular Ca2+ homeostasis under pathological conditions can contribute to various forms of cell death (Dong et al., 2006). There are two common forms of cell death with distinct morphological and biochemical features – apoptosis and necrosis (See table 1.1). Apoptosis is a tightly regulated, energy dependent process in which cell death is characterized by cell shrinkage, nuclear condensation, chromatin fragmentation
(Earnshaw et al., 1999, Nicholson, 1999). Necrosis is a passive form of cell death associated with inflammation resulting form cellular and organelle swelling, rupture of plasma membrane and spilling of cellular contents (Van Cruchten et al., 2002). Despite the differences between apoptosis and necrosis, there is strong evidence to suggest that both of these are interrelated (Leist et al., 2001).
31 Table 1.1 Summary of features between Necrosis and Apoptosis
Necrosis Apoptosis Membrane blebbing, no loss in Loss of membrane integrity intergrity Shrinkage of cytoplasm, condensed Swelling of cytoplasm nucleus No energy requirement Energy dependent (ATP) process
Inflammation No inflammation
Non random oligonucleosomal Random DNA degradation fragmentation of DNA Mitochondria swelling Mitochondria permeabilisation
No vesicle formation due to Formation of vesicles (apoptotic lysis bodies) Fragmentation of cells into smaller Total cell lysis bodies
Examples of these inter-relationships are exemplified in the sharing of signalling and regulatory mechanisms involving mitochondrial disruption
(Nieminen, 2003). Both of these processes can be induced by Ca2+ overload (Orrenius et al., 2003) and preservation of mitochondrial membrane integrity through Bcl-2 and Bcl-XL overexpression blocks both apoptosis and necrosis (Imahashi et al., 2004, Weisleder et al., 2004). In addition, there are data showing that overexpression of Bcl-2 led to a decrease in the the rate of ATP loss as well as reduction in acidification during ischaemia; the effects of metabolic protection by Bcl-2 appeared to be due to the decreased ATP uptake in the mitochondria by the F1F0-
ATPase (Imahashi et al., 2004). The factors contributing to the precise mode of cell death are not well defined and may depend on the intensity
32 and duration of the insults. However, under circumstances when ATP is absent, cells undergoing apoptosis will die by necrosis (Leist et al., 1997).
The mPTP (mitochondrial permeability transition pore) is a non-selective pore, is permeable to molecules with a size limit of ≤1.5kDa (Crompton et al., 1987), may form in the mitochondrial inner membrane under conditions of Ca2+ overload especially in the presence of oxidative stress (Crompton,
1999, Halestrap et al., 2004). Although mPTP induces the opening of the inner mitochondrial membrane, there are studies suggesting that it can also lead to pathological alterations in the outer membrane (Bernardi,
1999, Lemasters et al., 1998). Crompton and colleagues have shown that cyclosporine A inhibits the development of mPTP opening (Crompton et al., 1988), which acts by reducing the calcium sensitivity in the inner mitochondrial membrane instead of total inhibition (Halestrap et al., 1997). mPTP opening is associated with proteins like ANT (adenine nucleotide transporter), mitochondrial phosphate carrier, cycophilin D and VDAC
(voltage depdendent anion channel) (Crompton, 1999, Leung et al., 2008)
The use of an inhibitor of ANT such as BKA (bongkrekic acid) can decrease the sensitivity of the mPTP opening to Ca2+ (Halestrap et al.,
1990, Novgorodov et al., 1991).
The strong association between mPTP opening and cell death during IR injury has been extensively studied during the past decade (Di Lisa et al.,
2006, Duchen et al., 1993, Halestrap et al., 2004, Lemasters et al., 1997).
33 During ischaemia, the mitochondria in the cardiomyocytes are unable to produce ATP via oxidative phosphorylation due to the lack of oxygen and this leads to a considerable drop in ATP/ADP levels, adenine nucleotide depletion, lactic acid build up, a drop in intracellular pH, which then results in reversal of the Na+/Ca2+ and leads to an influx of Ca2+ at the same time (section 1.3.3.). This increase in intracellular Ca2+ can activate the mPTP but due to the low pH within the cell, the opening of the mPTP is inhibited and is only activated upon reperfusion when pH returns to normal through the loss of lactic acid (Di Lisa et al., 2003, Murphy et al., 2008).
There are defence mechanisms for the removal of excess ROS within the cell, however, during reperfusion these protective mechanism maybe severely impaired and lead to oxidative stress as these ROS accumulate in turn having adverse effects on mPTP (Becker, 2004, Orrenius et al.,
2007). In addition, the burst of ROS activity can have further inhibitory effects on the activity of the ion channel pumps and cause imbalance to the ionic activities within the cell by further reducing ATP levels (Hool,
2006).
The cause of mPTP opening induced apoptosis and necrosis during ischaemia and reperfusion can be attributed to the uncoupling of the mitochondria that occurred as a result of the increased permeability to H+
(protons). Mitochondria are then unable to synthesize ATP via oxidative phosphorylation (Halestrap, 2006, Halestrap et al., 2009). In addition to uncoupling of the mitochondria, the increased in permeability of the inner
34 mitochondria matrix leads to a massive swelling of the compartment as small molecules equilibrates across the inner membrane and results in increase in water that causes swelling of the mitochondria. This is followed by rupturing of the outer membrane of the mitochondria leading to loss of inner membrane components such as cytochrome c and AIF. The release of cytochrome c causes the activation of other pro-apoptotic proteins and caspases that lead to apoptotic cell death. Depending on the extent and severity of the opening of the mPTP, apoptotic cell death will predominate over necrotic cell death if the mPTP can maintain sufficient amount of ATP levels or in the case where the mitochondria is severely depleted of ATP, cell death will proceed via necrosis (Figure 1.5).
1.3.4.6 Reperfusion associated changes to the endothelium and
microcirculation
The vascular endothelium, as well as the cardiac myocytes suffer injury as a result of ischaemia and reperfusion and may be the major locus for initiation of neutrophil activation immediately post reperfusion (Lefer et al.,
1991). Under normal physiological conditions, the endothelium synthesises the vasodilator compounds prostacyclin (Armstrong et al.,
1978) and nitric oxide (Loscalzo et al., 1995).
Once it is synthesised in the endothelium, nitric oxide may rapidly diffuse into the adjacent smooth muscle layer or the lumen of the blood vessel. In
35 Stress to cell, e.g. Ischaemia/reperfusion
Mitochondrial Ca2+ overload and oxidative stress
Mitochondrial permeability Mitochondrial transition pore opens uncoupling
Mitochondrial swelling and outer membrane rupture
Release of cytochrome c
Activation of caspases Moderate insult, Severe insult, pores reseal pores open Extent of permeability mPTP pores close, ATP transition may mPTP pores open, production maintained determine whether cell ATP depleted death is necrotic or apoptotic following ischaemia and reperfusion APOPTOSIS NECROSIS
Figure 1.5 Overview of mPTP opening in apoptosis and necrosis. The extent and reversibility of mPTP opening may determine whether a cell dies by apoptosis or necrosis. Modified from (Halestrap, 2006).
36 the smooth muscle cells it binds to and activates guanylate cyclase producing a rise in cyclic GMP which initiates a cascade of reactions leading to vasodilatation. In the lumen of the blood vessel, nitric oxide may minimise platelet aggregation (again by a guanylate cyclase catalysed reaction) or may act back on the luminal wall of the endothelial cells to prevent neutrophil adhesion (Lefer et al., 1996).
Microvascular ischaemia - reperfusion injury was initially thought to involve an “endothelial trigger” and a “neutrophil amplification” step (Ratych et al.,
1987). Within minutes of reperfusion, release of nitric oxide from the endothelium is compromised (Tsao et al., 1990). Decreased nitric oxide in the vascular smooth muscle will result in vasoconstriction. At the same time, luminal levels of nitric oxide fall, which leads to appearance of the major families of adhesion molecules, the selectins, beta2 integrins and the immunoglobulin superfamily (Lefer et al., 1996). Appearance of these molecules on the luminal surface of the blood vessels attracts and activates white cells resulting in physical plugging of the microvasculature within the reperfused organ, termed the “no reflow” phenomenon (Rezkalla et al., 2002). The longer term development of the highly debilitating processes of chronic allograft vasculopathy and rejection can also in part be traced back to ischaemia – reperfusion induced endothelial cell
“activation” resulting in adhesion molecule production and luminal recruitment of a range of inflammatory cells (Al-Lamki et al., 2008,
Belperio et al., 2008).
37 1.4 PRESERVATION OF THE DONOR HEART
1.4.1 The need for donor heart preservation
The previous sections have detailed a number of damaging consequences of ischaemia reperfusion damage. Apart from the insult following donor brain death, the donor heart, of necessity undergoes a sequence of ischaemia – reperfusion related events:
(i) an extended period of cold ischaemic storage after donor heart
procurement when the donor heart is transported to the
recipient;
(ii) a short period of warm ischaemia during implantation of the
donor heart into the recipient and;
(iii) reperfusion of the donor heart when the recipient blood flow is
established through the donor heart.
Ischaemic storage times were minimised by necessity in early clinical transplants as the donor and recipient procedures were performed in adjacent operating theatres in the same institution. With the advent of cadaveric organ utilization and distant organ procurement, development of specialised storage solutions to minimise storage and reperfusion injury was essential. Minimisation of such injury is particularly important for the heart as it must do active work to support the recipient circulation.
38 1.4.2 Major elements of heart preservation
1.4.2.1 Immediate mechanical arrest of the heart
Ischaemia rapidly depletes high energy phosphates in the myocardial cells that initiates a series of biochemical, ionic and morphological processes resulting in irreversible tissue injury. Early strategies employed the use of rapid induction of asystole to minimize electromechanical activity in the heart and conserve myocardial energy reserves (Hearse et al., 1976,
Nelson et al., 1976). It was shown that this approach resulted in improved post-ischaemic functional recovery. Agents used to induce rapid asystole include potassium, calcium channel blockers and anaesthetic agents
(Lowe et al., 1977). Other arresting strategies also include the use of hyperpolarizing agents such as ATP sensitive potassium channel openers
(Lawton et al., 1997) and adenosine (Jovanovic et al., 1998).
Of all the options, potassium is the most commonly used arresting agent in preservation solutions used clinically. However, the high potassium concentrations commonly used are not without problems. Its use may lead to endothelial damage in the donor heart, graft dysfunction and episodes of chronic rejection (Parolari et al., 2002, Young, 2000). Other investigators have also shown that high potassium concentration can lead to coronary vasoconstriction affecting cardioplegia delivery (Leicher et al.,
1983), cause damage to vascular endothelium (Ruel et al., 2004), and lead to arrhythmias and myocardial stunning post reperfusion (Ellis et al.,
39 1980). Indeed, it has been recently pointed out that “depolarising potassium has no intrinsic cardioprotective properties beyond inducing cardioplegic arrest” (Rudd et al., 2009).
A recent study investigated a normokalemic nondepolarizing cardioplegic solution consisting of simple Krebs solution supplemented with adenosine and lignocaine. Recovery of isolated rat hearts arrested in this “AL” solution was compared to hearts arrested and stored in the commercially available Celsior solution. Results showed that hearts arrested and stored in AL solution demonstrated significantly improved cardiac output during reperfusion over those stored in Celsior solution at both 4oC and warmer arresting temperatures of 28oC and 30oC (Rudd et al., 2009). This approach may in time provide a viable alternative to the use of potassium
– based depolarising cardioplegia, especially as both adenosine and lignocaine have cardioprotective properties over and above their ability to arrest the heart (Cohen et al., 2008, Hinokiyama et al., 2003).
1.4.2.2 Hypothermia
Along with rapid arrest, cooling to temperatures ranging from 4oC to 6oC has been a long-standing principal element in successful preservation of the heart (Childs et al., 1969). The current acceptable cold ischaemic storage period for hearts in the clinical context is routinely between 4-6 hours compared to 24 - 48 hours for abdominal organs such as liver and kidney (Agarwal et al., 2006, Mangus et al., 2006). Cardiac preservation
40 times beyond 4 hours are associated with increased rate of primary graft failure and increased risk of mortality (Hosenpud et al., 2001, Taylor et al.,
2007).
The mechanism of the cardioprotective effects of hypothermia are centred on the reduction in metabolic rate and reduced oxygen consumption by the myocardial cells in the heart resulting in minimization of ischaemic and post-ischaemic injury to the myocardium. Hypothermia slows the metabolic rate in the organ or tissue but does not arrest metabolism completely
(Buckberg, 1991). According to the van’t Hoff equation, the rates of oxygen consumption, as well as all enzyme catalysed reactions and biological processes decrease by approximately 50% for every 10oC reduction in temperature (Jahania et al., 1999). In isolated heart preparation the greatest recovery of post-ischaemic function in the hearts was previously shown to be between 4 and 24oC with a rapid drop off in protection above 24oC (Hearse et al., 1976). Oxygen demands and preservation of high energy phosphate stores followed a similar temperature relationship (Buckberg et al., 1977, Rosenfeldt, 1982).
Although the benefits of profound hypothermia (< 24oC) maybe more beneficial in the context of prolonged ischaemia, these benefits may come at a cost. The major problems are i) intra-cellular swelling, ii) intracellular sodium accumulation; iii) derangements in calcium homeostasis and iv) intracellular acidosis. All these changes have their basis in decreased
41 metabolic rate and have been discussed in earlier sections. Increased cytokine production and the development of a pro-inflammatory phenotype have also been observed in cardiac tissue after cold storage (Jahania et al., 1999). Strategies used to minimise these problems will be discussed in later sections.
1.4.2.3 Minimisation of ischaemia reperfusion injury
Another key factor in the formulation of modern cardiac preservation solutions is the minimisation of the damaging effects of ischaemia- reperfusion. As will be detailed in subsequent sections, the evolving knowledge base of both ischaemia-reperfusion injury and more especially in the area of molecular events in preventing such damage has facilitated development of new protective strategies.
Inclusion of substrates for energy production: The importance of supplementation of amino acid substrates such as glutamate and aspartate has been highlighted (Arsenian, 1998). Both may provide substrates for the TCA cycle, glutamate by transamination to alpha- ketoglutarate and aspartate by conversion to oxalacetate, entering the
TCA cycle at the level of succinate (Sanborn et al., 1979).
Supplementation of blood cardioplegia with both glutamate and aspartate was shown to increase oxygen uptake in the myocardium during induction of cardioplegia and improve post-ischaemic functional recovery and
42 oxygen utilization in a canine simulated cardiac surgery model
(Rosenkranz et al., 1986) and later in a canine model of donor heart procurement after brain death (Tixier et al., 1991). More importantly, amino acid supplementation has been successfully employed in the clinical arena with aspartate – enriched St Thomas solution (Richens et al., 1993) and incorporation of glutamate into Celsior solution. Both approaches will be discussed in greater detail in section 1.4.3.
Maintenance of appropriate pH with buffering agent: Tissue acidosis during ischaemia occurs as a result of lactic acid production and metabolic activity which will further reduce ATP levels and lead to cellular injury
(section 1.3.2). Buffering agents such as histidine, bicarbonate, and phosphate are added exogenously to cardioplegic solutions to maintain the pH of the myocardial cells by counteracting tissue acidosis arising from hydrogen ion accumulation (del Nido et al., 1985, McConnell et al., 1975).
Studies have demonstrated good preservation of high energy phosphates stores and post-ischaemic functional outcome (del Nido et al., 1985), as well as maintenance of interstitial pH and improved post-cardioplegic contractile recovery with these buffers (Warner et al., 1988) .
Minimisation of cell swelling: Cell swelling or tissue oedema occurs during cold hypothermic ischaemic storage as the Na+/K+ ATPase is inhibited, allowing the entry of sodium and chloride ion into the cell followed by entry of water molecules passively at the same time to equilibrate the
43 intracellular and extracellular osmotic pressures (Belzer et al., 1988). The use of membrane impermeants such as the non-metabolisable monosaccharide, mannitol to prevent such damage may be traced back to early studies by Leaf and colleagues who showed that mannitol improved coronary blood flow and myocardial function in the ischaemic myocardium
(Powell et al., 1976, Willerson et al., 1972). The supplementation of preservation solutions with mannitol may have 2 advantages. Firstly, the effectiveness of mannitol lasts for at least 6 hours in cold storage which is well within the limits of the current ischaemic time for hearts in clinical heart transplantation; secondly, mannitol is a free radical scavenger and is able to remove the excess oxygen free radicals accumulated in the cell during cold ischaemic storage and reperfusion (Ferreira et al., 1989,
Maathuis et al., 2007). The same may apply to other polyhydroxylated sugars in clinical use as impermeants such as hydroxyethyl starch and raffinose. Examples of other impermeable substances used in preservation solutions are dextran, lactobionate and gluconate, (Conte et al., 2000).
Prevention of calcium overload: Calcium overload is a major complication during ischaemia and reperfusion injury that impairs post-ischaemic recovery and lead to myocardial cell death (section 1.3.4.3), which has also previously reported in hearts during cold storage (Hendry et al., 1991,
Kohno et al., 1987). The composition of preservation solutions can be manipulated by several different approaches to minimise the influx of
44 calcium in the myocardial cells. These include: (i) lowering of potassium concentration to minimise the depolarising conditions which elicits a rise in cytosolic free calcium (Steenbergen et al., 1990); (ii) maintenance of sodium concentration in the extracellular compartment of the cell to prevent calcium entry via the Na+/Ca2+ exchanger (section 1.3.4.3); (iii) the inclusion of magnesium to compete with calcium in the sarcolemmal and intracellular compartments of the cell (Geffin et al., 1989). Bernard and colleagues (Bernard et al., 1985), assessed the effect of extracellular pH on the cold, potassium-arrested heart. They found that hearts arrested and stored in slightly acidic (pH 7) preservation solutions had higher creatine phosphate and ATP levels at the end of the cold ischemic period and had a faster recovery of contractile function post reperfusion. They asserted that the accompanying mild intracellular acidosis contributed to a minimised metabolic load during cold ischaemia resulting in decreased intracellular calcium load.
Minimisation of oxidative damage: As described earlier, reactive oxygen
- species such as the superoxide radical (O2 •) and hydrogen peroxide
(H2O2) contribute to the exacerbation of myocardial damage during reperfusion of previously ischaemic tissue (McCord, 1985). Endogenous protective enzymes such as superoxide dismutase, were shown to lose activity during ischaemia (Gauduel et al., 1984). Proof of principle studies initially demonstrated that supplementation of St Thomas solution N° 2 with exogenous superoxide dismutase and catalase improved post-
45 ischaemic cardiac recovery following (normothermic) arrest (Chambers et al., 1987a). During reperfusion xanthine oxidase is a major source of ROS production (superoxide, hydrogen peroxide and the hydroxyl radical by iron catalysed Fenton chemistry, Figure 1.4) (Huang et al., 2002,
Kuppusamy et al., 1989). Free iron is a major cause of mitochondrial damage and its presence may induce apoptosis during hypothermia
(Lemasters, 2004).
The pro-drug allopurinol and its active metabolite oxypurinol are specific inhibitors of the xanthine oxidase enzyme (Day et al., 2007). The potential damaging role of oxidants derived from this source was emphasised by the finding that supplementation of St Thomas solution N°2 with either allopurinol or oxypurinol enhanced the cardioprotective effect of St
Thomas solution alone in a normothermic isolated working rat heart model of cardiopulmonary bypass and cardiac arrest (Chambers et al., 1987b).
Allopurinol is an important element in the commercially available University of Wisconson solution (Section 1.4.3). Another important endogenous
“anti-oxidant” is the tripeptide, glutathione. Studies have shown the importance of GSH (glutathione) playing a key role in the defense of tissues against oxidative damage that is based on the supply of thiol (SH) compounds to other enzymes (Biguzas et al., 1990, Ceconi et al., 1988).
The antioxidative effects of GSH are due to the following: (i) it can function as a co-factor for enzymic removal of oxidative species (glutathione peroxidise); (ii) it is used by thiol transferases to maintain protein bound
46 thiol groups in a reduced state, often a prerequisite for other enzymes to remain active and; (iii) direct scavenging of free radicals (Ceconi et al.,
1988, Singh et al., 1989). The importance of maintaining levels of the reduced form of glutathione was highlighted by studies from Hearse’s laboratory. They demonstrated that supplementation of St Thomas solution N° 2 with either 1 or 10 mM GSH improved post ischaemic cardiac function compared to hearts arrested in St Thomas solution alone in an isolated working rat heart model of cardiac arrest and cardiopulmonary bypass (Chambers et al., 1989a). GSH is currently used in preservation solutions such as Celsior (Section 1.4.3).
1.4.3 Development of current cardioplegic / preservation solutions
Many different myocardial preservation solutions have been developed and are in use for clinical heart transplantation. In one survey, it was reported that at least 167 different types of preservation fluids were used for heart transplantation in the United States (Demmy et al., 1997). This in itself is a reflection of the current uncertainties regarding the optimal strategy for myocardial preservation. Some for example Bretschneider solution (HTK, Custodial), Celsior, St Thomas solution (STS, Plegisol), and
University of Wisconsin solution (UW, Viaspan) are commercially available solutions, but many are locally produced non-commercial solutions
(Demmy et al., 1997, Richens et al., 1993). In some centres, the same solution is used for both flush (cardioplegia) and storage, whereas other centres have elected to use separate solutions for initial cardioplegia and
47 subsequent cold storage and transport of the cardiac allograft. With currently available preservation solutions, the maximum recommended storage time for cardiac allografts is approximately 6 hours.
Preservation solutions differ in both their electrolyte composition and additives and can be divided into two broad categories, extracellular and intracellular, based on their Na+ and K+ concentrations. Preservation solutions that mimic extracellular fluid contain a high Na+ concentration (>
70 mmol/L) and a K+ concentration in the range 5-30 mmol/L. Preservation solutions that mimic intracellular fluid contain a low Na+ concentration (<
70 mmol/L) and K+ concentration in the range 30-125 mmol/L. Examples of intracellular and extracellular preservation solutions are shown in Table
1.3. Celsior (Na+ 100 mM, K+ 15 mM) and UW solution (Na+ 30 mM, K+
120 mM) are examples of extracellular and intracellular preservation solutions which have been used for clinical heart transplantation
(Drinkwater et al., 1995, Remadi et al., 2002, Wildhirt et al., 2000).
The primary rationale for the development of intracellular preservation solutions is that the presence of similar concentrations of Na+ and Cl- in the intracellular and extracellular compartments minimizes the passive fluxes of these ions into the cell (and hence cell swelling) during hypothermia.
48 Table 1.2 Composition of some commercial preservation solutions.
Component UW Col HTK ST2 Celsior
Ionic Compostion Na+ (mmol/l) 30 10 10 120 100 K+ (mmol/l) 120 115 10 16 15 Cl- (mmol/l) 0 15 50 203 41.5 Mg2+ (mmol/l) 5 30 4 16 13 Ca2+ (mmol/l) 0 0 0.015 1.2 0.25
Acid-Base Buffers Bicarbonate (mmol/l) 0 10 0 10 0 Phosphate (mmol/l) 25 57.5 0 0 0 Sulphate (mmol/l) 4 30 0 0 0 Histidine 0 0 180 0 30
Impermeants Lactobionate (mmol/l) 100 0 0 0 80 Raffinose (mmol/l) 30 0 0 0 0 Hydroxyethyl starch (g/l) 50 0 0 0 0 Dextran 40 (g/l) 0 0 0 0 0 Mannitol (mmol/l) 0 0 30 0 60 Glucose (mmol/l) 0 140 0 0 0
Metabolic Agents Adenosine (mmol/l) 5 0 0 0 0 Glutamate (mmol/l) 0 0 0 0 20 Ketoglutarate (mmol/l) 0 0 1 0 0 Tryptophan (mmol/l) 0 0 2 0 0
Anti-Oxidants Glutathione (mmol/l) 2 0 0 0 3 Allopurinol (mmol/l) 1 0 0 0 0
pH 7.4 7.1 7.2 7.8 7.3
Osmolality (mOsm/l) 320 350 310 324 360
UW = University of Wisconsin
Col = Collins Solution
HTK = Histidine-Tryptophan-Ketoglutarate
ST2 = modified St. Thomas Hospital Solution 2
49 Another potential advantage of intracellular solutions is that the high K+ concentration in the preservation solution facilitates cardiac arrest while the low Na+ concentration reduces the drive for the Na-H exchanger. A significant concern with intracellular preservation solutions, particularly with regard to myocardial preservation, is the potential for high K+ concentrations to cause coronary endothelial cell injury. This is a controversial issue as there is contradictory experimental evidence
(Chambers et al., 1999, Sorajja et al., 1997, Q. Yang et al., 2004). Several investigators have noted that UW solution (an intracellular preservation solution) provided excellent endothelial cell preservation at 4°C, but caused endothelial injury at higher temperatures (Ou et al., 1999, von
Oppell et al., 1990). This observation suggests that if UW preservation solution is used to preserve the donor heart, it should be completely rinsed from the heart before any cardiac rewarming occurs at the time of implantation.
A further limitation of hyperkalaemic preservation solutions, whether intracellular or extracellular, relates to their depolarizing action, which may result in continuing transmembrane fluxes and the consequent maintenance of high energy phosphate metabolism, even during hypothermic ischaemia (Chambers et al., 1999). A potentially beneficial alternative to hyperkalemic cardioplegia for myocardial protection (as discussed earlier) is to arrest the heart in a "hyperpolarized" or "polarized" state, which maintains the membrane potential of the arrested
50 myocardium at or near to the resting membrane potential. At these potentials, transmembrane fluxes will be minimized and there should be little metabolic demand, resulting in improved myocardial protection
(Chambers et al., 1999, Dobson et al., 2004, Rudd et al., 2009). The use of compounds such as adenosine or ATP – sensitive potassium channel openers, which are thought to induce hyperpolarized arrest, have demonstrated improved protection after normothermic, or short periods of hypothermic, ischemia when compared to hyperkalemic (depolarized) arrest. Similarly, studies in which the sodium channel blockers, tetrodotoxin and lignocaine, were used to induce polarized arrest
(demonstrated by direct measurement of membrane potential during ischemia) was also shown to provide better recovery of function after long- term hypothermic storage (Chambers et al., 1999, Dobson et al., 2004,
Rudd et al., 2009). The combination of adenosine with lignocaine in the same cardioplegic solution, as proposed by Dobson and colleagues, has been shown to dramatically enhance myocardial protection during both normothermic and hypothermic ischaemia (Dobson et al., 2004, Rudd et al., 2009).
The following preservation solutions - Collins Solution, St. Thomas
Hospital Solution, University of Wisconsin (UW) Solution, Histodine-
Tryptophan-Ketoglutarate (HTK) and Celsior solution that have been employed in clinical heart transplantation (Jamieson et al., 2008, Yang et al., 2005) are evaluated below.
51 Collins Solution: This solution was initially developed for kidney preservation (Collins et al., 1969). Its formulation was later modified by the
Eurotransplant organization [Eurotransplant Foundation Annual Report,
1976] by taking out the magnesium supplementation to allow its use for heart preservation and was named the Eurocollins solution. Eurocollins solution is an intracellular crystalloid which contains strong phosphate buffer and glucose.
Experience with Eurocollins solution in clinical heart transplantation is limited. Konertz and colleagues reported a single centre series of 26 heart transplants over a period of approximately 18 months with an 89% survival over that period with no follow-up data (Konertz et al., 1988). Functional data at 1 year post transplant in a group of 30 patients whose donor hearts were stored in Eurocollins solution were reported to be equal to that of recipients whose hearts were stored in St Thomas solution (Weyand et al.,
1992).
Experimental studies have raised some concerns about using such intracellular based solutions for initial induction of cardiac arrest (Kohno et al., 1987). Another study showed that post-ischaemic recovery of function of hearts stored in Eurocollins solution was improved by flushing with extra-cellular based solution immediately before reperfusion (Toshima et al., 1992).
52 The major disadvantage with using this solution for long cold ischaemic preservation periods is that the added glucose will be a substrate for glycolysis with resultant lactate accumulation (Section 1.3.2.2) which will lead to tissue swelling, macrophage activation, cytokine production and initiation of inflammation (Jahania et al., 1999, Muhlbacher et al., 1999).
St. Thomas Hospital Solution (ST): St Thomas solution was initially developed as an extracellular based cardioplegic solution for open-heart surgery. Its development evolved from a landmark study in the isolated rat heart (Hearse et al., 1976). This study was the first to systematically evaluate and optimise a multi-component solution specifically designed to address the major issues thought to be important in preservation of the heart during surgery, i.e. rapid arrest and effective protection during ischaemia. This study demonstrated that the protective effects of potassium (the arresting agent), magnesium, procaine ATP and creatinine phosphate (to minimise ischaemic damage) were additive.
On the basis of these findings, cold cardioplegia with “St Thomas solution
N°1” was instituted for clinical cardiac surgical use at St. Thomas Hospital in 1975, with the initial report of its efficacy in 1977 (Braimbridge et al.,
1977). This study demonstrated that a 2 minute infusion of cold St Thomas
N°1 provided protection for 90 minutes. However, this solution was not ideal for extended periods of cold ischaemia, as the calcium level was relatively high (2 mM) and the solution was unbuffered. A series of further
53 studies to further optimise component composition and concentrations, resulted in the development of St Thomas solution N°2, marketed as
“Plegisol” by Abbott Laboratories in 1982 (Hearse, 1980). Differences between solutions N° 1 and N° 2 were i) the omission of procaine; ii)
Reduction of Ca concentration to 1.2 mM; iii) the inclusion of 10 mM sodium bicarbonate as a buffering agent; iv) A decreased potassium concentration (16 mM) (see Table 1.3).
The efficacy of this new formulation was first demonstrated in combination with hypothermia in the isolated rat heart and in situ canine heart (Jynge et al., 1981) and then its clinical usefulness demonstrated in series of 28 patients undergoing open heart surgery at St Thomas Hospital (Chambers et al., 1989b). St Thomas solution N° 2 provided adequate myocardial protection for periods of ischaemia ranging from 44 – 135 minutes.
A trial of the use of St Thomas solution N°1 in clinical donor heart preservation was begun in 1989 (Demertzis et al., 1993). In this protocol,
St Thomas was simply used as the cardioplegic solution, with hearts being stored in Ringer’s lactate solution. The major finding of this study was that cardiac preservation with UWS or St Thomas solution was equivalent for cold ischaemic times up to 4 hours. A survey of clinical outcomes of donor hearts arrested and stored by a range of approaches found that the use of cardioplegic solutions such as St Thomas solution for storage was associated with a 2.5 times increase in recipient deaths compared to
54 recipients receiving hearts stored in saline (Wheeldon et al., 1992). The experimental demonstration that significant post-ischaemic myocardial oedema was caused by the high chloride levels in St Thomas solution is in line with these clinical findings (Drewnowska et al., 1991).
University of Wisconsin (UW) Solution: This is an intracellular based solution first developed in 1986 for pancreas transplantation (Wahlberg et al., 1986). UW solution was also the result of a rational approach to design of organ preservation solutions. It contains components such as allopurinol, an inhibitor of xanthine oxidase and reduced glutathione to minimise oxidant damage at reperfusion, the colloid hydroxyethyl starch to prevent interstitial oedema and lactobionate and raffinose to prevent intracellular oedema. UW was found to have decreased the rate of delayed graft function and have proven excellent preservation results for cold storage of kidney, liver and pancreas and remains the gold standard for multi-organ retrieval (Muhlbacher et al., 1999). Efficacy of UW solution in heart and lung preservation was demonstrated experimentally (Rinaldi et al., 1995, Swanson et al., 1988) and its usefullness in clinical heart transplantation was first demonstrated in 1990 (Stein et al., 1991).
Interestingly, there is some experimental evidence and anectodal clinical evidence that, not withstanding the potential endothelial damage arising from its high potassium concentration, UW solution may be particularly efficacious for donor hearts exposed to extended periods of cold ischaemia (Kajihara et al., 2006). This may be because allopurinol also
55 enhances the rate of recovery of post ischaemic adenine nucleotide recovery in the post-ischaemic heart by enhancing salvage of hypoxanthine (Lasley et al., 1988).
Histidine-Tryptophan-Ketoglutarate (HTK): HTK solution was developed by
H.J. Brettschneider in Germany where it was employed as a cardioplegic solution from 1980 (Bretschneider, 1980). The formulation of HTK includes several potential protective features: i) the buffering agent is histidine, a particularly efficient buffer at low temperatures (Bernard et al., 1985); ii) low calcium and chloride concentrations; iii) inclusion of a non- metabolisable impermeant (mannitol). Clinical use of HTK in preservation has shown good results in liver, kidney and pancreas (Englesbe et al.,
2006, Groenewoud et al., 1994, Pokorny et al., 2004). Its use has also been demonstrated in heart and lung, although clinical usage is limited in this context (Agarwal et al., 2008, Human et al., 1993, Luh et al., 2004,
Reichenspurner et al., 1993, Saitoh et al., 2000). HTK has been compared with UW during clinical trials of multi-organ preservation and has been shown to be as effective in decreasing the rate of delayed graft function and more cost effective than UW solution (Agarwal et al., 2006, Agarwal et al., 2008, Mangus et al., 2006, Potdar et al., 2004).
Celsior solution: The previous preservation solutions evaluated for donor heart usage were either primarily developed for preservation of abdominal organs (Eurocollins, UW or HTK solutions) or for short-term cardiac stand-
56 still in open heart surgery (St Thomas solution). Celsior is an extracellular type solution that was developed specifically for heart preservation. It had its origins in 1993 report by Menasche and colleagues using 2 solutions
(Menasche et al., 1993). The first solution arrested the heart and the heart remained in this solution during the period of hypothermic storage after which it was flushed out and replaced by a second solution which served as a modified reperfusate.
Further refinements resulted in the combination of all the protective elements of the 2 solutions into a single formulation - Celsior (Menasche et al., 1994), which was designed to be used not only as a storage medium but also as a perfusion fluid during initial donor heart arrest, graft implantation and reperfusion.
The formulation of Celsior was based on the minimisation of the elements of ischaemia reperfusion damage introduced earlier. Included are: i) The impermeants mannitol and lactobionate, the omission of which significantly decreases the efficacy of UW solution (Sumimoto et al., 1990). ii) The antioxidant reduced glutathione to prevent oxygen derived free radical injury during reperfusion. Of particular relevance to the heart may be the ability of exogenous GSH in modulation of intra-mitochondrial calcium levels (Beatrice et al., 1984). Two other agents also possess some forms of anti-oxidant activity. Mannitol is a highly efficient scavenger of the hydroxyl radical, HO• and histidine is highly reactive towards singlet
57 excited oxygen, thought to inhibit myocardial Na+/K+ ATPase (Vinnikova et al., 1992). iii) Inclusion of glutamate may have a number of beneficial effects including a) enhanced post-reperfusion mitochondrial ATP production, through its ability to act as a substrate for α-ketoglutarate formation and its ability to increase malate-aspartate shuttle, b) decreased myocardial ammonia production, and c) decreased post ischaemic lactate levels (Arsenian, 1998). iv) Histidine as a buffering agent (discussed in previous section). v) The relatively high magnesium concentration, low calcium level and slightly acid pH was designed to minimise post- reperfusion calcium overload (Poole-Wilson et al., 1979).
A multi-centre randomised trial of Celsior as an arresting and storage vehicle against “Standard” single flush and storage solutions demonstrated that Celsior was as safe and effective as the other solutions in routine use (Vega et al., 2001). Another trial where donor hearts were also arrested and stored in Celsior showed an operative mortality (up to 30 day post transplant) of 8.6%, with 90% of patients having satisfactory hemodynamic assessments on day 2 and good cardiac function as measured by echocardiograms. Actuarial 5 year survival was 75±5%
(Remadi et al., 2002). Both this study and a subsequent study comparing a population of recipients receiving “high-risk” donor hearts (i.e. at least 2 of: high donor age, female sex, high post-brain death inotrope support, size mismatch > 20%, cold ischaemic time >180 min) with recipients
58 receiving “standard allografts” remarked on the effectiveness of Celsior for preservation of the “at risk” donor heart (De Santo et al., 2006).
Many studies have shown that Celsior is also an effective preservation solution for use in kidney, pancreas and liver transplantation (Faenza et al., 2001, Manrique et al., 2006, Ohwada et al., 2002). Celsior has also shown potential as a multi-organ preservation solution (Karam et al.,
2005). Celsior solution is now used as a preservation solution for clinical heart and lung transplantation at St Vincent’s Hospital Sydney and as the
“baseline” preservation solution in studies described in this thesis.
1.4.4 Need to improve and extend cardiac preservation
The heart is particularly sensitive to hypothermic ischemic injury and storage beyond 4 hours of ischemic time has shown to be strongly associated with significant rise of primary graft failure and death post- transplantation at 1 and 5 years (Hosenpud et al., 2001, Taylor et al.,
2007, Taylor et al., 2004). Data collected over the past decade showed an ongoing significant mortality in the 30 day period post-transplant and is mainly attributed to primary allograft failure due to ischaemia-reperfusion injury (IRI) and inadequate cardiac preservation acquired during the transplant process (Figure 1.6).
Because of the ongoing shortage of donors, “marginal” quality donor hearts (i.e. hearts from donors in the older age group and hearts from
59 donors managed on high levels of catecholamines post brain death) are now often being considered for transplantation (Maathuis et al., 2007,
Taylor et al., 2007). Therefore improvements in managing the process of
IRI and the use of fresh approaches to cardiac preservation would increase the available pool of donor hearts and further improve the already excellent post-transplant outcomes, at the same time reducing the number of patients who die waiting for a donor heart whilst on the ever increasing waiting list.
100 All comparisons significant at p < 0.0001 80
60
1982-1991 (N=18,844) 40 1992-2001 (N=34,987)
Survival (%) 2002-6/2005 (N=9,459) 20 HALF-LIFE 1982-1991: 8.9 years; 1992-2001: 10.3 years; 2002-6/2005: NA 0 0123456789101112131415 YearsYears
Figure 1.6 Comparision of adult heart transplants survival by era (1982 –
2005). Area circled in pink shows the significant mortality in the 30 day period post-transplant. Major causes of mortality are 33.2% from infection; 41.8% from graft failure; 11.8% from multiple organ failure and 13.2% from other causes.
Adapted from (Taylor et al., 2007).
60 1.5 NOVEL PHARMACOLOGICAL AND PHYSIOLOGICAL
APPROACHES TO DONOR HEART PROTECTION
Since the development and evaluation of Celsior in the mid to late 1990’s further insights into the mechanism of ischaemia reperfusion damage have suggested new pharmacological approaches for myocardial preservation.
Three important findings were i) the central role of nitric oxide homeostasis at a number of levels to normalise cardiac function; ii) the implications of
IR – induced activation of the sodium hydrogen exchanger and iii) the emerging role of activation of poly (ADP-ribose) polymerase in ischaemia reperfusion damage.
At another level, the role of activation of adaptive signalling pathways in the various “conditioning” protocols (preconditioning and more lately postconditioning) have lead to further discovery of pharmacological agents which may activate these important protective pathways. Each will be discussed in turn below.
1.5.1 Nitric oxide (NO)
It is recognised that the maintenance of NO homeostasis is important to the maintenance of cardiac function both at the myocyte and the endothelial cell level. Nitric oxide is synthesised from substrates and co- factors including L-arginine, oxygen, tetrahydrobiopterin and NADPH by either: i) the endothelial isoform (eNOS), ii) the neuronal isoform, (nNOS)
61 or iii) the inducible (iNOS) isoform. Although these isoforms may be found in a variety of cell types, the presence of eNOS in endothelial cells is central to normal cardiac physiology through its continual low-level formation of NO (Loscalzo et al., 1995).
The role of NO modulating the severity of ischaemia-reperfusion was also examined in non-preconditioned and preconditioned hearts (Bolli, 2001).
Bolli and colleagues (Bolli et al., 1997) were the first group to show that nitric oxide played a role in preconditioning against myocardial stunning in rabbit hearts. Since then several studies have demonstrated that the addition of exogenous NO mimics ischaemic preconditioning (IPC) (Hill et al., 2001, Takano et al., 1998, Zingarelli et al., 1995). The cardioprotective effects of IPC are discussed further in section 1.5.4.
Nitric oxide exerts its cardio-protective effects at a number of levels: i) NO is a powerful vasodilator that maximises perfusion of at-risk and injured myocardium (Ignarro et al., 1987). ii) NO reversibly inhibits the mitochondrial electron transport chain during the early minutes of reperfusion to minimise potential superoxide and ROS – induced damage
(Burwell et al., 2008). iii) NO is a strong inhibitor of adhesion molecule up- regulation and neutrophil adherence to vascular endothelial cells (Lefer et al., 1996). iv) NO prevents platelet aggregation (Azuma et al., 1986), which in concert with its anti-neutrophil effects will minimise post- reperfusion plugging of the micro-vasculature. v) NO may minimise post-
62 reperfusion apoptosis by S-nitosylation of a redox-active thiol group in the active site of caspase 3 (Maejima et al., 2005).
NO homeostasis during cardiac storage and reperfusion: Hypothermic ischaemic storage may severely compromise endogenous NO production by the effect of the low storage temperature on enzyme activity as well as the low availability of oxygen. Reperfusion after storage may lead to elevated levels of intracellular calcium, activation of eNOS and enhanced generation of NO which depletes the biopterin cofactor, BH4. This may result in uncoupling of NOS (Section 1.3.4.2 and Figure 1.4) with consequent formation of superoxide and peroxynitrite. Endothelial dysfunction which may follow was thought to be a major contributor to cardiac allograft vasculopathy after transplantation (Valantine, 2003).
Additives to cardioplegic solutions: The cardioplegic / storage solution provides the ideal vehicle for supplements which may augment endogenous nitric oxide production or modify its bioavailability.
i) Radical Scavengers: Rapid post-reperfusion removal of superoxide may increase the bioavailability of nitric oxide. One potential candidate to accomplish this is superoxide dismutase, however not all isoforms of this enzyme are suitable. The cytosolic (Cu/Zn) form has a short biological half life (6 – 10 mins) (Nelson et al., 2005). Recombinant human extracellular superoxide dismutase, (ECSOD), was shown to be cardioprotective in an
63 isolated (non-working) rat heart subjected to cold-arrest and warm reperfusion (Hatori et al., 1992). Addition of 30,000 IU/L ECSOD to the arresting perfusate (but not the Cu/Zn containing isoform) significantly improved recovery of left ventricular dP/dt max and decreased the LDH level in the coronary effluent. However, ECSOD has the disadvantage of being stripped from the endothelial vessel surfaces by high heparin concentrations, making it inappropriate for clinical (or most experimental) use.
Another approach was the development of a “chimeric” manganese containing SOD, which has a long (4 hour) half-life and binds avidly to the external surfaces of endothelial cells (Nelson et al., 2005). This agent was found to provide superior recovery of post-storage cardiac function, as well as reduced LDH release and reduced tissue accumulation of lipid hydroperoxides in a rabbit heart model of donor heart preservation after 4 hours hypothermic storage when compared to hearts stored in UW solution (Nelson et al., 2002).
ii) eNOS Substrate: The role of exogenous L-arginine in limiting myocardial ischaemia reperfusion injury has been established (Weyrich et al., 1992). Supplementation of both blood and crystalloid cardioplegic solutions with L-arginine has also been demonstrated to protect the heart from reperfusion injury after normothermic or mild hypothermic (28°C) arrest (Izhar et al., 1998, Sato et al., 1995). However, in an extended
64 period of hypothermic storage, (10 hours), more suitable as a model of donor heart preservation, L-arginine supplementation of the cardioplegic / storage solution showed little protective effect (Kevelaitis et al., 1996). In this isolated (non-working) model of donor heart preservation, recovery of
LV function of hearts arrested and stored in Celsior supplemented with 2 mM L-arginine alone was increased but not significantly different from hearts stored in Celsior alone and L-arginine supplementation failed to preserve endothelial function.
iii) Nitric oxide donors: Compounds which release nitric oxide spontaneously or after biological activation are able to substitute for nitric oxide synthase whose activity may be compromised during hypothermic storage or reperfusion. Importantly, exogenous NO inhibits native eNOS via a product inhibition related mechanism and may minimise the oxidant forming activity of uncoupled NOS (Griscavage et al., 1995).
The inclusion of glyceryl trinitrate (GTN) into UW solution and Celsior has improved cardiac function in a rat model of heterotopic transplantation after extended (16 hr) periods of cold storage (Baxter et al., 1999, Baxter et al., 2001). In an isolated working rat heart model of donor heart preservation, significant improvement in post-storage function was observed in hearts arrested and stored in (a) aspartate enriched St
Thomas’ solution (2) supplemented with 0.1 µM diethylamine NONOate, (a diazeniumdiolate), after 12 hours hypothermic storage (Du et al., 1998)
65 and (b) Celsior solution supplemented with 0.1 mg/ml GTN after 6 hours hypothermic storage (Gao et al., 2005).
A recently recognised family of nitric oxide donors are the S-nitrosothiols
(Al-Sa'doni et al., 2004). A high molecular weight S – nitroso human serum albumin has proved efficacious in an isolated working rabbit heart model of donor heart preservation. Hearts which had been perfused with Krebs solution supplemented with 2 µM S-nitroso compound, then arrested and stored in Celsior (also supplemented with 2 µM S-nitroso compound) for 6 hours at 4°C had significantly improved cardiac output (60% vs 20% of baseline) after 75 minutes of post-storage reperfusion than hearts arrested and stored in Celsior alone (Semsroth et al., 2005).
1.5.2 Inhibition of sodium hydrogen exchange
The mammalian sodium hydrogen exchanger, (NHE), is an integral membrane protein that catalyses the (electro-neutral) exchange of cytosolic protons for extracellular sodium ions using the energy from the sodium gradient across the cell membrane and is a major regulator of intracellular pH (Orlowski et al., 1997). To date, 9 isoforms of the exchanger have been identified (NHE1 – NHE9) (Fliegel, 2008). NHE1 is the predominant, most widely distributed isoform and is the major isoform expressed in the cell membranes of cardiac cells (Karmazyn et al., 1999).
Of interest in ischaemia reperfusion has been the demonstration of sodium
66 hydrogen exchange activity in the inner mitochondrial membrane (Numata et al., 1998) although its exact identity remains contentious.
The sodium hydrogen exchanger is quiescent under normoxic conditions, but there is disagreement as to whether it is active only at reperfusion or during both ischaemia and reperfusion (Allen et al., 2003). As described previously (section 1.3.3 and 1.3.4.1) the activation of the sodium- hydrogen exchanger (Na+/H+) by intracellular acidosis during ischaemia may lead to Na+ overload and this indirectly causes the Na+/Ca2+ exchanger to work in reverse mode leading to a potentially toxic intracellular influx of Ca2+. Reduction of myocardial calcium overload by pharmacological inhibition of the exchanger has been a popular approach to maximise myocardial protection during ischaemia reperfusion, especially after the development of HOE-694, and HOE-642, (now known as cariporide), both inhibitors specific for the NHE1 isoform (Scholz et al.,
1993). Other protective effects of NHE inhibition following on from or independent of minimisation of intracellular calcium include: i) a reduction in P-selectin expression and leukocyte adhesion (Buerke et al., 2008); ii) gradual normalisation of intracellular pH during reperfusion (Stromer et al.,
2000); iii) opening of ATP-sensitive K+ channels in mitochondria (Miura et al., 2001); iv) decreasing the open probability of the mitochondrial permeability transition pore (Javadov et al., 2008); v) direct decrease in mitochondrial superoxide production (Garciarena et al., 2008).
67 Studies have demonstrated the ability of a range of pharmacological inhibitors of Na+/H+ exchange to protect the heart from ischaemia reperfusion injury in numerous different animal models across a wide range of species (Masereel et al., 2003).
These promising pre-clinical findings have yet to be translated to the clinical arena. The “Sodium-Proton Exchange Inhibition to Prevent
Coronary Events in Acute Cardiac Conditions” (EXPEDITION) trial tested the efficacy of cariporide in high-risk patients undergoing CABG. The investigators found that patients receiving active treatment, (cariporide), had a highly significant reduction in the incidence of MI (14.4% vs 18.4% in placebo group) at the 5th post-operative day. However, mortality rose from 1.5% to 2.2% in the active treatment group due to an increased frequency of cerebrovascular events.
Mentzer and colleagues concluded that although cariporide was effective in minimising CABG-associated ischaemia reperfusion damage, it caused cerebrovascular events due to possible interference with pH regulation or rebound pro-coagulant effects when the platelet inhibitory effects of cariporide were withdrawn (Mentzer et al., 2008). In spite of these negative findings, the approach of sodium hydrogen exchange inhibition and cariporide may still be useful in cardiac preservation for transplantation as the usage of the NHEI would differ significantly from
EXPEDITION. In the EXPEDITION trial patients were exposed to a total of
68 1,620 mg of cariporide over 48 hours (loading dose 180 mg; infusion of 40 mg/hr over the 1st 24 hrs then 20 mg/hr over the 2nd 24 hour period).
Exposures are significantly less in the experimental cardiac transplantation setting, as outlined below.
The efficacy of the Na+/H+ exchange inhibitor, cariporide has been successfully demonstrated previously in a model of donor heart preservation and a translational large animal model of clinical heart transplantation in our laboratory. Cropper et al, using an isolated working rat heart model of donor heart preservation showed in hearts exposed to
10 µM (3.7 mg/L) cariporide prior to storage and/or at reperfusion was sufficient for improvement of post-storage improvement of cardiac functional recovery after 6 hours hypothermic storage compared to controls stored in St Thomas solution (2) alone (p<0.001) (Cropper et al.,
2003). Gao et al using the same model, showed that supplementation of
Celsior with 10 µM cariporide was sufficient to provide post-storage functional improvement after 6 hours hypothermic storage and addition of cariporide and 0.1 mg/ml GTN to Celsior enabled recovery of cardiac function after 10 hours hypothermic storage (Gao et al., 2005). Ryan et al, employing a porcine model of orthotopic heart transplantation incorporating donor brain death demonstrated that treatment of the donor animal with an IV dose of 2 mg/kg cariporide and the recipient with the same dose 10 minutes before donor heart reperfusion improved load
69 independent indices of cardiac function compared to controls receiving no treatment (p<0.0001) (Ryan et al., 2003a, Ryan et al., 2003b).
1.5.3 Inhibition of poly(ADP-ribose) polymerase
There are a number of post-translational protein modifications such as phosphorylation, acetylation, ubiquitination, and methylation which are essential for proper cellular function. Another such modification is poly(ADP-ribosyl)ation, a process where polymers of ADP-ribose are formed from molecules of NAD+ and are attached to target proteins via ester linkages to glutamate, aspartate or lysine (Althaus et al., 1987). This reaction is catalysed by the poly(ADP-ribose) polymerase (PARP) family of enzymes, the most common isoform and the isoform most important in the cardiac ischaemia reperfusion context being the nuclear enzyme PARP 1
(Ame et al., 2004).
PARP 1 is an important “house-keeping” enzyme under normal conditions and functions as a DNA nick-sensing enzyme, binding to single and double stranded DNA breaks and adding branched ADP-ribose polymers to a range of proteins including histones. This results in areas of high negative charge at the site of strand breaks that facilitates entry of a range of DNA repair enzymes to the site of damage (de Murcia et al., 1997). In addition to “normal” physiological functions, “hyper-activation” of PARP 1 by oxidative or nitrosative stress is thought to play a role in the initiation of damage to the reperfused myocardium (Figure 1.7).
70 Myocardial Ischaemia
Reperfusion
Burst of oxidants and peroxynitrite
Single strand breaks in DNA
PARP Inhibitors
Activation of PARP
Pro-inflammatory cytokines and Depletion of NAD+ & ATP chemokines
Endothelial Cell death by dysfunction necrosis
• Myocardial contractile dysfunction • Coronary vascular injury • Pro-inflammatory responses
Figure 1.7 Representive scheme of the PARP pathway in myocardial ischaemia and reperfusion injury. The restoration of oxygenated blood flow during reperfusion leads to production of reactive oxidant species including oxygen free radicals and peroxynitrite that induces single strand breaks in DNA which in turn activates PARP. PARP activation depletes NAD+ and ATP and leads to endothelial dysfunction, cell death and cellular injury. Promotion of pro- inflammatory cytokines and chemokines by PARP add to further cellular injury.
Pharmacologic use of PARP inhibitors preserve cellular NAD+ and ATP levels and prevent acute cellular dysfunction. Modified from (Szabo et al., 2004a).
71 The massive demand of NAD+ untilization in this process slows the rate of glycolysis and mitochondrial respiration resulting in cellular dysfunction and death (Zingarelli et al., 1996). PARP activation has been observed in the pathogenesis of various experimental cardiovascular contexts and inflammatory states such as organ transplantation, myocardial infarction, cardiomyopathies, colitis and arthritis (Liaudet et al., 2001a, Mabley et al.,
2001, Mazzon et al., 2001, Pacher et al., 2002, Pieper et al., 2000, Skuta et al., 1999, Virag et al., 2002).
Pharmacological inhibition or genetic deletion of PARP 1 is thought to preserve cellular NAD+ and ATP pools in oxidatively stressed cardiomyocytes thereby allowing them to function normally after ischaemia reperfusion, or, if tissue damage has begun, to recruit the apoptotic machinery to remove irreversibly injured cells (Bowes et al., 1999, Bowes et al., 1998, Fiorillo et al., 2006, Levrand et al., 2006). The pharmacological inhibition of PARP activation using a range of PARP inhibitors (Figure 1.7) in the context of cardiovascular disease has recently been reviewed (Pacher et al., 2007). This approach has been highly successful in a range of experimental ischaemia-reperfusion settings such as myocardial infarction, heart preservation during cardiac (heterotopic) transplantation, and bypass surgery (Pacher et al., 2005, Szabo, 2005a,
Szabo et al., 2005a, Ungvari et al., 2005). Protective effects after intervention with PARP inhibitors include i) normalisation of myocardial intracellular calcium levels and contractility; ii) decrease in cardiomyocyte
72 necrosis; iii) down-regulation of proinflammatory responses and neutrophil infiltration during myocardial reperfusion after a period of ischaemia
(Docherty et al., 1999, Farivar et al., 2005, Hauser et al., 2006, Pieper et al., 2000, Szabo et al., 2006a, Szabo et al., 2004b).
There are studies demonstrating the role of PARP activation in reperfusion injury in global ischaemia-reperfusion using the isolated perfused heart system (Szabados et al., 2000), in in vivo heart transplantation (Szabo et al., 2002) and after cardiopulmonary bypass with cardioplegic arrest
(Ramlawi et al., 2006).
While there have been many studies in vitro and in vivo showing deleterious effects of PARP activation on myocardial function and tissue injury, more work is required to understand the role of PARP in ischaemic preconditioning. A study by Liaudet (Liaudet et al., 2001b) showed that the protective effect of preconditioning disappears in PARP deficient mice or through the use of PARP inhibitor (3-amino benzamide) and suggested that low levels of PARP activation during preconditioning were necessary for its observed protective effect.
Previous studies have shown a potential anti-oxidant role for PARP inhibitors when these agents were shown to protect the mitochondria in the post-ischaemic myocardium by decreasing the extent of ROS production (Halmosi et al., 2001, Thiemermann et al., 1997). Inhibition of
73 PARP may also play an interesting role in protective cell signalling pathways (discussed further in Section 1.5.5). In a recent study, activation of the pro-survival PI3-k/Akt pathway by PARP inhibitors has shown to be cardioprotective in post-ischaemic hearts (Kovacs et al.,
2006). They showed that the presence of PARP inhibitors significantly increased the extent of phosphorylation of both Akt as well as one of its downstream targets, GSK-3β, Phosphorylation and deactivation of GSK is an important mechanism for the inhibition of the mitochondrial permeability transition pore opening (Section 1.5.5).
A recent study by Toth-Zsamboki (Toth-Zsamboki et al., 2006) demonstrated for the first time the presence of PARP activation in circulating leukocytes in patients with myocardial infarction during primary percutaneous coronary intervention. Several PARP inhibitors have entered clinical testing in Phase I and Phase II trials for cardiac disease and cancer (Pacher et al., 2007, Szabo, 2005b).
1.5.4 Ischaemic pre and post conditioning
Perhaps the most far-reaching recent advance in myocardial protection was the realisation that the heart could be induced to adapt itself to tolerate the damaging consequences of an acute ischaemia-reperfusion insult by the application of mechanical or pharmacological strategies
(“conditioning”). Such protection has been demonstrated upon exposure of the heart to a series of brief non-lethal cycles of ischaemia and reperfusion
74 before a long index ischaemic episode (ischaemic preconditioning) or at reperfusion (ischaemic postconditioning) and will be discussed in more detail below.
Ischaemic preconditioning
Originally described by (Murry et al., 1986), their protocol, employed four cycles of 5 minutes ischaemia and 5 minutes of reperfusion prior to 40 minutes of index ischaemia in a canine circumflex ligation model of cardiac ischaemia reperfusion injury. Although the observed protective effect was powerful, few insights into its mechanism were gleaned from these studies. This cardio-protective effect is now known as classic or early IPC.
IPC has subsequently been demonstrated in a variety of organs such liver
(Moses et al., 2005, Pang et al., 1995), intestines (Ishida et al., 1997) and in the kidney (Bonventre, 2002).
An important point to note from Murry’s initial study was that a significant reduction in infarct size occurred only when a relatively short (40 minute) ischaemic period is chosen. Ischaemic preconditioning did not confer protection when the index ischaemic period was extended to 3 hours.
Several early observations had important implications as to the mechanism of ischemic preconditioning. The protection afforded by classic preconditioning was not blocked by inhibitors of protein synthesis, which may imply that post-translational modifications of existing proteins are of
75 primary importance (Thornton et al., 1990). The protection afforded by IPC is also lost if the time between the initial IPC protocol and the period of sustained ischaemia is more than 1-2 hours. The protection is regained after about 24 hours and lasts for up to 2-3 days. This phenomenon is known as the “second window of protection” (Edwards et al., 2000, Millar et al., 1996). In contrast to early preconditioning, this delayed protection involves the upregulation of genes and de novo synthesis of a number of protective proteins (Bolli et al., 2007, Liu et al., 1999, Meng et al., 1996,
Yellon et al., 1995).
Shortly after the first description of IPC, there were independent reports showing preconditioned hearts experienced less anaerobic glycolysis less acidosis during index ischaemia (Murry et al., 1990, Steenbergen et al.,
1993). The exact mechanism is not known but was believed to be associated with a lower rate of ATP consumption in preconditioned hearts
(Fralix et al., 1993, Imahashi et al., 2004).
Further information on the mechanism of ischaemic preconditioning was quickly elucidated. Liu and colleagues found that activation of the adenosine A1 receptor could trigger IPC’s protective response, also demonstrating that IPC could be recapitulated with infusions of A1 specific agonists (Liu et al., 1991). Activation of ATP-sensitive potassium channels, (normally inhibited by ATP and opened during energy-depleted states), was also found to mediate the preconditioning response (Gross et
76 al., 1992) with further studies identifying the KATP channels located in the inner membrane of the mitochondria as key to the protective response
(Sato et al., 2000).
Subsequently other G-coupled receptor agonists, bradykinin and opioids could also mimic the IPC response (Schultz et al., 1995, Wall et al., 1994).
These findings led other investigators to postulate that activation of G coupled protein receptors led to opening of mitochondrial ATP-sensitive potassium channels with subsequent mitochondrial free radical generation which then activates protein kinase C and cellular signalling pathways
(Pain et al., 2000).
Apart from the KATP channel several other targets have been implicated in the development of the protective preconditioning response. Increasing the closed probability of the mitochondrial permeability transition pore has been recently recognised as a powerful protective approach against ischaemia reperfusion damage (discussed in section 1.3.4.5) and has recently been shown to play a role in the preconditioning response. A previous report has showed that the mitochondrial KATP channel opener, diazoxide, was able to minimise calcium-activated permeability transition pore opening (Hausenloy et al., 2002). It is also reported that the sodium hydrogen exchanger was inhibited by ischaemic preconditioning of isolated rat hearts (Xiao et al., 2000). This finding may also explain the observation that preconditioned hearts are less edematous post
77 reperfusion (Sanz et al., 1995). Inhibition of programmed cell death may also play a role in the preconditioning response. Preconditioning has been shown to lower intra-cardiac levels of pro-apoptotic Bcl-2 family member,
BAX, and prevents post-reperfusion activation of caspases (Piot et al.,
1999).
Ischemic preconditioning can improve many of the deleterious effects of donor heart preservation discussed earlier including intra-cellular and mitochondrial calcium overload (Wang et al., 2001), oxidative damage at reperfusion (Vanden Hoek et al., 2000), endothelial dysfunction (Kaeffer et al., 1996) and leukocyte adhesion (Kubes et al., 1998).
Ischaemic preconditioning or pharmacological preconditioning has proved effective in several transplant-related models. In an isolated working rat heart model of donor heart preservation, a single cycle of 5 minutes ischaemia and 5 minutes reperfusion immediately before arrest and storage has been shown to improve post-storage cardiac contractile function in hearts stored in UW solution and St Thomas’ solution 2, after
10 hours hypothermic storage as well as reducing creatine kinase leakage compared to non-preconditioned controls (Karck et al., 1996). Our laboratory examined the role of ATP-sensitive potassium channel opening in the mechanism of ischaemic and pharmacologic preconditioning, also in an isolated working rat heart model of donor heart preservation. Hearts exposed to a single cycle of 5 minutes ischaemia and 5 minutes
78 reperfusion or pre-treated with the KATP channel opener, pinacidil (200 µM) showed significantly better contractile function after 6 or 12 hours storage in aspartate-supplemented St Thomas solution compared to non- preconditioned or untreated controls. This functional improvement could be completely abolished in both the preconditioned and pinacidil treated hearts by pre-treatment with the KATP channel inhibitor, glibenclamide
(Hicks et al., 1999). Ischaemic preconditioning was also effective in other models of donor organ preservation such as a single rat lung transplant model after 6 or 12 hours storage (Du et al., 1996), and more recently in a rat liver transplant model (6 hours storage) (Rehman et al., 2008).
However, in the clinical transplant setting, the application of a potentially injurious series of clamping cycles to fragile donor organ artery may result in severe vascular complications (Koneru et al., 2005).
Ischaemic postconditioning
In 2003, Zhao and colleagues reported another protective strategy against reperfusion injury which they termed postconditioning (Zhao et al., 2003).
Intermittent periods of ischaemia and reperfusion were applied during early reperfusion, specifically after 60 minutes of ischaemia, three cycles of 30 seconds of reperfusion followed by 30 seconds of re-occlusion were applied at the commencement of reperfusion. They found that this technique reduced myocardial infarct size and attenuated reperfusion injury. Tsang and colleagues, (Tsang et al., 2004) showed that postconditioning is a form of “modified reperfusion”, first shown by
79 Okamoto and colleagues to limit post-ischaemic injury and did not enhance the protection afforded by IPC despite activating the same signalling pathways (Okamoto et al., 1986).
The optimum timing of delivery of the postconditioning stimulus is model dependent. In a rabbit heart, a delay of 10 minutes between the onset of reperfusion and postconditioning abolished protection (X.M. Yang et al.,
2004b). However, some investigators found that even a short delay of 1 minute between end ischaemia prior to the application the cycles of postconditioning was sufficient to block protection (Kin et al., 2004). The number of studies performed in recent years designed to elucidate the mechanism of postconditioning has highlighted its potential and the need to fully understand the phenomenon for successful application of postconditioning to clinical contexts.
Despite the differences in the timing of the protective stimuli of pre and postconditioning in relation to the index ischaemic insult, it has become increasingly apparent that there are a number of shared elements in the mechanisms of both processes. Just as adenosine has been implicated in triggering preconditioning, activation of adenosine receptors by endogenous adenosine has been implicated in the protective effect of postconditioning (Kin et al., 2005), as has a role for activation of mitochondrial KATP channels, protein kinase C and redox signalling
(Penna et al., 2006).
80 As was the case for ischaemic preconditioning, the postconditioning process also protects against several potentially deleterious changes which may occur in the donor heart during storage and reperfusion. These include minimisation of intra-cellular calcium overload and post reperfusion oxidative stress (Sun et al., 2005), as well as endothelial dysfunction and white cell adherence (Zhao et al., 2003).
To date, there are only two reports of the application of ischemic post conditioning in the setting of experimental donor heart preservation. In the first, it was demonstrated that rat hearts exposed to 3 cycles of 30 seconds ischaemia + 30 seconds reperfusion after a period of 4 hours of hypothermic storage regained about 20% of pre-storage baseline cardiac output compared to non-postconditioned hearts which had no post-storage recovery of function. The functional improvement was accompanied by a significant decrease in LDH efflux from postconditioned hearts (Lauzier et al., 2007). The second study examined the role of inhibition of the mitochondrial permeability transition pore in the protective effect post- conditioning after cardioplegia and 8 hours hypothermic storage in an isolated (non-working) rat heart model of donor heart preservation (Ferrera et al., 2007). These investigators found that 2 cycles of 30 seconds reperfusion and 30 seconds ischemia following the index cold ischaemia reduced infarct size and improved functional recovery post reperfusion with mitochondria extracted from post-conditioned hearts being more resistant to calcium-induced mPTP opening.
81 1.5.5 Pro-survival signalling pathways as a common mechanism to
ischaemic pre and post conditioning
1.5.5.1 Proximal elements
A protective role of pro-survival signalling pathways in ischaemic pre- and postconditioning has been identified in a number of models of cardiac ischaemia reperfusion. Hausenloy and Yellon have termed these pathways the Reperfusion Injury Salvage Kinase or RISK pathway
(Hausenloy et al., 2005b, Hausenloy et al., 2004b). Pharmacological recruitment of these pro-survival pathways may facilitate the translation of the cardio-protective benefits observed experimentally into efficient approaches to protect the human heart.
The most intensively studied pathways in relation to the RISK pathway to date have been the phosphatidylinositol 3’-kinase / Akt pathway and the p44 p42 mitogen activated kinase (or ERK 1/2) pathway. The phosphatidylinositol 3’-kinases (PI 3-k) are a group of three classes of protein / lipid kinases based on their substrate specificity (classes I, II or
III). In the context of RISK, the most important are the class I PI 3-k’s as they can be activated by growth factor receptors (tyrosine kinases) or G- protein-coupled receptors (Alessi et al., 1996) previously identified as key trigger factors for ischemic pre and postconditioning (Section 1.5.4).
82 Activation of PI 3-k catalyses the conversion of phosphatidylinositol-4,5- diphosphate to phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3).
Formation of PtdIns(3,4,5)P3 directs down-stream signaling through a number of effectors including Akt (known as protein kinase B) (Figure 1.8).
Akt binds this lipid tri-phosphate through its amino terminal “pleckstrin homology” domain, which recruits Akt to the plasma membrane and induces a conformational change that allows phosphoinositide-dependent kinase 1 (PDK 1) and the rictor-mTOR complex to phosphorylate (and activate) Akt at Thr308 and Ser473 respectively (Alessi et al., 1996,
Sarbassov et al., 2005). When activated, Akt phosphorylates a number of down-stream (protein) targets that control cell proliferation and survival
(discussed below).
The other signalling pathway proposed to be involved with myocardial protection at reperfusion was the p44/p42 mitogen activated kinase pathway (now known as the extracellular-signal-related-kinase, ERK pathway) (Yellon et al., 1999). This pathway is a hierarchy of a G-protein and 3 kinase tiers. Growth factor binding to the appropriate receptor initiates protein tyrosine kinase phosphorylation which in turn activates the
G-protein, Ras. Ras then activates Raf-1, (the MAP kinase kinase kinase) which then phosphorylates and activates the MAP kinase kinase, MEK1/2
(MAPK/ERK kinase) which in turn phosphorylates ERK1/2 (Seger et al.,
1995). Early evidence suggested that activation of the Ras – Raf-1 – MEK
83 – ERK pathway was protective against apoptotic cell death (H.G. Wang et al., 1996) (Figure 1.8).
In the context of ischaemic preconditioning, Murphy’s laboratory showed that the preconditioning stimulus itself activated (phosphorylated) Akt and phosphorylated GSK3ß, one of its down stream targets, as well as implicating an IPC – induced increase in eNOS activity via an increase in nitrite + nitrate levels (Tong et al., 2000, Tong et al., 2002). Importantly, they showed that the improvement in cardiac function derived from preconditioning could be reversed by Wortmannin, an inhibitor of the PI 3- k / Akt pathway. Concordant with these findings was the observation that a cardiac-specific knock-out of PDK-1, (one of the kinases responsible for activating Akt), is associated with increased sensitivity to hypoxia (Mora et al., 2003). In contrast, animal models overexpressing PI 3-k (Class Ia) and
/ or Akt were observed to have increased cellular survival and smaller infarct size after exposure to ischaemia reperfusion challenge (Matsui et al., 2002, Oudit et al., 2004).
The importance of pro-survival signalling at the time of reperfusion was first identified by Bell and Yellon (Bell et al., 2003b). They showed that bradykinin, an agent which could mimic ischaemic pre (and subsequently post) conditioning was efficacious in protecting an isolated perfused mouse heart infarct model of ischaemia / reperfusion injury when the agent was given immediately before reperfusion. This functional protection
84 Conditioning Stimulus
PI 3-k PIP2 PIP3 PIP3 Ras PTEN PDK1 Akt mTORC2 • • Raf
eNOS GSK-3ß • MEK 1/2 NO NHE1 •Phosphorylation sGC • • of pro-apoptotic cGMP ERK 1/2 proteins (BAD, • Bax) PKG
Bcl2 •PKCε •PLB
•Minimise Mitochondrial Damage & Apoptosis, Minimise Calcium Maximise Cell Survival Overload
Figure 1.8 Schematic representation of the mechanisms of the major pro- survival kinase pathways associated with pre- and postconditioning. For details see Section 1.5.5. PI 3-k: phosphatidylinositol 3’-kinase; PIP2: phosphatidylinositol-4,5-diphosphate; PIP3; phosphatidylinositol-3,4,5- triphosphate; PTEN: phosphatase and tensin homologue deleted on chromosome 10; PDK1: phosphoinositide-dependent kinase 1; mTORC2: rictor- mTOR complex 2; eNOS: endothelial nitric oxide synthase; NO: nitric oxide; sGS: soluble guanylate cyclase; cGMP: cyclic GMP; PKG: protein kinase G; PKCε: protein kinase Cε; PLB: phospholamban; NHE1: sodium hydrogen exchanger
(class 1). (Dark green spots- phosphorylation; Blue arrows – activation; Red arrows - inhibition)
85 was accompanied by an increase in Akt phosphorylation along with a transient increase in eNOS phosphorylation 10 minutes post reperfusion. eNOS had previously been shown to be another down-stream target of Akt
(Dimmeler et al., 1999).
Subsequently, Hausenloy and colleagues showed a bi-phasic increase in both Akt and ERK 1/2 phosphorylation with the first increase immediately after the preconditioning stimulus in a rat infarct model of ischaemia reperfusion damage. The extent of phosphorylation waned during the period of extended lethal ischaemia but increased again after reperfusion of the preconditioned hearts with no phosphorylation increases observed in the non-preconditioned group (Hausenloy et al., 2005a). Inhibition of either pathway with LY-294002 (Akt pathway inhibitor) or PD98059 (an inhibitor of MEK-ERK) abolished the protective effect of the preconditioning stimulus.
The same proximal elements of the RISK pathway were found to be operating in the setting of postconditioning. Tsang and colleagues identified increases in Akt phosphorylation, as well as eNOS and p70S6K phosphorylation in a rat heart infarct model (Tsang et al., 2004). Hearts were exposed to 35 minutes of global ischaemia with one group being exposed to a postconditioning stimulus of 6 cycles of 10 seconds reperfusion + 10 seconds ischaemia and a control group having no intervention. Infarct size was reduced by about 50% in the postconditioned
86 group, with the protective effect being abolished by inhibition of the Akt pathway with Wortmannin or LY-294002. Although the extent of activation of the ERK 1/2 pathway was not examined in this study, it was showed in a rabbit heart model of postconditioning that exposure of the hearts to the
ERK inhibitor, PD98059, immediately before reperfusion abolished the protective effect of postconditioning, thus implicating a protective role for activation of the ERK pathway (X.M. Yang et al., 2004b).
1.5.5.2 Downstream targets of RISK pathway
The ultimate targets for these pro-survival signalling pathways in the heart have been postulated to be i) protection of the mitochondria; ii) minimisation of cell death (both apoptosis and necrosis) and iii) normalisation of calcium handling by the endoplasmic reticulum. None of these effects is mutually exclusive.
Activation of eNOS by Akt, mentioned above, is a key precursor to a number of disparate protective consequences (Figure 1.8). Nitric oxide may activate guanlyate cyclase leading to production of cyclic GMP and activation of protein kinase G (Burley et al., 2007). Activation of protein kinase G can lead directly to activation of the sacoplasmic reticulim calcium dependent ATP-ase via phospholamban (PLB) phosphorylation which minimizes excessive activation of the contractile apparatus
(Abdallah et al., 2005). The resultant decrease in cytosolic calcium levels
87 along with activation of PKCε will result in opening of the KATP channel and inhibition of mitochondrial transition pore opening (Costa et al., 2005).
S-Nitrosylation, the reversible coupling of NO to a sulphur atom may also play a role in mitochondrial protection. Complexes 1 and 4 of the electron transport chain are reversibly inhibited by nitrosylation (Nadtochiy et al.,
2007). At reperfusion, this inhibition will be slowly relieved as NO is displaced, thus avoiding a rapid flux of electrons through the electron transport chain, and thereby minimising production of superoxide and inhibiting transition pore opening. Reduction of permeability transition pore opening by any means will lower the chances of myocyte necrosis.
Another potential target for S-nitrosylation is a redox-sensitive cysteine residue at the active site of the “executioner caspase”, caspase 3, which results in loss of its protease activity (Maejima et al., 2005). Such nitrosylation was thought to inhibit cleavage of the pro-apoptotic Bcl-2 family protein, Bid, thereby blocking release of cytochrome c from the mitochondria (Kim et al., 2000).
Both activated Akt and the ERK 1/2 pathway can phosphorylate (and inactivate) glycogen synthase kinase (GSK) 3ß on Ser 9 (a highly conserved regulatory site on the N terminal region of the enzyme) (D.A.
Cross et al., 1995, Ding et al., 2005). The involvement of the inhibition of mitochondrial permeability transition pore opening by phosphorylated
GSK-3ß was suggested for a variety of cardioprotective agents
88 (Juhaszova et al., 2004). However, the actual mechanism by which phospho-GSK-3ß modulates pore activity is still unclear as the nature of the pore-forming unit of the mPTP is contentious (Halestrap et al., 2009).
Activation of the PI 3-k /Akt or ERK 1/2 pathways modify the effects of a number of pro-apoptotic and anti-apoptotic members of the Bcl-2 family
(Adams et al., 2001). The anti-apoptotic protein, Bcl-2 can be phosphorylated by ERK 1/2 at a number of sites in the flexible loop region of the molecule (Ruvolo et al., 2001). Phosphorylation of Bcl-2 causes a conformational change within the protein that was thought to serve as a survival sensor against stress stimuli (Figure 1.8). ERK may also directly inhibit the apoptotic pathway by phosphorylation and inhibition of caspase
9 (Allan et al., 2003).
BAD is a pro-apoptotic “BH3-only” member of the Bcl-2 family. In its unphosphorylated state it binds to Bcl-XL, at the mitochondrial surface and prevents the intrinsic anti-apoptotic activity of Bcl-XL (Yang et al., 1995).
Activated Akt directly phosphorylates BAD (Datta et al., 1997).
Phosphorylation of BAD on Ser 136 creates a binding site for 14-3-3 proteins that facilitates release of BAD from its target proteins and negates its pro-apoptotic activity (Zha et al., 1996). In the same vein, phosphorylation of pro-apoptotic Bax by Akt prevents the conformational change in this molecule that is necessary for its pore-forming interaction with the outer mitochondrial membrane (Yamaguchi et al., 2001).
89 Another recently-identified target for Akt – mediated phosphorylation, particularly relevant to previous discussion is the sodium hydrogen exchanger (NHE isoform 1) (Figure 1.8). Snabaitis and colleagues demonstrated that NHE1 is phosphorylated on Ser 648 which inhibits binding of calcium-calmodulin and markedly inhibits NHE1 activity in intact ventricular myocytes in response to intracellular acidosis (Snabaitis et al.,
2008). Xiao and Allen previously noted that sodium hydrogen exchange activity in pre-conditioned hearts at reperfusion was decreased (Xiao et al., 1999). Snabaitis’ findings suggest a rational explanation for this observation – ischaemic preconditioning activated Akt at reperfusion, leading to phosphorylation and inactivation of NHE1. The resultant slight cytosolic acidosis at reperfusion with a delay in restoration of physiological pH will maintain the mitochondrial permeability transition pore in the closed state and may contribute to the observed cardioprotection of both pre- and post-conditioning (Fujita et al., 2007).
1.5.5.3 Endogenous “Anti-RISK” elements
The tempo and extent of protein phosphorylation by protein and lipid kinases are controlled by specific phosphatases, which catalyse the removal of phosphate groups. Most important for the proximal elements of the RISK pathway are: i) the lipid 3’ phosphatase PTEN, (phosphatase and tensin homologue deleted on chromosome 10), predominantly responsible for the removal of the 3’ phosphate from phosphatidylinositol
(3,4,5) triphosphate thus preventing recruitment, phosphorylation and
90 activation of Akt (Oudit et al., 2004); ii) protein phosphatase A2, which catalyses dephosphorylation (deactivation) of Akt and ERK 1/2 (Millward et al., 1999).
Cardiac PTEN activity has been shown to be modulated by ischaemic preconditioning. Cai and Semenza (2005), using an isolated rat heart ischaema reperfusion model (30 minutes ischaemia followed by 45 minutes reperfusion) preceded by a single preconditioning stimulus of 15 minutes ischaemia + 30 minutes, showed that preconditioning led to loss of PTEN activity which directly to increased Akt phosphorylation immediately post reperfusion and significantly increased functional recovery compared to nonpreconditioned controls. In addition, they showed that IPC lead to dephosphorylation of PTEN which made it sensitive to rapid proteasomal degradation and interestingly, prolonged reperfusion showed maximum Akt activation and minimal PTEN after 60 minutes followed by gradual loss of phospho-Akt and increase in PTEN levels (Cai et al., 2005).
In addition, potential “anti-RISK” role for protein phosphatases in ischaemic postconditioning has recently been inferred (Bouhidel et al.,
2008). These investigators compared the protective ability of 6 cycles of
10 seconds reperfusion + 10 seconds ischemia in a coronary artery occlusion model of IRI in wild type and obese (ob/ob) mice.
Postconditioning reduced infarct size by 58% in wild type mice and was
91 accompanied by increased phosphorylation of Akt and ERK as well as a decrease in PTEN, MAP kinase phosphatase and protein phosphatase
2A. Postconditioning produced no reduction of infarct size in the obese mice and the phosphorylation status or RISK pathway elements were not increased. There was, however a significant increase in the levels of all three phosphatases.
Other studies are concordant with a negative regulatory role of PTEN in PI
3-k signalling. Haploinsufficiency of PTEN was demonstrated to reduce the threshold for IPC protection in an isolated mouse heart model of IRI
(Siddall et al., 2008). Rho kinase, previously shown to specifically phosphorylate and activate PTEN, has also been recently suggested as a target for the treatment of cardiovascular disease (Z. Li et al., 2005).
Hamid and colleagues have shown that administration of the Rho kinase inhibitors Fasudil or Y-27632 to an isolated rat heart infarct model of ischaemia reperfusion immediately before reperfusion, significantly decreased infarct size and apoptosis. The protective effect could be blocked by inhibition of PI 3k or nitric oxide synthase, inferring again that
PTEN maximised the protective effect of the PI 3k / Akt pathway (Hamid et al., 2007).
1.5.5.4 Pharmacological modulation of pro-survival pathways
The pathways underpinning myocardial protection discussed above may be divided into several phases: i) the trigger phase, consisting of G-protein
92 coupled receptors or tyrosine kinase receptors, ii) mediators, the pathways themselves and iii) targets such as mitochondria and endoplasmic reticulum. Each of these phases is amenable to pharmacological modulation in various experimental models of ischaemia reperfusion damage with a number of experimental approaches being trialled in clinical settings. G-protein coupled receptor agonists that have been trialled include adenosine, bradykinin and opioids. Of these agents, adenosine has been the most intensively studied, but has shown discordant results in infarct limitation studies (Goto et al., 1991, Olafsson et al., 1987). However, endogenous adenosine produced during preconditioning protected the heart early in reperfusion by binding to the adenosine 2b receptor and activating Akt (Solenkova et al., 2006). Both bradykinin and opioids were shown to minimise infarction through activating Akt at reperfusion (Gross et al., 2006, X.M. Yang et al., 2004a).
Tyrosine kinase receptor agonists include fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin, insulin-like growth factor (IGF) and urocortin. FGF, insulin, IGF-1 and urocortin have all been shown to be cardioprotective at reperfusion subsequent to a period of extended ischaemia with activation of both the PI 3-k / Akt and ERK 1/2 pathways in the case of insulin, IGF-1 and urocortin and ERK 1/2 for FGF
(Hausenloy et al., 2004b). Inhibition of poly(ADP-ribose) polymerase has also been shown to result in activation of the PI 3-k /Akt pathway leading to mitochondrial protection after oxidative stress (Tapodi et al., 2005).
93 Interestingly, a recent study has shown that inhibition of PARP in cultured human endothelial cell cultures exposed to oxidant stress, (peroxynitrite), resulted in activation of VEGR receptor 2 with resultant activation of the PI
3-k Akt pathway (Mathews et al., 2008). The effect of PARP inhibition on improvement of donor heart preservation and recruitment of RISK elements will be discussed in detail in Chapters 3 and 5.
Many pharmacological agents, autacoids and hormones administered at reperfusion can activate ERK 1/2 or the Akt pathway. Statins and atrial natriuretic peptide were demonstrated to reduce infarct size in mouse and rabbit models respectively (Bell et al., 2003a, Yang et al., 2006).
Involvement of RISK pathways was inferred from the abolition of the cardioprotective effect of these drugs by specific inhibitors of the PI 3-k pathway (wortmannin) and the ERK 1/2 pathway (PD98059).
Pharmacological preconditioning with the phosphodiesterase-5 inhibitor, sildenafil, results in activation of endothelial and inducible nitric oxide synthases and the pro-survival PI 3-k / Akt pathway. The resultant increase in intra-cellular nitric oxide activates distal elements of the Akt pathway (Figure 1.8), including protein kinase G (A. Das et al., 2008).
Other agents showing promise as pharmacological activators of survival signalling are erythropoietin (Bullard et al., 2005) and volatile anaesthetic agents (Pagel, 2008).
94 The mitochondrion has emerged as the major target for pro-survival signalling pathways, with a major focus on regulation of permeability transition pore activity by ischaemic and pharmacological preconditioning and postconditioning (Hausenloy et al., 2009). Crompton and colleagues first demonstrated that the pore was a discrete entity with a molecular weight cut-off of about 1,500Da (Crompton et al., 1987). However, the exact nature of the pore-forming unit and the exact mechanism of modulation of pore opening by RISK elements remain to be elucidated.
Soon after his initial description of the pore, Crompton’s group showed that cyclosporine A was a potent inhibitor of mPTP opening (Crompton et al., 1988). Subsequent studies showed that cyclosporine binding to cycophilin D was a key component of inhibition of mPTP opening
(Halestrap et al., 1990).
In a recent study, cyclosporine has been successfully used as a cardioprotective agent against ischaemia reperfusion injury in an in vivo mouse LAD ligation model of cardiac infarction (Lim et al., 2007). This study demonstrated that the protective effect of cyclosporine given at reperfusion was as efficacious as pre-or postconditioning and that these protective effects were lost in mice in which cycophilin D was knocked out.
Importantly, a pilot study in a group of patients about to undergo percutaneous coronary intervention for the relief of acute myocardial infarct has shown that that introduction of cyclosporine A immediately before PCI significantly reduced infarct size as measured by creatine
95 kinase release over the first 3 days post reperfusion of the culprit artery
(Piot et al., 2008).
Phosphorylation and inactivation of GSK-3ß by the ERK 1/2 pathway or
Akt as a mechanism for involvement of RISK pathway activation has been widely examined (Juhaszova et al., 2004), although the exact role of phospho-GSK-3ß (Ser 9) in cardiac myocyte protection is yet to be resolved. Genetic manipulation of GSK-3ß in the mouse has shown that mice lacking the Ser-9 phosphorylation site (ie animals lacking the ability to inhibit GSK by Akt phosphorylation) still resulted in cardioprotection by pre and postconditioning (Nishino et al., 2008). Interestingly, using the same mice (a substitution of alanine for serine at position 9) showed that these mutant mice postconditioning was without protective effect, but cyclosporine A added at reperfusion reduced infarct size (Gomez et al.,
2008). Specific inhibitors of GSK-3ß such as SB216763 have also been shown to be as efficacious as opioid-induced protection in a rat infarct model of ischaemia reperfusion damage (Obame et al., 2008).
In light of the variability of ischaemic pre and postconditioning as shown in a number of experimental studies and the potential hazards involved in physical manipulation of major vessels during clinical heart transplantation to achieve the potential protective effects of ischaemic pre or postconditioning, pharmacological therapies that may harness the
96 protective effects may be the best way forward in translating experimental cardiac salvaging to the clinical arena.
1.6 EXPERIMENTAL MODEL CHOSEN FOR CURRENT STUDY
Isolated cardiac myocytes have been widely employed to examine cardiac preservation injury at the cellular to molecular level. Cellular systems have been developed as a screening model for cardiac preservation solutions(Schmid et al., 1991) or elucidating the mechanism of ischaemic preconditioning (Diaz et al., 2006), but the performance of isolated cells to what is often “simulated” ischaemia reperfusion protocols may not necessarily be a reflection of the response of the whole organ to an authentic ischaemia reperfusion challenge. Orthotopic transplantation of a preserved donor heart in a large animal model (preferably incorporating brain death of the donor before procurement of the donor heart) represents the ultimate method of choice for assessment of cardiac preservation strategies, as it incorporates all the aspects of clinical heart transplantation. However, the cost of large animals, the requirement for specialised operating facilities and the large teams required to carry out these procedures, have meant that this model is used as a final translational test of a protocol before clinical use, rather than a proof-of- principle screening method.
97 The experimental model used to assess cardiac preservation in this thesis is the isolated working rat heart. This approach strikes a balance between the more clinically relevant but resource intensive large animal model and the relatively cost effective cellular model which may provide consistent, although perhaps far less clinically relevant results.
The isolated heart perfusion circuit, first described by Langendorff
(Langendorff, 1895), was a preparation that allowed assessment of cardiac function in a non-working (often termed “Langendorff”) system.
Essentially, the model involved cannulation of the aorta to allow retrograde perfusion of the coronary arteries by a perfusate (commonly Krebs-
Henseleit buffer). Neely (Neely et al., 1967) introduced a modification to this system with the placement of a left atrial cannula. Perfusate flow through this cannula allows the heart to generate antegrade flow (aortic flow and cardiac output) and perfuse its own coronary arteries (the so- called “working” mode). Importantly, this modification now allows assessment of left sided heart function (or pump function), a parameter unable to be measured in the non-working heart preparation or a heterotopic heart transplant model.
In the setting of (donor) heart preservation the isolated working rat heart model was an essential screening tool in the development of St Thomas’ solution (Hearse, 1980) and later Celsior solution (Menasche et al., 1994).
98 A key strength of the model is that the whole organ may be studied with comparatively low cost and high throughput.
It must be realised that the conditions under which the heart functions in this model differ substantially from normal physiology. The isolated heart no longer has normal input from the nervous system or hormones that can modify its in vivo function. In addition, the heart is routinely perfused by on oxygenated crystalloid buffer rather than whole blood (Galinanes et al.,
1990). An oxygenated crystalloid buffer seems to be sufficient to meet the demands of the rat heart for oxygen at the high coronary flows typically observed in this model. However, the non-physiological nature of this model results in a decline of cardiac function over time (typically between
5 – 10% per hour) (Sutherland et al., 2000). However, the absence of white cells and other blood borne products which may be deleterious to the heart at reperfusion, allows the assessment of potentially cardioprotective strategies directly on the heart.
99 1.7 Thesis Hypothesis and Aims
The literature reviewed in this chapter reveals that there has been significant progress made in the elucidation of mechanisms mediating cardiac ischaemia reperfusion injury and protection against these insults that can potentially be applied to the donor heart. However, the recent insights into adaptive mechanisms of cardiac protection such as the recruitment of pro-survival signalling pathways at reperfusion remain to be systematically assessed in a model of donor heart preservation incorporating extended periods of hypothermic ischaemia. Given the complex interplay of elements of ischaemia reperfusion injury likely to be operating during donor heart preservation and reperfusion, it is likely that a combination of therapeutic approaches may be required to maximise cardiac protection post reperfusion.
Numerous pharmacological agents have been shown to mimic the protective effects of ischaemic pre- and postconditioning when given immediately before index ischaemia or at reperfusion by activating pro- survival signalling, however, it is not immediately obvious that this mode of protection can be elicited by adding such pharmacological supplements to an arresting and storage solution routinely used for clinical cardiac preservation.
100 Hypotheses
The two Hypotheses that underpin this thesis are as follows:
i) Supplementation of a standard cardioplegic / storage solution (Celsior) with agents that inhibit poly(ADP-ribose) polymerase, optimise bioavailability of nitric oxide or inhibit intracellular calcium efflux can improve post-reperfusion cardiac function in an isolated working rat heart model of donor heart preservation. The specific indices of cardiac function to be examined are (a) aortic flow (a measure of the ability of the heart preparation to act as a pump); (b) coronary flow; (c) cardiac output
(another index of contractile and pump function) and (d) heart rate.
ii) Exposure of the heart to these agents at arrest and during index hypothermic storage recruits and activates pro-survival kinase signalling and suppresses cell death pathways post reperfusion. The pro-survival pathways to be examined will be the MEK / ERK 1/2 and PI 3-k / Akt pathways and their down stream targets phospholamban, GSK3ß and
Bcl2. Phosphorylation status of these elements will serve as a surrogate marker for pathway activity. The presence of cleaved caspase 3 will serve as a measure of programmed cell death.
101 Aims
The overall aim of the study is to develop a preservation strategy using an isolated working rat heart model of donor heart preservation that optimises recovery of the stored heart after “short-term” (6 hours) hypothermic storage as well as viable recovery of cardiac function after “prolonged” (10 hours) hypothermic storage.
The specific aims are:
Using the agents (INO-1153) a PARP inhibitor, GTN (a nitric oxide donor) and Cariporide (an inhibitor of sodium hydrogen exchange):
i) To identify the pharmacological supplements or combinations of such supplements required to maximise recovery of the aforementioned indices of cardiac function after 6 or 10 hours hypothermic storage.
ii) To determine the extent to which these supplements activate the ERK
1/2 and / or Akt pro-survival signalling pathways.
iii) To assess whether recovery of the heart after hypothermic storage is dependent of activation of the above pro-survival signalling pathways.
iv) To assess the role of these pharmacological supplements on modifying the extent of apoptosis in hearts after reperfusion.
102 CHAPTER 2
General Materials and Methods
103 2.1 MATERIALS
The following section lists all the drugs, chemicals and experimental compounds used in the study along with their suppliers.
Table 2.1 Anaesthesia and Pre-medications.
Drug Source Parnell Laboratories, Aust Ketamine (Alexandria, NSW) Troy Laboratories, Aust Xylazine (Smithfield, NSW) Pfizer Pty Ltd, Heparin (North Ryde, NSW)
The above agents were given to induce anaesthesia and to anticoagulate the animals in preparation for surgery (Details of dosage and timing of administration are given in Section 2.4).
Table 2.2 Perfusion buffer components.
Chemical / Purity Source Potassium Chloride (AR) Sigma-Aldrich (St Louis, Mo) Sodium Chloride (AR) Sigma-Aldrich (St Louis, Mo) Magnesium Sulphate (AR) Sigma-Aldrich (St Louis, Mo) Potassium Dihydrogen Phosphate (AR) Sigma-Aldrich (St Louis, Mo) Sodium Bicarbonate (AR) Sigma-Aldrich (St Louis, Mo) Calcium Chloride (AR) Sigma-Aldrich (St Louis, Mo) Glucose (AR) Sigma-Aldrich (St Louis, Mo)
104 Preparation of perfusion buffer
This perfusate solution was based on that described by Krebs-Henseleit
(1932) as modified by Hearse and colleagues (Hearse et al., 1975). Its composition was as follows: NaCl 118.0 mM; KCl 4.7 mM; MgSO4 1.2 mM;
KH2PO4 1.2 mM; NaHCO3 25.0 mM; CaCl2 2.5 mM, glucose 11.0 mM
(Table 2.1). The components were dissolved in water purified through a
MilliQ water purification system (Millipore, Australia). The resultant solution was bubbled continuously with Carbogen (95% O2 and 5% CO2) at 37°C for an hour prior to the experiment. The final pH of the Krebs solution was between 7.3 and 7.4. The perfusate was filtered through an inline filter
(5µm pore size) during the course of pre-storage perfusion and post- storage reperfusion.
Preservation solution
Celsior solution was used for cardioplegia and storage of the isolated rat heart. For composition of Celsior, see section 1.4.3, Table 3. It is supplied as a sterile solution and was manufactured by Imtix Sangstat (France) and now by Genzyme (Netherlands). It was stored at 4°C in the dark in an air- tight bag.
105 Table 2.3 Pharmacological supplements.
Name Class Source Cariporide Sodium Hydrogen Aventis Pharma (Frankfurt (Lot N° B004) Exchange Inhibitor a. M., Germany) David Bull Laboratories Glyceryl Trinitrate Nitric Oxide Donor (Australia) INO – 1153 Poly (ADP-ribose) Inotek Pharmaceuticals (Lot N° DP-3-16) Polymerase Inhibitor Inc (Beverly, MA) Sigma Aldrich Wortmannin PI 3-kinase Inhibitor (St Louis, Mo) Cayman Chemical PD98059 MEK Inhibitor (Ann Arbor, MI)
The above agents were added to either the perfusion buffer or cardioplegic / storage solution to modify effectiveness of heart preservation. Cariporide and INO-1153 were obtained as pure substances under Materials Transfer Agreements from Aventis Pharma, and Inotek
Pharmaceuticals respectively and were stored in a dry environment at
4°C. Purity of Wortmannin and PD98059 were specified as ≥ 98% by the suppliers. These chemicals were stored at –30°C. Glyceryl trinitrate was supplied in glass ampoules as a sterile solution and was stored at 4°C in the absence of light.
Preparation of pharmacological supplements
All drug supplements for addition to pre- or post-storage perfusate or cardioplegic / storage solution were prepared fresh on the day of use.
Appropriate amounts of cariporide, INO 1153, Wortmannin or PD98059 were weighed out on a 6 place microbalance (Sartorius MC 210 S,
Goettigen, Germany), then dissolved in 0.5 ml DMSO to facilitate their
106 dissolution. These solutions were maintained at room temperature until they were added to the perfusate or Celsior. The GTN was supplied as a
50 mg/10 ml solution, and the appropriate amount was drawn up and added directly to Celsior.
Table 2.4 Chemicals and agents used in the preparation of lysis buffer for Western Blotting (Section 2.8).
Compound / Purity Source Sodium Chloride / AR Sigma-Aldrich (St Louis, Mo)
Trishydroxymethyl aminomethane Sigma-Aldrich (St Louis, Mo) (HCl salt) / AR Triton X-100 / AR Sigma-Aldrich (St Louis, Mo) Sodium Orthovanadate / AR Sigma-Aldrich (St Louis, Mo)
ß – Glycerophosphate / AR Sigma-Aldrich (St Louis, Mo) Dithiothreitol / AR Sigma-Aldrich (St Louis, Mo)
Roche Diagnostics Aust Pty Ltd Protease Inhibitor Cocktail (Castle Hill, NSW)
107 Table 2.5 Reagents and equipment used in protein preparation, electrophoresis and transfer (Section 2.10).
Reagents / equipment Source Bio – Rad Laboratories Pty. Ltd., Laemmli sample buffer Australia Media unit, Victor Chang Cardiac SDS (sodium dodecyl sulphate) Research Institute 30% Acrylamide/bis solution Bio – Rad Laboratories Pty. Ltd., (polyacrylamide) Australia Molecular weight marker (Precision Bio – Rad Laboratories Pty. Ltd., prestained maker) Australia Electrophoresis system (Mini Bio – Rad Laboratories Pty. Ltd., protean 3 ) Australia Bio – Rad Laboratories Pty. Ltd., Transfer system Australia Polyvinylidene difluoride (PVDF) Millipore, Australia membrane Sigma-Aldrich (St Louis, Mo) APS (Ammonium persulphate)
TEMED Bio – Rad Laboratories Pty. Ltd., (Tetramethylethylenediamine) Australia Tris-hydrochloride Sigma-Aldrich (St Louis, Mo)
2.2 ETHICAL CONDUCT OF EXPERIMENTAL STUDIES
All procedures were approved by the Animal Ethics Committee of the
Garvan Institute of Medical Research, Sydney, Australia (Animal Research
Authority reference numbers 03/24 and 06/25). All animals received humane care in compliance with the guidelines set down by the National
Health and Medical Research Council (Australia) and the “Guide for the
Care and Use of Laboratory Animals” (National Institute of Health,
Bethesda, MD).
108 2.3 ANIMALS
Adult male wistar rats weighing 300 - 380grams were used throughout all studies reported here. Animals were purchased from the Animal Resource
Centre in Western Australia and were housed in the Biological Testing
Facility at the Garvan Institute or more recently in the Biocore Facility attached to the Victor Chang Cardiac Research Institute until the day of surgery. They were housed two per cage and fed with standard rat pellets and sterile water was provided ad libitum. The animals were weighed at arrival and regularly thereafter until they had attained a weight of at least
300grams. This ensured an acclimatisation period of at least 1 week prior to their experimental use.
2.4 SURGICAL PROCEDURES
The following is an outline of the surgical methodology common to all studies.
2.4.1 Surgical instruments
Below is an illustration of the surgical instruments used for isolated heart preparations throughout the studies in this thesis.
109 1
6 2 3 4 5
Figure 2.1 Surgical instruments. Top: 1. 4.0 Silk black braided non absorbable suture. From left to right: 2. Medium toothed forceps; 3. Small forceps with serrated tip; 4. Microsurgical scissors, 5. 2 sets of dissecting scissors, 6.
Operating scissors curved, 14cm long.
2.4.2 Pre-medication and anaesthesia
Rats were anaesthetised with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg), followed by 200IU dose of heparin given intravenously via the right renal vein for anti-coagulation (Figure 2.2). To assess depth of anaesthesia, a toe pinch was performed to check for absence of withdrawal reflexes. Any “top-up” anaesthestic was achieved with 0.1ml dose of ketamine.
Figure 2.2 Rat was anti-coagulated by an injection of heparin into left renal vein.
110 2.4.3 Surgical removal of heart
Once the animal was anaesthetized and heparin administered, access to the heart was obtained via a transabdominal incision and the thoracic cavity was exposed by making lateral incisions along both sides of the rib cage. The anterior chest wall was folded back to expose the pericardium and tissues of the mediastinum (Figure 2.3).
Figure 2.3 Surgical exposure of the heart (arrowed).
Timing of duration of warm ischaemia was commenced at this point. The heart and the lungs were picked up with other chest contents pushed aside, then the heart was excised by transecting the great vessels, taking care to leave enough length on the aorta to facilitate insertion of the aortic cannula (Figure 2.4).
Figure 2.4 Excision of the heart – lung block.
111 The heart – lung block was immediately submerged in a beaker of ice cold perfusion buffer to arrest the heart. The heart was gently manipulated with the forceps to wash out as much blood as possible (Figure 2.5, below).
Figure 2.5 Hypothermic arrest of the heart.
The heart was then placed on a moistened, ice cold cotton swab, and the aorta was trimmed such that a 4 mm vertical stub remains. The patency of the aorta was then checked and unwanted connective tissue and thymus were then trimmed away (Figure 2.6).
Figure 2.6 Trimming the aorta.
112 2.4.4 Attaching the heart to perfusion circuit
The aortic arch was trimmed if necessary to increase the circumference of the aorta and allow easier insertion of the Langendorff perfusion cannula.
The heart was positioned on a bed of ice underneath the aortic cannula.
Using fine tipped forceps, the aortic arch was gently eased over the
Langendorff cannula (Figure 2.7a). The aorta was then clamped to the cannula with a small artery clip while a ligature was securely tied around after which the clamp was removed (Figure 2.7b).
a b
Figure 2.7 Cannulation of the aorta.
113 Retrograde perfusion was commenced at a hydrostatic pressure of 100 cm H2O as soon as the heart was securely positioned. Once retrograde flow had properly been established, timing of the warm ischaemic period was concluded. A small incision was further made in the pulmonary artery to allow adequate drainage of the coronary effluent (Figure 2.8).
Figure 2.8 Venting the pulmonary artery.
Once heart was securely attached to the cannula, the hila of the lungs were tied off and then excised and discarded (Figure 2.9a and b).
a b
Figure 2.9 (a) Ligation and (b) removal of lungs.
114 The heart was then gently rotated on the aortic cannula to expose the left atrial appendage (Figure 2.10a). An incision was made in the left atrial appendage for the working heart cannula (Figure 2.10b).
a b
Figure 2.10 (a) Rotation of the heart to gain access to left atrial appendage and
(b) Preparation of the cannulation site on the left atrial appendage (left atrial cannula arrowed).
The primed left atrial perfusion cannula was then gently inserted and temporarily secured with a small artery clip (Figure 2.11a) then secured with another 0 silk tie (Figure 2.11b).
a b
Figure 2.11 (a) Clipping and (b) securing the left atrial cannula with a silk tie.
115 With both cannulae now in place and secured, the flows could now be changed to allow perfusate to travel in the physiological direction through the left atrium and left ventricle after which it was ejected through the aorta
(the working heart preparation) (Figure 2.12). After antegrade flow has been initiated, the heart is carefully inspected for leaks around both cannulation sites that may artefactually increase the coronary flow. If a leak is found, repair is attempted. If attempt is unsuccessful, the heart is excluded from analysis.
Figure 2.12 Completed working heart preparation showing aortic cannula (white arrow), left atrial cannula (red arrow). The coronary effluent of the ejecting heart
(blue arrow). Under experimental conditions, the water-jacketed chamber (yellow arrow) surrounds the heart to maintain a constant temperature. The coronary effluent is collected in the chamber and recycled.
116 2.4.5 Cardiac Functional Parameters Measured in a Typical Donor
Heart Preservation Study
Measurements of cardiac functional parameters common to all studies are outlined below. After aortic cannulation, all hearts were perfused in
Langendorff mode for a period of 10 minutes during which time left atrial cannulation was completed and the hearts were given time to equilibrate.
Hearts were then perfused in working mode for 15 minutes. The specific indices of cardiac function that were measured were aortic flow, coronary flow, cardiac output and heart rate.
Aortic Flow
Aortic flow was measured by an in-line ultrasonic flow probe positioned at the outlet of the aortic cannula which was connected to a flow meter
(Transonics Instruments Inc. Ithaca, NY) (Figure 2.13).
a b
Figure 2.13 (a) Transonics flow probe (arrowed) positioned at the outlet of the aortic cannula and (b) the flow meter to which it was connected.
117 The data were sent to a MacLab / 4E realtime acquisition system and displayed using Chart 3 solfware (ADInstruments Pty Ltd, Sydney,
Australia) (Figure 2.14).
Figure 2.14 MacLab data acquisition system (arrowed) connected to flowmeter above. Aortic flow is displayed in the centre third of the screen.
Pressure
The pressure generated by the heart was monitored throughout by a pressure transducer (Ohmeda, Pty Ltd., Singapore), located level with the heart and was also displayed using Chart 3 software.
Coronary Flow
Coronary flow during working heart mode was measured by timed collection (1 minute intervals) of the effluent draining from the apex of the heart. The volume collected is recorded on the trace at the conclusion of the collection period.
118 Cardiac Output
Cardiac output during working mode, a measure of cardiac contractility or left sided heart function was derived from the sum of aortic flow and coronary flow.
Timing of Reported Observations and Exclusions
Throughout all the studies, “Baseline Observations” were taken at the end of the initial 15 minutes of working heart mode, immediately prior to arrest and storage of the heart. If at this point, aortic flow was less than 30 ml/min, coronary flow was less than 10 ml / min or heart rate was less than
200 beats / min, hearts were excluded from the study. Additionally, hearts with irreparable leaks at the ligature lines were also excluded.
After the storage period, the heart was remounted on the rig then perfused for 15 minutes in Langendorff mode then working mode for a further 30 minutes. At the end of this post-storage working mode period, functional observations were repeated. Recovery of cardiac function was then expressed as a percentage of the pre-storage baseline.
2.5 CARDIOPLEGIA AND STORAGE
At the end of 15 minutes of working heart mode, the left atrial cannula, afterload and Langendorff preload cannulae were clamped off.
Cardioplegic solution (Celsior) was then infused via a sidearm in the
Langendorff preload tubing. Celsior was infused under gravity from an
119 open topped-chamber that is filled with a 50ml volume. This chamber was located 70cm above the heart, giving an initial infusion pressure of 70 cm
H2O. The first 15 ml of cardioplegic solution was bled off from the pressure sidearm to quickly remove remaining perfusion buffer in the tubing. The heart was then infused for 3 minutes and the time to arrest, final flow rate at 3 minutes, and total volume infused was noted. The trace recording was stopped when asystole was seen on the flow trace.
Following cardioplegia, the aortic and left atrial cannulas were clamped off and the heart was stored in a 100ml beaker (Figure 2.16) of Celsior for 6 or 10 hours at 1-4°C, depending on specific protocol. See Chapters 3, 4 and 5 for specific details.
Figure 2.15 Arrested heart still mounted to the perfusion cannulae is stored in a beaker of 100 ml of pre-chilled Celsior storage solution. The beaker and heart is then immediately transferred to an esky of ice.
120 2.6 COLLECTION AND PREPARATION OF HEART FOR MOLECULAR
STUDIES AND HISTOLOGY
After completion of collection of the post-storage cardiac functional indices, the left ventricular free wall of the heart was dissected out and bisected on an ice – cold moist surface. Half was frozen by immersion in liquid nitrogen then stored at – 80°C for Western blot analysis and the other half was fixed in 4% paraformaldehyde (Sigma-Aldrich, St Louis,
MO) for subsequent histological analysis. The timing of the collection of individual experimental treatments is outlined in the Methods sections of
Chapters 3, 4 and 5.
2.7 PREPARATION OF TISSUE LYSATES FROM CARDIAC TISSUE
A quantity of 60mg of left ventricular wall tissue from each heart was homogenized separately (Omni International GLH homogenizer, Marietta
GA, USA) in ice cold lysis buffer (150 mM NaCl, 50 mM Tris HCl, 1%
Triton X-100, 1 mM sodium orthovanadate, 1 mM ß-glycerophosphate, 5 mM DTT, 15% Roche cocktail of protease inhibitors, pH 7.4), see Table
2.4. Samples were then centrifuged (Eppendorf AG, model 5415R,
Hamburg, Germany) at 10,000 rpm for 5 minutes at 4°C. Protein concentration of each lysate was measured using a Bradford assay kit, with bovine serum albumin used as standard (Pierce Biotechnology Inc,
Rockford IL).
121 2.8 SDS-PAGE AND IMMUNO (WESTERN) BLOTTING CONDITIONS
Protein samples were boiled in laemmli sample buffer for 5 minutes before loading onto either 8% or 10% SDS-polyacrylamide gels (40 µg per lane).
SDS-polyacrylamide gels of 0.75 mm thickness were cast using a mini
Protean 3 electrophoresis system with a 15 well comb. Table 2.6a and
2.6b show the components used to cast SDS-polyacrylamide stacking and resolving gels. After electrophoretic separation on the mini Protean 3 system, proteins were transferred to polyvinylidene difluoride (PVDF) membrane. Conditions for electrophoretic separation and transfer process are listed in table 2.7. The molecular weight marker used was a precision plus protein pre-stained standard of sizes between 10 – 250 kDa.
Membranes were blocked for 1 hour in Tris buffered saline, (pH 7.4) which contained 1% BSA and 0.1% Tween20. Membranes were probed overnight with a range of antibodies to either PI3-K /Akt, ERK 1/2, or their down stream targets. All antibodies used in the thesis are listed below
(Table 2.8). See Methods sections in Chapters 3, 4 and 5 for the particular primary antibodies and dilutions used in each study. The secondary antibodies used were a horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG. The protein bands were visualized using enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL). Band intensities were digitised with Image J freeware (National Institute of Mental Health,
Bethesda, MA).
122 Table 2.6 Components for casting of SDS-polyacrylamide (a) separation and (b) stacking gels.
(a)
Components for 8% gel 10% gel Separation gel (10mL, total 2 gels) (10mL, total 2 gels)
H2O 4.6 mL 4.0 mL 30% acrylamide/bis 2.7 mL 3.3 mL solution 1.5M Tris-HCL pH 8.8 2.5 mL 2.5 mL 10% SDS 0.1 mL 0.1 mL 10% APS 0.1 mL 0.1 mL TEMED 0.006 mL 0.004 mL
(b)
5% gel Components for Stacking gel (5 mL, total 2 gels)
H2O 3.4 mL 30% acrylamide/bis solution 0.83 mL 1.0M Tris-HCL pH 6.8 0.63 mL 10% SDS 0.05 mL 10% APS 0.05 mL TEMED 0.005 mL
Table 2.7 Conditions of electrophoresis and transfer process.
Electrophoretic Conditions Transfer process (Wet) separation Power 130 Volts 105 Volts
Run time 70 minutes 80 minutes Temperature Room temperature 4oC or with ice pack
123 Table 2.8 List of antibodies used throughout study.
Antibodies Source Akt Cell Signaling Technology, MA, USA Phosphorylated Akt (Ser 473) Cell Signaling Technology, MA, USA
Erk1/2 (p44/p42 MAPK) Cell Signaling Technology, MA, USA
Phosphorylated Erk1/2 (phospho- Cell Signaling Technology, MA, USA p44/p42 MAPK, Thr202/Tyr204)
Bcl2 Cell Signaling Technology, MA, USA Phosphorylated Bcl2 (Ser 87) Santa Cruz Biotechnology, CA, USA Bad Cell Signaling Technology, MA, USA Phosphorylated Bad (Ser 136) Cell Signaling Technology, MA, USA GSK3β Cell Signaling Technology, MA, USA Phosphorylated GSK3β (Ser9) Cell Signaling Technology, MA, USA Caspase 3 Cell Signaling Technology, MA, USA Cleaved caspase 3 (Asp175) Cell Signaling Technology, MA, USA
eNOS Cell Signaling Technology, MA, USA
Phosphorylated eNOS (Ser113) Cell Signaling Technology, MA, USA
ERM (Ezrin/Radixin/Moesin) Cell Signaling Technology, MA, USA
Phosphorylated Ezrin (Thr567) / Cell Signaling Technology, MA, USA Radixin (Thr564) / Moesin (Thr558)
Phospholamban (2D12) Abcam Inc, MA, USA
Phosphorylated phospholamban Abcam Inc, MA, USA (phospho S16)
β-actin Cell Signaling Technology, MA, USA
HRP conjugated IgG (anti-rabbit) Amersham Biosciences Australia
HRP conjugated IgG (anti-mouse) Amersham Biosciences Australia
124 2.9 PROCESSING OF TISSUE FOR IMMUNOHISTOCHEMICAL AND
IMMUNOFLUORESCENCE ANALYSIS
Immunohistochemistry
Accumulation of poly(ADP-ribose), the product of poly(ADP-ribose) polymerase, was used as an indicator of PARP activity. Samples of left ventricular free wall of the heart were fixed in 4% paraformaldehyde and embedded in paraffin after which 5 µM sections were cut. These sections were deparifinised in xylene then rehydrated through a series of decreasing concentrations of ethanol. Antigen was retrieved by incubating sections in 0.1M sodium citrate (pH 6.0) in a microwave oven for 4 minutes
(“high” setting) then 5 minutes (“medium” setting). Sections were then cooled for 20 minutes.
Non-specific binding was blocked by incubating slides in 2% (w/v) skimmed milk / 2% (v/v) Triton X-100 in Tris buffered saline (137mM NaCl,
10mM Tris, pH 7.4) for 30 minutes at room temperature. Sections were then treated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. Sections were then incubated in a monoclonal anti-PAR antibody (isotype mouse IgG) (Alexis Biochemicals,
Lausen, Switzerland) at a dilution of 1:200 for 1 hour at room temperature, then treated with a secondary HRP-labelled anti-mouse IgG anti-body for
30 minutes (also at room temperature) (Novolink Polymer Detection
Systems, Vision BioSystems Ltd, Mount Waverley, Australia). The
125 presence of the PAR antibody was detected by the brown staining chromophore, diaminobenzadine (DAB). Sections were counterstained with haematoxylin, dehydrated and mounted in VECTASHIELD HardSet mounting medium (Vector Laboratories, Burlingame, CA).
Photomicrographs were taken using an Olympus BX51 microscope equipped with an Olympus DP70 digital camera.
Immunofluorescence
Rat hearts were fixed in 4% paraformaldehyde and embedded in paraffin blocks. Sections (5 µm thick) were cut and de-paraffinized in xylene then rehydrated through a series of decreasing concentrations of ethanol then phosphate buffered saline (pH 7.4). Sections were then treated with 10% goat serum in phosphate buffered saline containing 0.2% Triton X-100 (pH
7.4) for 30 minutes. Sections were then incubated in a cleaved caspase 3 primary antibody (Cell Signaling Technology, Danvers, MA) (diluted 1:150) at 4°C for 12 hours. The primary antibody was detected with goat anti- rabbit IgG labeled with Alexa Fluor 555 (diluted 1:300) (Molecular Probes,
Invitrogen). Sections were then incubated with mounting media (Vector
Shield) containing 4’, 6-diamidino-2-phenylindole (DAPI) and coverslips applied onto the slides and sealed with nail polish. The labeled antibody was observed by fluorescence microscopy. For quantification of cleaved caspase 3 immunofluorescence, the area of caspase 3 positive staining was scored in each of 10 random 60x magnification fields in each experimental group.
126 2.10 STATISTICAL ANALYSIS
All functional data and antibody intensities were expressed as mean ± SE unless otherwise specified. Data were analyzed by the StatView 4.5 statistical software package. Differences among groups were compared by one-way ANOVA analysis followed by Fisher’s PLSD post hoc test, a P value of less than 0.05 was considered significant.
127 CHAPTER 3
Improved Post-storage Cardiac Function by Poly (ADP-
ribose) Polymerase Inhibition:
Role of Phosphatidylinositol 3-kinase Akt Pathway
128 3.1 INTRODUCTION
Heart transplantation has become established as an effective therapy for patients with end – stage heart disease. However, success of the procedure is limited by early and late failure of the transplanted heart. It is now recognized that ischemia – reperfusion injury sustained by the allograft during the transplant process is a major determinant of both short and long term outcome (Hosenpud et al., 2001). Importantly, of all the factors associated with early allograft failure, ischemia – reperfusion injury is one of the few factors that is amenable to therapeutic intervention (Hicks et al., 2006). Although disparate mechanisms leading to tissue and organ dysfunction after ischemia/reperfusion have been proposed, formation of oxygen and nitrogen centered radicals as well as peroxynitrite at reperfusion may underpin a number of observed pathological sequelae
(Zweier et al., 2006). Peroxynitrite has been shown to be responsible for formation of single stranded DNA and resultant activation of poly (ADP- ribose) polymerase (PARP) (Szabo et al., 2004a).
The PARP family of enzymes has many roles in cellular homeostasis, including transcriptional regulation of a number of proteins, detection of
DNA strand breaks and initiation of repair to damaged DNA (Burkle,
2005). In addition to these physiological functions, sustained PARP activation after ischemia reperfusion is thought to play a role in initiation of cell death through apoptosis (Yu et al., 2002) or by profound depletion of
NAD+ and ATP resulting in necrosis (Virag et al., 2002).
129 Proof of principle of the role of PARP activation in ischemia – reperfusion injury has been demonstrated in PARP knock – out mice, where deletion of the PARP gene provided significant protection against molecular and functional changes following myocardial ischemia – reperfusion and infarction (Grupp et al., 1999, Zingarelli et al., 1998) as well as cerebral ischemia (Eliasson et al., 1997), lung inflammation (Liaudet et al., 2002) and shock (Oliver et al., 1999).
During the past decade, structure based drug design has facilitated the synthesis of a series of highly potent PARP inhibitors which have verified the protective effects demonstrated in PARP knock – out models (Jagtap et al., 2005). Pharmacological inhibition of PARP has been shown to be beneficial in a number of models of cardiac ischemia reperfusion injury.
Improved left ventricular function, decreased oxidative stress and decreased apoptosis was observed in a global ischemia reperfusion model in the rat supplemented with 3-aminobenzidine (Yamazaki et al., 2004). In a Fisher rat model of age - associated heart failure, chronic exposure to the PARP inhibitor INO 1001 resulted in improved left ventricular systolic and diastolic left ventricular function and improved acetylcholine – responsive relaxation of aortic rings (Pacher et al., 2004). In a porcine model of regional cardiac ischemia produced by ligation of the left anterior descending coronary artery, post reperfusion infarct size and cardiac function was improved when INO1001 was given at the time of reperfusion
(Khan et al., 2003). This agent also slowed cardiac rejection and improved
130 allograft function in a rat heterotopic model of acute rejection(Szabo et al.,
2006b). Limited information has been obtained in the area of the application of PARP inhibitor as strategy to optimize donor organ preservation during organ transplantation (Szabo et al., 2005b).
Whilst the prevention of severe, acute energy depletion was initially thought to be the prime cause of the protective effect of PARP inhibition
(Virag et al., 2002), recent studies have also shown that inhibition of PARP is associated with activation of Akt (Pacher et al., 2004, Tapodi et al.,
2005, Veres et al., 2003). Interest in recruitment and activation of pro- survival kinase pathways as an underlying protective principle against ischemia reperfusion injury has been heightened since it has been found that a number of protective physiological and pharmacological pre – and post-conditioning strategies involve up-regulation of the phosphatidylinositol 3-kinase (PI 3-kinase) /Akt pathway (Hausenloy et al.,
2005b, Hausenloy et al., 2004b).
The aims of the present study, using an isolated working rat heart model of donor heart preservation are to 1) measure the recovery of post-storage function of hearts treated with the PARP inhibitor, INO-1153, 2) to optimise timing of the delivery of INO-1153 and 3) to determine whether the activation of PI 3-kinase / Akt pathway is involved in the protective effects of INO-1153 administration.
131 3.2 MATERIALS AND METHODS
Animals
As described in Chapter 2, section 2.3.
Ethical conduct of studies
As described in Chapter 2, section 2.2.
Chemicals and pharmacological agents
As described in Chapter 2, section 2.1.
Preparation of perfusion buffer
As described in Chapter 2, section 2.1.
Isolated working heart preparation
A detailed account of the surgical steps in preparing the isolated heart appears in Chapter 2, section 2.4. Briefly, the rats were anaesthetised with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). After bolus injection of 500 IU heparin into the renal vein, the heart was rapidly excised and arrested by immersion in chilled (2 – 3°C) perfusion buffer. The aorta was cannulated and immediately perfused retrogradely on a Langendorff perfusion apparatus with Krebs buffer at a hydrostatic pressure of 100 cm H2O. During this time, a small incision was made in the left atrial appendage into which another cannula was inserted
132 and tied off. This non-working preparation was stabilised for 10 minutes and then converted to working mode by switching the supply of perfusate from the aorta to the left atrial cannula at a hydrostatic pressure of 20 cm
H2O (pre-load). The working heart ejected perfusate via the aortic valve into the aortic cannula. The hydrostatic pressure in the aortic cannula was maintained at 100 cm H2O (after-load) throughout the working phase for all rat hearts.
Aortic pressure was monitored in a side arm of the aortic cannula with a pressure transducer (Ohmeda, Pty Ltd., Singapore). Aortic flow was measured by an in-line flowmeter (Transonics Instruments Inc. Ithaca,
NY). Aortic pressure and flow were recorded using MacLab/4e
(ADInstruments Pty Ltd, Sydney, Australia) and heart rate was calculated from the flow trace. Coronary flow was measured by timed collection of the effluent draining from the apex of the heart.
Experimental protocol (Figure 3.1)
All hearts remained in working mode for 15 min prior to storage.
Measurements of heart rate (HR), aortic flow (AF), coronary flow (CF), and cardiac output (CO) were made at 10 minutes after conversion to working mode and used as pre-storage baseline.
Exclusion criteria: Any hearts having a baseline aortic flow less than 35 ml/min, heart rate of less than 200 beats/min, or coronary flow less than
10 mL/min were excluded.
133 After collection of baseline hemodynamic data, the heart was arrested by infusion of cold Celsior preservation solution (at 2–3°C) into the coronary circulation for 3 minutes from a reservoir 60 cm above the heart. All hearts were stored in 100 mL of the same solution for 6 hours under hypothermic conditions (2–3°C). Hearts were then remounted on the perfusion apparatus and reperfused in Langendorff mode for 15 minutes. Hearts were then switched to working mode then stabilized for 30 minutes. The indices of cardiac function measured at baseline were then rerecorded.
Recovery of each parameter was expressed as a percentage of its pre- storage baseline. After the 30 minutes functional observations were taken,
(i) sections of the left ventricular free wall of each heart was rapidly frozen by immersion in liquid nitrogen and stored at -80 °C for Western blot analysis; (ii) a transverse section of each heart was collected and stored in paraformaldehyde prior to embedding for histology analysis.
Experimental groups (Figure 3.1)
Rat hearts were randomly assigned to 1 of the 5 groups according to the treatment they received.
Group 1: Control group, (n = 9) in which rat hearts received no interventional treatment and were stored in Celsior preservation solution for 6 hours.
Group 2: Pre-storage, (n = 8), in which 1 µM INO-1153 was added to
Krebs pre-storage perfusion buffer only. This concentration of INO-1153 was based on pilot studies performed to determine the maximum
134 concentration of the agent which could be used without direct effect
(positive or negative) on aortic flow, coronary flow, cardiac output or heart rate.
Pre-storage Phase Storage Post-Storage Phase Phase Langendorff (2-3°C) Langendorff Working Heart Working Heart
10 min 15 min 6 hrs 15 min 30 min Treatment Groups
1. Control Krebs Celsior Krebs Celsior Krebs 2. INO-1153 pre-storage Krebs+1µM INO-1153 3. INO-1153 cardioplegia Krebs Celsior + 1µM INO-1153 Krebs & storage
4. INO-1153 post-storage Krebs Celsior Krebs + 1µM INO-1153 5. Wortman’ prestorage Krebs + Celsior + +INO-1153 cardioplegia 0.1µM Wortmannin 1µM INO-1153 Krebs & storage *
⇑ ⇑
Figure 3.1 Experimental protocol and treatment groups. Arrows indicate times at which measurements of cardiac function were made, i.e. heart rate (HR), aortic flow (AF), coronary flow (CF), and cardiac output (CO). ( * ) indicates the point where the heart tissue was harvested for histology and western immunoblotting analysis.
Group 3: Storage, (n = 9), in which 1 µM INO-1153 was present in the
Celsior storage solution during cardioplegia and 6 hours of hypothermic storage.
Group 4: Post-Storage, (n = 8), in which 1 µM INO-1153 was added to
Krebs perfusion buffer during post-storage reperfusion.
135 Group 5: Wortmannin / INO-1153 (n = 6), in which hearts were exposed to
0.1 µM Wortmannin (an inhibitor of PI 3-kinase) during pre-storage perfusion then arrested and stored in Celsior solution supplemented with 1
µM INO-1153.
Western immunoblotting
Briefly, a subgroup of 3 hearts was chosen from each experimental group for Western blot analysis and sixty milligrams of tissue from each heart were homogenized in cold lysis buffer (Chapter 2, section 2.7). Protein samples (30 µg) were boiled in sample loading buffer for 5 minutes before loading onto 8% SDS-polyacrylamide gel under reducing conditions. After electrophoretic separation, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Australia) and membranes were blocked for 1 hour in Tris buffered saline, (pH 7.4) which contained 1%
BSA and 0.1% Tween 20 (described in Chapter 2, section 2.8).
Membranes were then incubated overnight with primary rabbit polyclonal antibodies (1: 3000 dilution) against total Akt, phospho – Akt (Ser 473) and
β-actin (all obtained from Cell Signaling Technology, Danvers, MA).
Membranes were then exposed to a horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Life Sciences) secondary antibody. The protein bands were visualized using enhanced chemiluminescence (Amersham
Life Sciences). Band intensities were normalized against β-actin loading control and digitised.
136 Immunohistochemistry
To assess the extent of PARP activation in group 1 (control hearts) and group 3 (INO 1153 treated hearts), a monoclonal antibody to poly(ADP- ribose) was used to detect the product of PARP reaction (Chapter 2, section 2.9).
Statistical Analysis
All functional data and antibody intensities were expressed as mean ± SE unless otherwise specified. Data were analyzed by the StatView 4.5 statistical software package. Differences among the groups were compared by one-way ANOVA analysis followed by Fisher’s PLSD post hoc test, a P value of less than 0.05 was considered significant.
3.3 RESULTS
3.3.1 Effect of pharmacological agents on baseline measurements of
cardiac function
Table 3.1 shows the prestorage baseline measurements of cardiac function for the control and each of the 4 treatment groups. All groups were equivalent at baseline with no significant differences were observed between groups for any functional parameter. Importantly, the inclusion
INO-1153 (group 2) or Wortmannin (group 5) in the Krebs-Henseleit perfusion buffer had no direct effect on cardiac function at the concentrations at which they were used.
137 Table 3.1 Pre-storage baseline cardiac measurements and values.
AF CF CO Treatment Groups HR (bpm) n (ml/min) (ml/min) (ml/min)
1. Control 45 ± 9.1 21 ± 2.9 66 ± 7.6 256 ± 29.0 9
2. INO-1153 pre-storage 51 ± 6.2 20 ± 1.6 71 ± 6.9 260 ± 30.0 8
3. INO-1153 storage 46 ± 4.9 20 ± 1.3 65 ± 5.1 262 ± 18.7 9
4. INO-1153 post-storage 42 ± 5.6 19 ± 1.8 61 ± 5.5 266 ± 29.0 8
5. Wortmannin + INO-1153 44 ± 8.5 19 ± 1.9 63 ± 9.6 255 ± 19.7 6
AF=Aortic flow, CF=Coronary flow, CO=Cardiac output, HR=Heart rate,
N=number of rat hearts. Data are expressed as means ± SD in each parameter.
3.3.2 Effect of INO-1153 treatment during heart preservation
improves post-storage cardiac function
The recovery of function of control hearts (no treatment) after 45 minutes reperfusion was uniformly poor (Figures 3.2 and 3.3). Previous work in our laboratory with an identical experimental model showed DMSO had no significant effect on heart recovery (Cropper et al., 2003).
After 6 hours of cold storage, post storage function of hearts was better preserved by INO-1153 treatment. Rat hearts receiving INO-1153 either before storage, at cardioplegia and during storage or at reperfusion demonstrated significantly improved recovery of coronary flow compared with the control group (79.9 ± 4.3 %, 77.6 ± 2.7 % & 67.4 ± 6.1 % vs 41.4
± 11.8 % P < 0.05) (Figure 3.2 (a)). Similarly, recovery of heart rate also
138 improved significantly independently of the timing of INO-1153 exposure
(99.1 ± 4.3 % & 96.5 ± 3.9 % vs 54.2 ± 14.2 %) (Figure 3.2 (b)). Left-sided heart function (aortic flow and cardiac output) was improved in all INO-
1153 treatment groups, however the improvement only reached significance when INO-1153 was present before cardiac storage or at cardioplegia and during storage when compared to control levels (43.6 ±
5.6 % & 41.3 ± 8.6 % vs 14.9 ± 7.7 %, P < 0.05 for aortic flow and 54.2 ±
4.3 % & 52.4 ± 6.4 % vs 24.1 ± 9.5 %, P < 0.05 for cardiac output)
(Figures 3.3 (a) and 3.3 (b)).
139 (a) Coronary flow * * 80 *
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Figure 3.2 Coronary flow (a) and Heart rate (b) represented as a percentage of baseline values in hearts after 45 minutes post-storage reperfusion following 6 hours hypothermic storage. Recovery of post-storage cardiac function for INO-1153 treatment groups 1 – 4 (control, pre-storage, cardioplegia and storage, post-storage). Values are expressed as mean ± SE. * P < 0.05 vs
Control, # P = 0.05 vs Control, ANOVA.
140
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0 Control Pre- Cardioplegia Post- storage & Storage storage
(b) Cardiac output
60 * *
) 50
e
y
lin
r 40
e
e
s
v
a 30
o
b
c
f e 20
o
R
% 10
(
0 Control Pre- Cardioplegia Post- Storage & Storage Storage
Figure 3.3 Aortic flow (a) and Cardiac output (b) represented as a percentage of baseline values in hearts after 45 minutes post-storage reperfusion following 6 hours hypothermic storage. Recovery of post-storage cardiac function for INO-1153 treatment groups 1 – 4 (control, pre-storage, cardioplegia and storage, post-storage). Values are expressed as mean ± SE. *
P < 0.05 vs Control, ANOVA.
141 3.3.3 Effect of INO-1153 on cardiac poly(ADP-ribose) deposition
The purpose of Figures 3.4 a and b was to demonstrate that at the concentration used in the study, (1µM), INO-1153 was acting as an inhibitor of polyADP-ribose polymerase. Control hearts that were not exposed to INO-1153 at any stage during the study, showed enhanced nuclear immunoreactivity for the poly(ADP-ribose) antibody which demonstrated PARP activation after hypothermic storage and reperfusion
(Figure 3.4a). Hearts that had been arrested and stored in Celsior preservation solution supplemented with 1µM INO-1153 showed markedly attenuated nuclear poly(ADP-ribose) deposition after hypothermic storage and reperfusion (Figure 3.4b). The brown staining of all nuclei is indicative of PAR accumulation (from poly(ADP-ribose) polymerase activation). The decrease in the number of brown staining nuclei and the presence of a large number of blue (counterstained with Hematoxylin) nuclei indicated that the addition of INO-1153 has markedly attenuated PAR deposition and decreased PARP activity.
142 (a)
(b)
Figure 3.4 Representative immunohistochemical sections from control
(group 1) and INO-1153 treated hearts (group 3) subjected to 45 minutes post-storage reperfusion following 6 hours hypothermic storage. (a) control heart and (b) 1 µM INO-1153 present during cardioplegia and storage. Positive staining is indicated by the brown stained nuclei of cardiomyocytes in the reperfused myocardium. Magnification 40x.
143 3.3.4 Effect of Wortmannin pretreatment against improved post-
storage cardiac function by INO-1153 treatment
When rat hearts were pretreated with the phosphatidylinositol 3-kinase inhibitor Wortmannin before cardioplegia and storage in INO-1153 – supplemented Celsior, all cardio-protective effects observed with INO-
1153 treatment alone were completely abolished. Figure 3.5 shows representative traces of arterial flow and heart rate for untreated (control) group and hearts arrested and stored in Celsior supplemented with 1µM
INO-1153 in the absence and presence of Wortmannin pretreatment. The typical control heart shows no post-reperfusion recovery of either aortic flow or heart beat, whilst the heart stored in INO-1153 – supplemented
Celsior shows immediate recovery of both aortic flow and heart beat upon post-storage reperfusion. Where the heart pre-treated with Wortmannin prior to storage, the functional improvement was decrease to untreated control levels.
Figure 3.6 shows the group data for all indices of cardiac function. Again, the significant improvement brought about by the PARP inhibitor was lost after the PI 3-k / Akt pathway was blocked.
144 Aortic Flow Heart Rate
Group1
Control
Group 3 INO-1153 in cardioplegia & storage
Group 5 Wortmannin pre- treated + INO-1153 in cardioplegia & storage
pre- post reperfusion pre- post reperfusion storage storage
Figure 3.5 Representative trace recordings of Aortic Flow (ml/min) and
Heart Rate (bpm) at baseline and after 45 minutes post-storage reperfusion following 6 hours hypothermic storage for treatment groups. Control hearts
(group 1); 1 µM INO-1153 treated hearts during cardioplegia and hypothermic storage (group 3); Hearts pretreated with 0.1 µM Wortmannin during prestorage perfusion then arrested and stored in 1 µM INO-1153 (group 5).
145
100 *
* 80
60 * * 40
20
(% baseline) Recovery of
0 Aortic Coronary Cardiac Heart Flow Flow Output Rate
Figure 3.6 Effect of PI3-k inhibition using Wortmannin on recovery of post- storage cardiac function. Treatment groups are: Control hearts (group 1);
1 µM INO-1153 present at cardioplegia and during hypothermic storage
(group 3); Hearts pretreated with 0.1 µM Wortmannin during prestorage perfusion then arrested and stored in 1 µM INO-1153 (group 5). Values are expressed as mean ± SE. * P < 0.05 vs Control, ANOVA.
146 3.3.5 Effects of Wortmannin and INO-1153 treatment on Akt
activation after post storage
Figure 3.7 (a) shows typical phosphorylation status of Akt in untreated controls, (lane 1), hearts exposed to INO-1153 during cardioplegia and storage (lane 2) and hearts pre-treated with Wortmannin before cardioplegia and storage in INO-1153 (lane 3). Quantitation of band intensity is shown in Figure 3.7 (b). Any differences in lane loadings were accounted for by dividing band intensities for phospho and total Akt by β- actin intensities for each blot (ie I(phospho-Akt) / I (β-actin) and I(total Akt) / I (β-actin)).
A ratio of normalised phospho-Akt / total Akt was then calculated for a random sub-set of 3 hearts per experimental treatment. The presence of
INO-1153 during cardioplegia and storage resulted in a 4.3 fold increase in phospho-Akt compared with untreated control (1.16 ± 0.37 vs 0.27 ± 0.07
P < 0.05). This increase was completely inhibited by the presence of the
PI 3-kinase / Akt pathway inhibitor, Wortmannin.
147 (a)
Phospho-Akt
Total Akt
β-Actin
(b)
* 1.5
1.0
phospho / 0.5 Total AktTotal Ratio
0
1µM INO-1153 - + +
0.1µM Wortmannin -- +
Figure 3.7 Representative Western immunoblots and histogram. (a) Akt phosphorylation, Akt and β–actin levels respectively in hearts and (b) Intensities quantification after 45 minutes post-storage reperfusion following 6 hours hypothermic storage. Quantification of band intensities showing ratio of phos-Akt
/ Akt (n = 3 each). Values are means ± SE. * P < 0.05 vs Control, ANOVA.
148 3.3.6 Effects of Wortmannin and INO-1153 treatment on Erk1/2
activation after post storage
Figure 3.8 (a) shows phosphorylation status of ERK in a subset of 3 untreated control hearts, (lane 1), hearts exposed to INO-1153 during cardioplegia and storage (lane 2) and hearts pre-treated with Wortmannin before cardioplegia and storage in INO-1153 (lane 3). Quantitation of band intensities is shown in Figure 3.8 (b). Any differences in lane loadings were accounted for by dividing band intensities for phospho and total ERK
1/2 by β-actin intensities for each blot (as described for Figure 3.7 (b)).
The presence of INO-1153 during cardioplegia and storage resulted in a
2.4 fold increase in phospho-ERK 1 compared with untreated control (0.39
± 0.20 vs 0.16 ± 0.09 P < 0.05) with no apparent increase observed in the phosphorylation status of ERK 2. Again this increase was completely inhibited by the presence of the PI 3-kinase / Akt pathway inhibitor,
Wortmannin.
149 (a)
Phospho-Erk1/2
Total Erk1/2
(b) 1
0.8
* 0.6
Total Erk1/2 0.4
Ratioof Phospho-Erk1/2 / 0.2
0
1µM INO-1153 - + +
0.1µM Wortmannin --+
Figure 3.8 Representative Western immunoblots showing (a) Erk1/2 phosphorylation and Total Erk1/2 levels in hearts and (b) intensities quantification after 45 minutes post-storage reperfusion following 6 hours hypothermic storage.
Quantification of band intensities showing ratio of phos-Erk1/2 / total Erk1/2 (n =
3 each). Values are means ± SE. * P < 0.05 vs Control, ANOVA.
150 3.3.7 Effects of Wortmannin and INO-1153 treatment on
phospholamban phosphorylation after post storage
The phosphorylation status of phospholamban and its interaction with the sarcoplasmic reticulum Ca2+ - ATPase is an important determinant of cardiac contractility (MacLennan et al., 2003). It has also been identified as a down-stream target of the PI 3-k / Akt pathway (Abdallah et al.,
2006).
Figure 3.9 (a) shows immunoblots of phospholamban phosphorylation levels in a sub-set of 3 control hearts (group1), hearts arrested and stored in INO-1153 - supplemented Celsior (group 3) and hearts pre-treated with
Wortmannin before arrest and storage in INO-1153 - supplemented
Celsior (group 5). Quantification of band intensities (expressed as a ratio of p-PLB / total PLB) is shown in Figure 3.9 (b). This shows a small but significant increase in the p-PLB / total PLB ratio for hearts stored in INO-
1153 in the absence of Wortmannin pre-treatment compared to untreated control hearts or Wortmannin pre-treated hearts.
151 (a)
Phospho- phospholamban Total
Phospholamban
(b) 1
0.8 *
0.6
0.4
phospholamban total / 0.2
of Phos-phospholamban Ratio 0
1µM INO-1153 - + +
0.1µM Wortmannin -- +
Figure 3.9 Representative Western immunoblots showing (a) phospholamban phosphorylation and Total phospholamban levels in hearts and (b) intensities quantification after 45 minutes post-storage reperfusion following 6 hours hypothermic storage. Quantification of band intensities showing ratio of phospho- phospholamban / total phospholamban (n = 3 each). Values are means ± SE. * P
< 0.05 vs Control, ANOVA.
152
3.3.8 Effects of Wortmannin and INO-1153 treatment on eNOS
activation after post storage
Figure 3.10 (a) shows immunoblots of phosphorylated and total endothelial nitric oxide synthase for a sub-set of 3 control hearts (group1), hearts arrested and stored in INO-1153 - supplemented Celsior (group 3) and hearts pre-treated with Wortmannin before arrest and storage in INO-
1153 - supplemented Celsior (group 5). Quantification of the phospho eNOS / total eNOS ratios for each of these treatment groups (Figure 3.10
(b)) showed no differences between any of the groups at the time the hearts were sampled (ie 45 minutes after post-storage reperfusion).
153
(a)
Phos-eNOS
Total eNOS
(b) 1
0.8
0.6
0.4 Total eNOS
0.2 Phospho-eNOS Ratio of /
0
1µM INO-1153 - + + 0.1µM Wortmannin --+
Figure 3.10 Representative Western immunoblots showing (a) eNos phosphorylation and Total eNOS levels in hearts and (b) intensities quantification after 45 minutes post-storage reperfusion following 6 hours hypothermic storage.
Quantification of band intensities showing ratio of phospho-eNOS / total eNOS (n
= 3 each). Values are means ± SE.
154 3.4 Discussion
PARP is activated in the reperfused myocardium during ischaemia- reperfusion (IR) in major procedures such as experimental heart transplantation and cardiopulmonary bypass surgery (chapter 1, section
1.5.3). Increased PARP activation can be induced by oxidative stress after
IR which is a main determinant of myocardial cell death and inflammatory reactions (Szabo et al., 2004a). Many studies have shown that PARP inhibition is protective against oxidative injury in cardiomyocytes in vitro as well as ex vivo and in vivo animal models of regional or global IR
(Szabados et al., 2000, Szabo, 2005a, Szabo et al., 2002).
The major findings in this study were: i) The functional data (reflected in Figures 3.2 and 3.3) showed that control hearts had the lowest cardiac contractility and recovery compared to the rest of the groups which had INO-1153 treatment, in particular the post- storage recovery of CF was 79% vs 40%; AF was 43% vs 15%; CO was
55% vs 23% and HR was 100% vs 50% of pre-storage baseline levels compared to the recovery of control hearts arrested and stored in unsupplemented celsior storage solution (P<0.05, treated vs control).
ii) The results of poly(ADP-ribose) (PAR) immunohistochemistry illustrated that INO-1153 was indeed acting as an inhibitor of poly(ADP-ribose) polymerase in this model at the concentration employed throughout the study (1 µM). Control hearts that were not treated with the PARP inhibitor,
155 INO-1153, showed markedly increased PAR deposition (Figure 3.4A) compared to hearts arrested and stored in the presence of INO-1153
(Figure 3.4B).
iii) Activation of PI3-k/Akt signalling by PARP inhibitor was essential for protection against ischaemia reperfusion injury and the functional recovery of post-storage cardiac function. Functional recovery was accompanied by a 4.3 fold increase in Akt phosphorylation and 2.4 fold increase in Erk1/2 phosphorylation (Both Figures 3.7 and 3.8, P<0.05 vs control) in presence of INO-1153. Importantly, hearts pre-treated with the PI 3-k inhibitor wortmannin before arrest and storage in INO-1153 supplemented celsior, showed significant reduction in Akt, Erk1/2 and phospholamban phosphorylation in figures 3.7 – 3.9 and was accompanied by a complete loss of the post-storage recovery of cardiac function observed in hearts arrested and stored in INO-1153 alone.
iv) Inclusion of INO-1153 in Celsior failed to increase the phosphorylation status of eNOS compared to control hearts stored in Celsior alone (Figure
3.10). This was despite a significant increase in Akt phosphorylation and activation (Figure 3.7) and an increase in phosphorylation status of phospholamban (down-stream of eNOS) (Figure 3.9).
INO-1001, a PARP inhibitor, currently undergoing clinical trials (Pacher et al., 2007) for various cardiovascular indications was used earlier in a
156 canine model of orthotopic heart transplantation which significantly improved post transplant cardiac contractility when given at reperfusion of the donor’s heart (Szabo et al., 2005b). Whilst we have shown a non significant benefit of addition of INO-1153 at reperfusion maximal benefit was most pronounced when INO-1153 was administered prior to or during cardioplegia and cardiac storage, as reflected in the post-storage functional parameters (Figures 3.2 and 3.3). This finding is in line with the finding that the infarct – sparing effects of post-conditioning in rat models was less potent than in the dog (Zhao et al., 2003) and the infarct – sparing effect of preconditioning in the rat was more sustained than that achieved with postconditioning (van Vuuren et al., 2008).
Efficacy of PARP inhibition at cardioplegia and during storage is logistically attractive in clinical heart transplantation. At multi-organ procurement, the pharmacological PARP inhibitor needs only to be added to the cardiac preservation solution at the time of cardioplegia, whilst the other organs are isolated from exposure to the agent. At present little information has been published on the effect of PARP inhibitors on lungs or abdominal organs and this may be of importance to the clinical arena as future research evolves. It has been reported that PARP inhibitors such as
3 - aminobenzamide and PJ34 were shown to increase post-storage injury during hypothermic IR injury when kidneys were cold stored using
University of Wisconsin solution as the preservation solution (Mangino et al., 2004).
157 The results of poly(ADP-ribose) (PAR) immunohistochemistry illustrated that control hearts that were not treated with our PARP inhibitor, INO-
1153, showed marked PAR deposition (Figure 3.4A). INO-1153 treated hearts during cardioplegia and hypothermic storage had substantially less
PAR accumulation which again is suggestive that Celsior solution alone is not sufficient to protect the heart against IR injury after 6 hours of cold storage. Indeed the functional data (reflected in Figures 3.2 and 3.3) showed that control hearts had the lowest cardiac contractility and recovery compared to the rest of the groups which had INO-1153 treatment. The present observation was consistent with another study which demonstrated that the use of another PARP inhibitor, 3- aminobenzamide, prior to reperfusion reduces the levels of in vivo accumulated PAR substantially when the rat hearts were subjected to regional IR (Pieper et al., 2000).
PAR is synthesized when PARP is activated in response to DNA strand interruptions or formation of DNA breaks. The cellular NAD+ and ATP depletion that occurs in association with PARP activation can lead to cell death and necrosis (Bouchard et al., 2003). Pharmacologic inhibition of
PARP reminiscent of the ischaemic preconditioning response was observed when the hearts were treated with PARP inhibitor 3- aminobenzadine prior to myocardial ischaemia and reperfusion in an in vivo infarct model (Liaudet et al., 2001b). This observation was further strengthened when they showed that cardioprotection induced by
158 ischaemic preconditioning (IPC) was associated with decreased deposition of PAR similar to that pbserved in the reperfused myocardium after administration of 3-aminobenzadine. This was associated with preservation of myocardial NAD+ levels, indicating that IPC attenuates
PARP activation. In the same study the investigators and reports from other laboratories (Vispe et al., 2000, Zahradka et al., 1982) hypothesized that IPC itself induces low levels of oxidative stress and subsequent low degree PARP activation which may lead to auto-ribosylation (i.e. auto- inhibition) of PARP. This process may protect against the deleterious effects of ischaemia and reperfusion associated with the large amount of
PARP activation in non-preconditioned hearts during reperfusion.
The results in this chapter show that pharmacological inhibition of PARP triggers a protective response to subsequent ischaemia reperfusion injury by activating the PI3-k/Akt survival pathway. In line with the present results
(Figures 3.5 - 3.7) it has been shown that activation of PI3-k/Akt signalling by PARP inhibitor is essential for protection against ischaemia reperfusion injury and it has been demonstrated that Akt phosphorylation can be inhibited by the addition of Wortmannin, a PI3-k inhibitor (Tong et al.,
2000). Kovacs and colleagues (Kovacs et al., 2006) in a recent study demonstrated that they were able to activate the PI3-k/Akt pathway through the use of PARP inhibitor which led to a significant recovery of heart function and preservation of the reperfused myocardium in isolated langendorff perfused hearts. There is evidence suggesting that other
PARP inhibitors such as PJ34 and 3-amino benzoic acid effectively
159 prevent PARP activation and myocardial dysfunction in hearts after 1 hour of cardiac preservation and reperfusion (Fiorillo et al., 2003, Szabo et al.,
2002).
A recent study sheds some light on the mechanism by which inhibition of
PARP may activate the PI 3-k / Akt pathway. Matthews and Berk (2008) showed PARP 1 in human umbilical vascular endothelial cells could be activated by exposure to hydrogen peroxide and peroxynitrite (Mathews et al., 2008). Resultant cell death and apoptosis was attenuated with upon exposure to the PARP inhibitor, PJ34 or PARP1 siRNA. PARP inhibition was associated with increased phosphorylation of Akt, pro-apoptotic BAD and importantly VEGF receptor 2, whose associated tyrosine kinases recruit the PI 3-k / Akt pathway. Their theory was that poly (ADP- ribosylation) of a cytoplasmic protein associated with the VEGF signalling pathway may prevent appropriate receptor phosphorylation and PARP inactivation may facilitate receptor phosphorylation and subsequent pro- survival signalling.
The findings (Figures 3.7 and 3.8) in our study also parallel another report
(Palfi et al., 2005) in which they administered a novel PARP inhibitor –
L2286 during IR and observed a significant increase in Akt and Erk activation in both Langendorff perfused rat heart and an in vivo cardiac injury model. Inhibition of PAR synthesis by another PARP inhibitor, PJ34, has shown to be cytoprotective in vitro that led to the sustained activation
160 of Erk1/2 and improving cell survival (Ethier et al., 2007). In addition, recent reports have suggested an alternative mechanism of PARP activation via interaction with Raf-MEK-Erk signalling pathway and other downstream targets to promote cell growth and differentiation which does not involve DNA damge or cause DNA breaks, however, the exact mode of activation is unclear at present (Cohen-Armon et al., 2007, Kauppinen et al., 2006). Other investigators also reported that PARP gene deletion reduced myocardial post-ischaemic injury (Pieper et al., 2000, Zingarelli et al., 2004) and pharmacological inhibition of PARP protected against hypertrophy, heart failure and improved functional and metabolic recovery of the reperfused myocardium (Liaudet et al., 2001a).
Signalling events downstream of PARP activation are not fully identified and understood at present, although in general the activation of PARP occurs as a result of severe oxidative or nitrosative stress (Pacher et al.,
2005). The cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) plays a crucial role in cardiac muscle contraction and relaxation by regulating intracellular Ca2+ concentration (Frank et al., 2003). The activity of SERCA2a is regulated by phospholamban (PLB) and sarcolipin
(SLN), with the role of PLB more extensively studied and changes in expression of both PLB and SLN have been reported in many cardiac related pathological states (Bhupathy et al., 2007, Frank et al., 2003).
PLB is an inhibitor of SERCA2a in its dephosphorylated form and phosphorylation of PLB results in an enhanced calcium re-uptake into the
161 sarcoplasmic reticulum that improves contractility (Periasamy et al., 2008,
Schwinger et al., 2003).
Several investigators have demonstrated that increasing SERCA2a expression led to improved myocardial contractility and calcium handling under normal physiological conditions as well as during diseased conditions (Chen et al., 2004, del Monte et al., 2004, He et al., 1997,
Muller et al., 2003). A recent report by Hassan-Talukder and team observed an increased SERCA2a/PLB ratio in transgenic mice and suggested that the enhanced baseline contractility could be due to
SERCA2a upregulation or overexpression under physiological conditions
(Talukder et al., 2007). They and along with other investigators in the past
(Chossat et al., 2001, Frank et al., 2003, Loukianov et al., 1998, Sumbilla et al., 1999) also demonstrated that overexpression of SERCA2a resulted in an increased sarcoplasmic reticulum Ca2+ uptake activity which protected the heart against ischaemia reperfusion injury by efficient removal of Ca2+ overload due to oxidative stress, which led to the preservation of cardiac contractile function following ischaemia- reperfusion.
In line with our result (Figure 3.9) showing a siginificant increased in phospholabam activity through the use of INO-1153, it was previously shown that the use of PARP inhibitor can improve cardiac contractility and myocardial Ca2+ handling in diabetes mellitus rats in vivo (Pacher et al.,
162 2002) as well as in a model of severe myocardial dysfunction (Szenczi et al., 2005). The correlation between myocardial Ca2+ handling ability and contractility function appears to be attributed to the altered expression of
SERCA2a/PLB interplay, however, the exact role of PARP inhibitors in disturbances of calcium handling is yet to be elucidated in later stages of diseases involving substantial oxidative free radical components especially during ischaemia-reperfusion induced injury (Moens et al., 2005).
The observed post-storage recovery of contractile function in the INO-
1153 treated groups may be secondary to phospholamban phosphorylation and SERCA activation via activation of Akt and PKG
(Abdallah et al., 2006). Two other important proteins which may modulate intracellular calcium levels have recently been shown to be Akt substrates.
Phosphorylation of the inositol 1,4,5-triphosphate receptor by Akt has recently been shown to inhibit calcium release from the endoplasmic reticulum and inhibit apoptosis (Szado et al., 2008). The sodium hydrogen exchanger has recently been shown to contain 3 phosphorylation sites identical to the optimal Akt target motif, inferring possible regulatory interactions between Akt and NHE 1. Snabaitis and colleagues have indeed shown that activated Akt phosphorylates NHE 1 at the Ser648 position and importantly that phosphorylation at this site inhibits sodium hydrogen exchange during acidosis (Snabaitis et al., 2008).
163 The present findings of improved post-storage cardiac function, reduced
PAR formation and activation of the PI3-k/Akt survival kinase following IR support the notion that the use of PARP inhibitor has potential beneficial effects for donor heart post reperfusion. Endothelial nitric oxide synthase
(eNOS) has been previously shown to be phosphorylated and activated by an Akt dependent process (Dimmeler et al., 1999, Fulton et al., 1999,
Michell et al., 1999).
However, in the present study inclusion of INO-1153 in Celsior failed to increase the phosphorylation status of eNOS compared to control hearts stored in Celsior alone (Figure 3.10). This was despite a significant increase in Akt phosphorylation and activation (Figure 3.7) and an increase in phosphorylation status of phospholamban (down-stream of eNOS) (Figure 3.9).
The inability to observe an increase in eNOS phosphorylation here may be a reflection of the rapid and transient nature of this process.
Phosphorylayion of eNOS was assayed here and routinely by others with an antibody directed towards a phosphorylated Serine at position 1177
(human sequence numbering) of the eNOS peptide, a well defined site of
Akt phosphorylation (Dimmeler et al., 1999). Erwin and colleagues showed that exposure of a bovine endothelial cell line to VEGF resulted in rapid phosphorylation at this site, (maximal phosphorylation within 5 minutes of
164 exposure to VEGF stimulus), which returned to basal levels 30 minutes exposure (Erwin et al., 2005).
Where significant eNOS phosphorylation was demonstrated in the setting of pharmacological postconditioning samples were also taken early after reperfusion, at 2 minutes (Abdallah et al., 2006), 5 – 15 minutes (Bell et al., 2001) and 7 minutes (Tsang et al., 2004) after the reoxygenation stimulus. Where the time course of eNOS phosphorylation was examined
(Bell et al., 2001), maximum eNOS phosphorylation was observed at 10 minutes post reperfusion. In the present study hearts were frozen for analysis at the end of post storage functional evaluation, (45 minutes post reperfusion). By this time point any increase in phosphorylation of eNOS present in the early stages of reperfusion may well have disappeared.
Conclusion
The salient findings from this study are as follows:
i) Significant post-storage improvement in all the assessed indices of cardiac function, i.e. coronary flow, heart rate, aortic flow and cardiac output (P<0.05 vs control), were observed when hearts were exposed to the PARP inhibitor INO-1153 either before hypothermic storage, or when hearts were arrested and stored in Celsior solution supplemented with
1µM INO-1153.
165 ii) Immunoblotting studies showed that the functional improvement in the group of hearts arrested and stored in INO-1153 – supplemented Celsior were accompanied by a significant increase in the phosphoylayion status of Akt, a modest but significant increase in ERK phosphorylation and a significant increase in phospholamban phosphorylation (All P<0.05 vs control). This is the first report of post reperfusion activation of RISK pathway elements after a period of hypothermic ischemia by appropriate pharmacological supplementation of the cardioplegic / storage solution.
iii) Exposure of the hearts to the phosphatidylinositol 3-kinase inhibitor,
Wortmannin, before arrest and storage in INO-1153 supplemented
Celsior, completely blocked the protective effect of INO-1153. A significant decrease in recovery of all the above aforementioned indices of cardiac function was observed post reperfusion (P<0.05 vs control). This functional decline was accompanied by a significant decrease in expression of phosphorylated Akt, Erk1/2 and phospholamban (all P<0.05 vs control). Taken together, these findings imply that activation of the Akt pro-survival pathway was crucial to the observed functional improvement.
Importantly for the logistics of the potential use of PARP inhibition in the context of clinical donor heart retrieval, simple supplementation of the commercially available cardioplegic / storage solution, Celsior with the agent is sufficient to provide maximum cardiac protection.
166 CHAPTER 4
Improved Post-storage Cardiac Function by the Presence of Glyceryl Trinitrate and Cariporide in Celsior Solution at
Arrest and during Hypothermic Storage: Role of the ERK
1/2 Pathway
167 4.1 INTRODUCTION
Minimisation of ischaemia-reperfusion injury incurred during organ procurement, storage and implantation is a key factor in improving short- and long-term outcomes of heart transplantation (Hicks et al., 2006).
Numerous approaches to reduce reperfusion damage to the donor heart have been employed including formulation of purpose – designed cardioplegic / storage solutions such as Celsior®, that is now in routine clinical use (Menasche et al., 1994, Vega et al., 2001).
Experimental studies indicate that protective strategies against myocardial infarction such as ischemic and pharmacological preconditioning are also effective in models of donor heart preservation. Previous work from our laboratory using an isolated working rat heart model of donor heart preservation has shown that ischemic preconditioning or the presence of pharmacological preconditioning agents such as nitric oxide donors
(diazenium diolates or glyceryl trinitrate), KATP channel openers or the sodium hydrogen exchange inhibitor cariporide at cardioplegia and during hypothermic storage significantly improved post-storage cardiac function in an isolated working rat heart model of donor heart preservation (Du et al., 1998, Gao et al., 2005, Hicks et al., 1999).
The search for an over-arching mechanism of protection against ischemia reperfusion damage has been advanced by the realisation that cardio- protective strategies such as ischemic pre- and postconditioning and
168 pharmacological interventions that mimic these physiological strategies can recruit and activate pro-survival kinase pathways such as the PI3K /
Akt pathway and the p42 / p44 extracellular signal-related MAP kinase
(ERK 1/2) at reperfusion (Hausenloy et al., 2007b, Hausenloy et al.,
2007c). Both pathways constitute the Reperfusion Injury Survival Kinase
(RISK) pathway.
Consistent with this mechanism, are the results from the previous chapter demonstrating that hearts arrested and stored in Celsior solution supplemented with the poly(ADP-ribose) polymerase inhibitor INO-1153 for 6 hours had significantly increased levels of phosphorylated Akt post reperfusion. This was associated with a significant improvement in post- storage cardiac function which was lost if hearts were pre-treated with wortmannin, an inhibitor of the PI3K / Akt pathway (Gao et al., 2007).
These observations prompted an examination of the extent to which the proximal RISK pathway elements ERK 1/2 and PI3K / Akt and potential down stream targets were activated in hearts arrested and stored in
Celsior supplemented with glyceryl trinitrate and cariporide. Two other recent findings lend weight to this re-evaluation of the potential role of these pathways in the protective effects of GTN and cariporide. p21 Ras, an upstream regulator of the ERK 1/2 and (potentially) the Akt pathway has been shown to be activated by NO through S-nitrosylation of the
Cys118 of Ras (Oliveira et al., 2003). The Ras / Raf / MEK / ERK pathway
169 was also shown to be activated by transient intracellular acidosis in cardiac myocytes exposed to cariporide (Haworth et al., 2006).
Thus, the aims of the present study were in an isolated working rat heart model of donor heart preservation 1) to assess the recovery of post- storage cardiac function of hearts arrested and stored in Celsior supplemented with glyceryl trinitrate or cariporide alone or in combination compared to Celsior alone; 2) to ascertain whether these pharmacological supplements activate and phosphorylate ERK 1/2 and PI3K / Akt compared to the unsupplemented control, 3) to assess the extent of activation of an anti-apoptotic marker (phosphorylated Bcl-2) and a marker of apoptosis (cleaved caspase 3) and 4) Examine the extent to which
PD98059, an inhibitor of the MEK /ERK pathway, modified the protective effect of GTN and cariporide.
170 4.2 MATERIALS AND METHODS
Animals
As described in Chapter 2, section 2.3.
Ethical conduct of studies
As described in Chapter 2, section 2.2.
Chemicals and pharmacological agents
As described in Chapter 2, section 2.1.
Preparation of perfusion buffer
As described in Chapter 2, section 2.1.
Isolated working heart preparation
A detailed account of the surgical steps in preparing the isolated heart appears in Chapter 2, section 2.4. Briefly, the rats were anaesthetised with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). After bolus injection of 500 IU heparin into the renal vein, the heart was rapidly excised and arrested by immersion in chilled (2 – 3°C) perfusion buffer. The aorta was cannulated and immediately perfused retrogradely on a Langendorff perfusion apparatus with Krebs buffer at a hydrostatic pressure of 100 cm H2O. During this time, a small incision was made in the left atrial appendage into which another cannula was inserted
171 and tied off. This non-working preparation was stabilised for 10 minutes and then converted to working mode by switching the supply of perfusate from the aorta to the left atrial cannula at a hydrostatic pressure of 20 cm
H2O (pre-load). The working heart ejected perfusate via the aortic valve into the aortic cannula. The hydrostatic pressure in the aortic cannula was maintained at 100 cm H2O (after-load) throughout the working phase for all rat hearts.
Aortic pressure was monitored in a side arm of the aortic cannula with a pressure transducer (Ohmeda, Pty Ltd., Singapore). Aortic flow was measured by an in-line flowmeter (Transonics Instruments Inc. Ithaca,
NY). Aortic pressure and flow were recorded using MacLab/4e
(ADInstruments Pty Ltd, Sydney, Australia) and heart rate was calculated from the flow trace. Coronary flow was measured by timed collection of the effluent draining from the apex of the heart.
Experimental protocol (Figure 4.1)
All hearts remained in working mode for 15 min prior to storage.
Measurements of heart rate (HR), aortic flow (AF), coronary flow (CF), and cardiac output (CO) were made at 10 minutes after conversion to working mode and used as pre-storage baseline.
Exclusion criteria: Any hearts having a baseline aortic flow less than 35 ml/min, heart rate of less than 200 beats per minute, or coronary flow less than 10 mL/min were excluded.
172 After collection of baseline hemodynamic data, the heart was arrested by infusion of cold Celsior preservation solution (at 2–3°C) into the coronary circulation for 3 minutes from a reservoir 60 cm above the heart. All hearts were stored in 100 mL of the same solution for 6 hours under hypothermic conditions (2–3°C). Hearts were then remounted on the perfusion apparatus and reperfused in Langendorff mode for 15 minutes. Hearts were then switched to working mode then stabilized for 30 minutes. The indices of cardiac function measured at baseline were then re-measured.
Recovery of each parameter was expressed as a percentage of its pre- storage baseline. After the 30 minutes functional observations were taken,
(i) the left ventricular free wall of each heart was rapidly frozen by immersion in liquid nitrogen and stored at -80 °C for Western blot analysis;
(ii) a transverse section of each heart was collected and stored in 4% paraformaldehyde prior to embedding for histology analysis.
Experimental groups
Figure 4.1 illustrates the experimental timeline and treatment groups. Rat hearts were divided into four groups according to the formulation of the cardioplegic and storage solution. Compositions of arresting and storage solutions were as follows:
Group 1, unsupplemented Celsior;
Group 2, Celsior supplemented with 0.1 mg/ml glyceryl trinitrate (GTN);
Group 3, Celsior supplemented with 10 μM cariporide;
173 Group 4, Celsior supplemented with a combination of 0.1 mg/ml glyceryl trinitrate (GTN) and 10 μM cariporide;
Group 5, Celsior supplemented with a combination of 10µM PD98059 and
0.1 mg/ml GTN and 10 μM cariporide;
Group 6, Celsior supplemented with a combination of 25µM PD98059 and
0.1 mg/ml GTN and 10 μM cariporide.
Cardioplegia Reperfusion
Pre-storage Storage phase Post-Storage Phase (2-3°C) Langendorff Langendorff Working Heart Working Heart
10 min 15 min 6 hrs 15 min 30 min Treatment Groups Group 1 Krebs Celsior alone Krebs
Group 2 Krebs Celsior + 0.1mg/ml GTN Krebs
Group 3 Krebs Celsior + 10µM Cariporide Krebs
Group 4 Krebs Celsior + 0.1mg/ml GTN + Krebs 10µM Cariporide
Group 5 Krebs 10µM PD98059 + Celsior + 10µM PD98059 + Krebs 0.1mg/ml GTN + 10µM Cariporide
Group 6 Krebs 25µM PD98059 + Celsior + 25µM PD98059 + Krebs 0.1mg/ml GTN + 10µM Cariporide
#
Figure 4.1 Study protocol and treatment groups. Arrows indicate times at which cardiac function were assessed, i.e. heart rate (HR), aortic flow (AF), coronary flow (CF), and cardiac output (CO). # indicates the point where the heart tissue was harvested for histology and western immunoblotting analysis.
174 Western immunoblotting
A subgroup of 4 hearts was chosen from each experimental group for
Western blot analysis and sixty milligrams of tissue from each heart were homogenized in cold lysis buffer (section 2.7). Protein samples were boiled in sample loading buffer for 5 minutes before loading onto 10%
SDS-polyacrylamide gel under reducing conditions (40 µg protein per lane) as described in section 2.8. Membranes were blocked for 1 hour in
Tris buffered saline, (pH 7.4) which contained 1% BSA and 0.1% Tween
20. Membranes were probed with primary rabbit polyclonal antibodies
(1:3000 dil) raised against total and phospho-p44/42 MAPK (Erk ½), total and phospho-Akt (Ser 473), total Bcl-2, Caspase 3 and Cleaved Caspase
3, β-actin (Cell Signaling Technology, Danvers, MA), phospho-Bcl2 (Ser
87) antibody (Santa Cruz Biotechnology). The secondary antibody was a horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Life
Sciences). The protein bands were visualized using enhanced chemiluminescence (Pierce Biotechnology) with bands visualised by autoradiography and intensities were digitised with Image J freeware
(National Institute of Mental Health, Bethesda, MA).
Immunofluorescence
To assess the extent of apoptosis throughout the treatment groups, a primary antibody to cleaved caspase 3 was used as a marker of apoptosis as described in chapter 2, section 2.9.
175 Statistical analysis
All values were expressed as mean ± SE unless otherwise specified. Data were analyzed by the StatView 4.5 statistical software package.
Differences among groups were compared by one-way ANOVA analysis followed by Fisher’s PLSD post hoc test, a P value of less than 0.05 was considered significant.
176 4.3 RESULTS
4.3.1 Prestorage baseline measurements of cardiac function
The pre-storage baseline measurements of cardiac function for control and all treatment groups are shown in Table 4.1. The groups were well matched with no significant differences between any of the experimental groups for any of the parameters measured.
Table 4.1 Pre-storage baseline cardiac measurements and values.
AF CF CO HR Groups n (ml/min) (ml/min) (ml/min) (bpm)
Control (Celsior alone) 48 ± 5 20 ± 2 68 ± 5 255 ± 37 8
Celsior + 0.1mg/ml GTN 51 ± 11 21 ± 2 72 ± 12 248 ± 43 9
Celsior + 10µM Cariporide 53 ± 10 18 ± 2.7 72 ± 11 245 ± 29 6
Celsior + 0.1mg/ml GTN + 46 ± 4 21 ± 1 67 ± 4 245 ± 34 6 10µM Cariporide Celsior + 10µM PD98059 0.1mg/ml GTN + 10µM 50 ± 6 20 ± 1 70 ± 6 235 ± 31 3 Cariporide Celsior + 25µM PD98059 0.1mg/ml GTN + 10µM 52 ± 6 18 ± 2 70 ± 11 239 ± 27 6 Cariporide
AF=Aortic flow, CF=Coronary flow, CO=Cardiac output, HR=Heart rate,
N=number of rat hearts. Data are expressed as means ± SD in each parameter.
177 4.3.2 Effect of cardioplegia / storage solution composition on post-
reperfusion recovery of cardiac function
Figure 4.2 shows representative traces of aortic flow and heart rate before and after 6 hours of hypothermic storage. Post-storage recovery of the hearts stored in unsupplemented Celsior was poor. In hearts arrested and stored in Celsior supplemented with GTN or cariporide, either singly or in combination, heart rate and aortic flow showed robust recovery immediately upon reperfusion. The grouped data bear out these initial observations (Figure 4.3). All indices of cardiac function (aortic and coronary flow, cardiac output and heart rate) showed significant post- storage improvement in hearts arrested and stored in supplemented
Celsior, whether the supplement was GTN or cariporide alone or the combination of the two.
178 Aortic Flow Heart Rate
Group 1 Celsior alone
Group 2 Celsior + 0.1mg/ml GTN
20 min
Group 3 Celsior + 10µM Cariporide
Group 4 Celsior + 0.1mg/ml GTN + 10µM Cariporide
pre- post reperfusion pre- post reperfusion storage storag
Figure 4.2 Effect of cardioplegic / storage solution composition on indices of post-storage cardiac function. Representative trace recordings of aortic flow and heart rate diuring pre-storage phase and during reperfusion subsequent to 6 hours of hypothermic storage.
179 140
* 120
* * 100 * * 80 * * * * * 60 * *
40 (% of pre-storagebaseline)(% Post ReperfusionRecovery 20
0 Aortic Coronary Cardiac Heart Flow Flow Output Rate
Figure 4.3 Summary comparision of various treatment groups showing recovery of aortic flow, coronary flow, cardiac output and heart rate after 45 minutes post-reperfusion phase (after 15 mins non-working and 30 mins working mode) following 6 hr hypothermic storage. Data are expressed as mean ± SE. *P < 0.05 vs unsupplemented Celsior control, ANOVA.
Treatment groups:
Group 1, Celsior alone (control);
Group 2, Celsior supplemented with 0.1mg/ml GTN;
Group 3, Celsior supplemented with 10µM cariporide;
Group 4, Celsior supplemented with 0.1mg/ml GTN +
10µM cariporide
180 4.3.3 Effect of cardioplegia / storage solution composition on post-
reperfusion phosphorylation status of ERK 1/2 and Akt.
Figure 4.4 (a) shows the typical phosphorylation status of ERK 1/2 in hearts arrested and stored in Celsior alone (Lane 1), GTN-supplemented
Celsior (Lane 2), cariporide-supplemented Celsior (Lane 3) and doubly supplemented Celsior (Lane 4). Total ERK 1/2 was robustly expressed across all experimental groups, indicating that neither the storage conditions nor the GTN or cariporide supplements had any effects on ERK
1/2 protein expression.
In contrast, a low level of phosphorylated ERK 1/2 was observed in hearts arrested and stored in Celsior alone. Inclusion of either GTN or cariporide or a combination of both increased the levels of phosphorylated ERK 1/2.
The ratio of phospho to total ERK 1/2 was quantified from band intensities for each experimental group (Figure 4.4 (b)). The ratios were normalised against ß-actin intensity to account for any differences in lane loading.
Supplementation of Celsior with GTN, cariporide or both resulted in a 3.1 fold, 5.1 fold or 7 fold increase in the level of phosphorylated ERK 1/2 respectively.
To determine whether the PI3K / Akt pathway was activated, we examined the phosphorylation status of Akt (Figures 4.5 a and b). In common with
ERK 1/2, there was strong expression of total Akt in all groups. In contrast to ERK 1/2, there was no increase in phosphorylation of Akt in any of the
181 experimental groups above that of the unsupplemented Celsior control in
Lane 1. The absence of any increase in the phosphorylation status of Akt was confirmed by quantitation of the phospho / total Akt ratios for each group (Figure 4.5 (b)).
182
(a) Lane 1 Lane 2 Lane 3 Lane 4
Phospho Erk 1/2
Total Erk 1/2
Actin
(b) 1
0.8
0.6 * * 0.4 #
Erk1/2 Total
0.2 Ratio of Phospho –Erk1/2 / 0
0.1mg/ml GTN - + - +
10µM Cariporide - - + +
Figure 4.4 Effect of cardioplegic / storage solution composition on the extent of Erk1/2 activation 45 minutes post-reperfusion phase following 6 hr hypothermic storage. (a) Representative western immunoblots from each group showing levels of Erk1/2 phosphorylation (activation), total Erk1/2 and β-actin. (b)
Histogram showing ratio of phosphorylated Erk1/2 to total Erk1/2 (n=4 in each group), values are means ± SE. # P = 0.005; * P < 0.001 vs control (Celsior alone), ANOVA.
183 (a)
Phospho-Akt
Total Akt
Actin
(b)
0.8
0.6
0.4 / Total Akt / Total 0.2 Ratio of Phospho-Akt 0
0.1mg/ml GTN - + - +
10µM Cariporide - - + +
Figure 4.5 Effect of cardioplegic / storage solution composition on the extent of Akt activation 45 minutes post-reperfusion phase following 6 hr hypothermic storage. (a) Representative western immunoblots from each group showing levels of Akt phosphorylation (activation), total Akt and β-actin. (b)
Histogram showing ratio of phosphorylated Akt to total Akt (n=4 in each group), values are means ± SE.
184 4.3.4 Effect of cardioplegia / storage solution composition on post-
reperfusion phosphorylation status of Bcl-2
Having demonstrated that GTN or cariporide supplementation to Celsior either individually or in combination improves post-storage cardiac function and activates the ERK 1/2 pathway, evidence of anti-apoptotic signalling down-stream of ERK 1/2 was investigated. Recently it was reported that
Bcl-2 was a substrate for ERK phosphorylation and that mono or multi-site phosphorylation of Bcl-2 enhanced its anti-apoptotic function (Deng et al,
2000 and 2004), so a commercially available antibody directed against phosphorylated Bcl-2 at Ser87 as a probe for “anti-apoptotic” status.
Figure 4.6 (a and b) show total Bcl-2 expression and extent of phosphorylation of Bcl2. There was little variation in the expression of total
Bcl-2 between experimental groups (Figure 4.6 (a)). The phosphorylation status of Bcl-2 paralleled that of ERK 1/2. The least phospho Bcl-2 was observed in hearts arrested and stored in Celsior alone, with significant increases observed in hearts arrested and stored in Celsior supplemented with GTN and cariporide either individually or in combination (Figure 4.6
(b)).
185 (a)
Phospho-Bcl2
Total Bcl2
Actin
(b) 1
0.8 * * 0.6 #
0.4
Total Bcl2 0.2
Ratio of Phospho-Bcl2 / of Phospho-Bcl2 Ratio
0 0.1mg/ml GTN - + - +
10µM Cariporide - - + +
Figure 4.6 Effect of cardioplegic / storage solution composition on the extent of Bcl-2 phosphorylation 45 minutes post-reperfusion phase following 6 hr hypothermic storage. (a) Representative western immunoblots from each group showing levels of Bcl-2 phosphorylation, total Bcl-2 and β-actin.
(b) Histogram showing ratio of phosphorylated Bcl-2 to total Bcl-2 (n=4 in each group), values are means ± SE. * P < 0.001; # P = 0.01 vs control (Celsior alone),
ANOVA.
186 4.3.5 Effect of GTN and Cariporide in Celsior solution on apoptotic
marker post-reperfusion
Caspase 3 activation (i.e. the presence of cleaved caspase 3) is a definitive marker of apoptosis where many pathologic stimuli converge.
Caspase 3 exists as an inactive 35 kDa peptide that is cleaved to an active 17-20 kDa peptide. To further examine the potential anti-apoptotic role of Erk1/2 activation of GTN / Cariporide supplementation to Celsior solution in the recovery of heart function, the extent of caspase 3 activation was examined using western immunoblotting and immunofluorescence microscopy.
Figure 4.7 (a) shows typical Western blots of pro- and cleaved caspase 3 as well as a ß-actin loading control. A 19 kD cleaved caspase 3 fragment
(active caspase 3) was observed in hearts arrested and stored in Celsior alone (Figure 4.7 a and b, lane 1). Hearts arrested and stored in Celsior supplemented with GTN and cariporide demonstrated decreased levels of cleaved caspase 3 that was non-significant in the case or cariporide alone but highly significant in the cases of GTN alone or the GTN cariporide combination (Figure 4.7 b).
Immunofluorescent detection of cleaved caspase 3 in histological sections agrees with the above Western blot findings. Figure 4.8 (a) – (d) shows representative immunofluorescence micrographs of hearts arrested and stored in Celsior alone (a), Celsior supplemented with GTN (b), cariporide
187 (c) or GTN and cariporide (d). The presence of the cleaved caspase 3 antibody has been visualised with Alexa Fluor 555 (red) and the nuclei counterstained with DAPI (blue). The area of red staining apparent in the
Celsior alone – stored hearts (a) is substantially reduced by inclusion of
GTN, cariporide or both in the arresting and storage solutions (Figure 4.8 b-d). Quantified relative areas of cleaved caspase 3 staining for each experimental group, presented in Figure 4.8 (e), reaffirms the significant decrease in cleaved caspase 3 levels in the presence of all supplements.
188 (a) Lane 1 Lane 2 Lane 3 Lane 4
pro-Caspase 3
Cleaved Caspase 3
Actin
(b) 200 180 160 140
120 100 * 80 * (arbitary units) 60
Band Intensity Means 40 of Cleaved Caspase 3 20 0
0.1mg/ml GTN - + - +
10µM Cariporide - - + +
Figure 4.7 Effect of cardioplegic / storage solution composition on the extent of cleaved caspase 3 (pro-apoptotic marker) 45 minutes post- reperfusion phase following 6 hr hypothermic storage. (a) Representative western immunoblots from each group showing levels of cleaved caspase 3, pro- caspase 3 and β-actin. (b) Histogram showing intensity of cleaved caspase 3 bands (n=4 in each group), values are means ± SE. * P < 0.001 vs control
(Celsior alone), ANOVA.
189
(a) (b)
(c) (d)
(E) 20
15 *
10
Caspase 3 5 *
oiiiy(riur units) (arbituary positivity
Relative Area of Cleaved Cleaved of Area Relative * 0 (a) (b) (c) (d) 0.1mg/ml GTN - + - + 10µM Cariporide - - + +
Figure 4.8 Effect of cardioplegic / storage solution composition on the immunohistochemical appearance of cleaved caspase 3. Representative immunofluorescence images (Magnification 600x) of the amount of cleaved
(activated, red) caspase 3 (a – d). (E) Histogram showing quantification of the extent of cleaved caspase 3 immunofluorescence. Relative area determined in 10 random fields per group. Values are expressed as mean ± SD. *P < 0.0001 vs
Group 1, ANOVA.
190 4.3.6 Effect of MEK /ERK Inhibitor, PD98059 on post-storage recovey
of hearts arrested and stored in Celsior supplemented with
GTN and Cariporide
In order to examine the relationship between post-storage recovery of function and activation of the ERK pathway, hearts arrested and stored in
GTN + Cariporide – supplemented Celsior were exposed to increasing concentrations of PD98059, an inhibitor of MEK 1/2 and the ERK 1/2 pathway. Hearts were exposed to PD98059 at arrest and during storage as well as during post-storage reperfusion up to a concentration of 25 µM.
Figure 4.9 shows recovery of heart rate, coronary flow, aortic flow and cardiac output after 45 minutes reperfusion. Unlike the loss of cardiac recovery observed in the presence of wortmannin after PARP inhibition observed in the previous chapter, the presence of PD98059 had no effect on recovery of heart rate and coronary flow (Figure 4.9 a and b) over the concentration range examined. There was a (non-significant) concentration dependent loss of contractile function (aortic flow - Figure
4.9 c, and cardiac output - Figure 4.9 d) over the same range of PD98059 concentrations.
191 (a) (b) Heart Rate Coronary Flow 100 100
75 75
50 50
25 25 Recovery (% of Baseline) (%Recovery of Baseline) (% Recovery of 0 0 0 5 10 15 20 25 0 5 10 15 20 25
[PD98059] (µM) [PD98059] (µM)
(c) (d) Aortic Flow Cardiac Output 75 75
50 50
25 25 Recovery (% of Baseline) (%Recovery of Baseline) (%Recovery of 0 0 0 5 10 15 20 25 0 5 10 15 20 25 [PD98059] (µM) [PD98059] (µM)
Figure 4.9 Effect of PD98059 exposure during storage and reperfusion of recovery of post-storage function of hearts arrested and stored in Celsior supplemented with GTN and cariporide.
192 4.3.7 Effect of MEK /ERK Inhibitor, PD98059 on post-storage
phosphorylation status of ERK 1/2 and Akt
An examination of the phosphorylation status of ERK and Akt may explain the apparent inability of PD98059 to completely abolish the post-storage recovery of cardiac function by GTN and cariporide observed in Figure
4.9. Figure 4.10 shows immunoblots of phospho and total Akt and ERK
1/2 in the 5 hearts exposed to 25 µM PD98059 in the presence of GTN and cariporide and one heart arrested and stored in GTN + cariporide for comparison. Figure 10 b shows that even at a concentration of 25 µM,
PD98058 the presence of phospho-ERK was still apparent. Interestingly,
Figure 10 a shows no Akt phosphorylation in the absence of PD98059, variable but apparent phosphorylation of Akt can be seen in the hearts exposed to 25 µM PD98059. Activation of Akt by PD98059 has been observed before in an infarct model of ischaemic preconditioning where hearts were exposed 10 µM PD98059 during reperfusion (Hausenloy et al., 2004a). In addition, down-regulation of the PI 3-kinase / Akt pathway by activation of the ERK 1/2 pathway in a cell culture model of growth factor signalling has been recently observed (Hayashi et al., 2008).
193 (a) (b) Control PD98059 Control PD98059
Phospho-Akt Phospho-Erk1/2
Total Akt Total Erk1/2
1 1
0.8 0.8
0.6 0.6
Total Akt Total 0.4 0.4 Total Erk1/2 0.2 0.2 Ratio of of Phospho-Akt / Ratio Ratio of Phospho-Erk1/2 / Phospho-Erk1/2 of Ratio 0 0 0.1mg/ml GTN + + + + + + 0.1mg/ml GTN + + + + + + 10µM Cariporide + + + + + + 10µM Cariporide + + + + + + 25µM PD98059 - + + + + + 25µM PD98059 - + + + + +
Figure 4.10 Effect of using 25µM PD98059 inhibitor on the extent of Akt and
Erk1/2 phosphorylation after 45 minutes post-reperfusion following 6 hours hypothermic storage. Representative western immunoblots showing (A) Akt phosphorylation and (B) Erk1/2 phosphorylation using PD98059 inhibitor. Each lane in the PD98059 treatment group represents a separate heart.
194 4.4 DISCUSSION
Previous studies have demonstrated that maintenance of an active “nitric oxide – cGMP pathway” and minimisation of sodium hydrogen exchange activity were essential for optimum post-reperfusion cardiac function after ischemic hypothermic storage (Myers, 1999, Pinsky, 1995). Previous studies from this laboratory and others have shown the efficacy of inclusion of nitric oxide donors such as glyceryl trinitrate and the sodium hydrogen exchange inhibitor cariporide on donor heart recovery in various experimental models (Baxter et al., 2001, Cropper et al., 2003, Gao et al.,
2005, Myers et al., 1996). These studies were designed to demonstrate functional improvement after cardiac storage as proof of principle, rather than a more detailed elucidation of the molecular mechanisms involved.
The present study was designed to give some molecular insights into the observed functional improvements. The principle findings reported were:
i) Significant improvement in post-storage functional recovery across all indices of cardiac function (aotic flow, coronary flow, cardiac output and heart rate in hearts arrested and stored in Celsior supplemented with GTN or Cariporide alone or in combination of the two compared to hearts stored in unsupplemented Celsior (P<0.05 vs control or unsupplemented Celsior only) (Figures 4.2 and 4.3).
195 ii) Significant Improvement in post-storage functional recovery of hearts stored in Celsior supplemented with a combination of GTN and Cariporide was accompanied by a 7 fold increase in Erk1/2 phosphorylation and 2 fold increase in Bcl-2 phosphorylation (Both P<0.001 vs control, Figures
4.4 and 4.6).
iii) The extent of apoptosis as a result of supplementing Celsior preservation solution with GTN and Cariporide together was examined and revealed a 3 fold decrease in expression of pro-apoptotic marker, cleaved caspase 3, assessed by either Western blotting or immunofluorescence microscopy (P<0.001 vs control) (Figures 4.7 and
4.8).
iv) Hearts stored in Celsior solution supplemented with GTN and
Cariporide were exposed to PD98059, an inhibitor of Erk1/2, and resulted in a non-significant decrease of all indices of cardiac function post reperfusion (Figure 4.9). The presence of PD89059 increased the phosphorylation status of Akt, whilst producing only partial inhibition of phosphorylated Erk1/2 (Figure 4.10). These findings suggest that
PD98059 may not be a suitable agent for dissecting out the role of survival kinase activation on functional recovery in this experimental model.
The present findings are consistent with a recent report showing that functional cardiac improvements are associated with increase in Erk1/2
196 phosphorylation during reperfusion in isolated rat heart models of ischaemia and reperfusion when the hearts were exposed to GTN before index ischaemia (Li et al., 2006). We observed a similar extent of Erk1/2 phosphorylation (or activation) during reperfusion when GTN was present in the cardioplegic and hypothermic storage (Figure 4.4). Activation of
Erk1/2 has been shown to be anti-apoptotic (Tibbles et al., 1999) and is implicated in drug induced and ischemic preconditioning (Fryer et al.,
2001, Punn et al., 2000); our results (Figures 4.7 & 4.8) agree with the observations by these investigators.
As outlined in the general introduction, the actions of nitric oxide are mediated by cGMP-dependent and independent mechanisms. Important among these independent pathways is the activation of ERK 1/2, which has been shown to occur via S-nitrosation and activation of the Cysteine
118 residue of Ras, the small GTP-ase immediately upstream of of the 3 kinase tiers of the ERK pathway (Lander et al., 1997). Under the hypoxic conditions likely to occur during hypothermic storage of the heart in the present study, xanthine oxidase has been shown to catalyse conversion of
GTN to nitric oxide and nitrosothiols (H. Li et al., 2005).
Proton accumulation caused by glycolysis has been thought to activate the sodium hydrogen exchanger during ischemia, with consequent accumulation of intracellular sodium. This process is then coupled to
“reverse mode” exchange of sodium for calcium, resulting in high levels of
197 intracellular calcium, a key element in the development of reperfusion injury(Mentzer et al., 2003). Inhibition of sodium hydrogen exchange either by ischemic preconditioning (Xiao et al., 1999), or the use of specific inhibitors such as cariporide is cardioprotective (Mentzer et al., 2003), although cellular and molecular mechanisms underpinning functional improvement are yet to be clarified. However, a consequence of the presence of cariporide during ischemic storage is a transient acidosis and delay in the restoration of normal pH in the first minutes post reperfusion, a process thought to minimise the open probability of the mitochondrial transition pore in ischemic preconditioning (Hausenloy et al., 2007a). The activation of ERK 1/2 in hearts arrested and stored in Celsior supplemented with the sodium hydrogen exchange inhibitor, cariporide observed in the present study is a novel finding (Figure 4.4). However, this finding is consistent with the recent finding that the Ras / Raf / MEK / ERK pathway was activated by transient intracellular acidosis in cardiac myocytes (Haworth et al., 2006).
Phosphorylation and activation of Akt, the other major proximal element of the RISK pathway, was not increased by either GTN or cariporide alone or in combination (Figure 4.5). Although activation of the Akt pathway results in an increase of endogenous nitric oxide via phosphorylation and activation of endothelial nitric oxide synthase (Mount et al., 2007), there is little evidence of the role of exogenous nitric oxide on the phosphorylation status of Akt. What evidence there is supports our finding. Exposure of
198 INS cells (a pancreatic beta cell line) to the nitric oxide donor S-nitroso acetyl penicillamine, (SNAP), resulted in a time and concentration dependent decrease and complete depletion of phospho-Akt (measured by Ser473 phosphorylation) (Storling et al., 2005).
Previous studies examining the effect of sodium hydrogen exchange inhibition on Akt phosphorylation status are also in agreement with our observation. Amiloride, a drug possessing sodium hydrogen exchange inhibitor activity was shown to exacerbate TRAIL-induced tumor cell apoptosis (as evidenced by an increase in cleaved caspase 3 levels) by a mechanism involving intracellular acidification – linked inhibition of Akt phosphorylation (Cho et al., 2005). Interestingly, in the present study the hearts arrested and stored in Celsior supplemented with cariporide only also show relatively high levels of cleaved caspase 3 compared to hearts arrested and stored in GTN or GTN and cariporide (Figure 4.7 and Figure
4.8).
In parallel with ERK 1/2 activation (Figure 4.4), increases in the extent of phosphorylation of Bcl-2 at Ser 87 were observed in hearts arrested and stored in GTN and cariporide alone or in combination (Figure 4.6). Similar increases have also been seen in an isolated rat heart model after pharmacological preconditioning with the polyphenolic phytoalexin, resveritrol (Das et al., 2005). In fact, Bcl-2 was identified as a down-stream target of phosphorylated ERK 1/2 (Deng et al., 2000). Mono or multi-site
199 phosphorylation in the flexible loop region of the Bcl-2 molecule (at
Threonine 69, Ser 70 or Ser 87) has been demonstrated to enhance the anti-apoptotic function of Bcl-2 (Deng et al., 2006). Recently, ERK 1/2 – dependent phosphorylation of BimEL, another pro-apoptotic Bcl-2 protein, was shown to be key to its ubiquitination and subsequent destruction
(Ewings et al., 2007).
Hearts arrested and stored in unsupplemented Celsior were found to have a significant increase in the level of cleaved (activated) caspase 3 post- reperfusion which was reduced to levels found in freshly excised hearts by
GTN and GTN + cariporide supplements (Figures 4.7 and 4.8). Activation of the ERK pathway has been shown to inhibit procaspase 3 cleavage and activation, although the mechanism of action of ERK was not elucidated at the time (Erhardt et al., 1999). Subsequently it was shown that activated
ERK was able to phosphorylate caspase 9 at Thr 125, a conserved MAP kinase consensus site (Allan et al., 2003). Phosphorylation at Thr 125 inhibited normal caspase 9 processing thus preventing procaspase 3 cleavage and activation.
Another recently identified target of ERK, particularly germane to cardiac protection and the RISK pathway was Kir6.2, the pore-forming subunit of the KATP channel (Lin et al., 2008). This study was performed on a neuronal KATP channel isoform, Kir6.2/SUR1 expressed in human embryonic kidney 293 cells and showed that activated ERK2
200 phosphorylated the T314 and S385 residues of the Kir6.2 subunit. The open probability and open frequency of the channel were increased and the closed duration reduced. The potential importance of this finding for myocardial protection lies in the fact that the inward rectifying K+ channel,
Kir6.2, was recently identified as a component of the human cardiac mitochondrial KATP channel (Jiang et al., 2006). In line with these findings, we have previously demonstrated that inhibition of KATP channel opening with glibenclamide abolished the cardioprotective effect of GTN (Gao et al., 2005).
The inability of the MEK 1/2 – ERK 1/2 inhibitor PD98059 to completely abolish the protective effect of GTN and cariporide (Figure 4.9) highlights the inconsistent effect of this agent in different models of ischaemia reperfusion injury. Whilst its effect in models of normothermic ischaemia reperfusion injury is generally to inhibit cardioprotective effects of ERK activation (Hausenloy et al., 2005a), results after hypothermia and ischaemia are different. In a Langendorff (Sprague-Dawley) rat heart model of hypothermic ischaemia reperfusion, Clanachen and colleagues showed that exposure of hearts to 20 µM PD98059 in St Thomas solution
(2) during a period of 8 hours hypothermic storage and reperfusion actually improved post-reperfusion cardiac function compared to hearts stored in St Thomas solution (2) only (Clanachan et al., 2003). The phosphorylation status of ERK 1/2 was not assessed in this study.
201 Another study in a range of cultured cells from different sources demonstrated that exposure to cultures extended periods (4 hours) at 4°C produced transient ERK phosphorylation and activation over the first 5 minutes of rewarming (Chan et al., 1999). Ballif and Blenis, examined the
PD98059 concentration required to inhibit the ERK pathway under different states of activation and found that in the basal minimally activated state, the pathway could be inhibited with 2 – 10 µM PD98059, whilst in the activated state (as might be the case in the heart after a period of cold storage), the pathway required > 100 µM PD98059 to adequately inhibit the pathway (Ballif et al., 2001). In a working heart model, this concentration of MEK inhibitor would be expected to have many off-target effects and may have a direct effect on cardiac function. Thus, together with the PD98059 – associated activation of Akt (Figure 4.10), it is not surprising that 20µM PD98059 produced only partial inhibition of protection in the present model.
Conclusion
In summary, this study demonstrates that –
i) Hearts arrested and stored in Celsior supplemented with GTN and cariporide together for 6 hours showed significant recovery of post-storage cardiac function, i.e 80% vs 30% of baseline CF, 58% vs 16% of AF, 70% vs 20% CO and 95% vs 47% of HR compared to control hearts (P<0.05 vs control or unsupplemented Celsior).
202 ii) Pro-survival kinase Erk1/2 and downstream anti-apoptotic marker Bcl-2 were significantly phosphorylated post reperfusion when Celsior was supplemented with GTN, cariporide or a combination of both (both
P<0.001 vs control).
iii) A significant decrease in the level of cleaved caspase 3, a marker of apoptosis was observed in hearts arrested and stored in Celsior supplemented with GTN and cariporide alone or in combination, in this model of donor heart survival (P<0.001 vs control).
Importantly for the potential application of this approach to cardiac preservation in clinical cardiac retrieval and transplantation, maximal protection and ERK activation were observed when the agents were added to the arresting and storage solution. Together with the demonstration of recruitment and activation of Akt by supplementation of the arresting and storage solution with the poly(ADP-ribose) polymerase inhibitor, INO 1153 in the previous chapter, the capacity now exists for recruiting both major elements of the RISK pathway. Combination of agents to recruit the RISK pathway in an infarct model has recently received favourable comment (Mocanu et al., 2007). Such an approach may be particularly powerful in the context of clinical cardiac preservation given the increasing use of “marginal” donor hearts in response to a shrinking donor pool and an ever expanding waiting list for transplantation.
203 CHAPTER 5
Enhanced Cardiac Recovery Post-Reperfusion after a
Prolonged Period of Hypothermic Storage by
Pharmacological Recruitment of Pro-survival Kinases Akt
and Erk1/2
204 5.1 INTRODUCTION
Although heart transplantation is now well established therapy for patients with end-stage cardiac disease, effective long term heart preservation remains a problem that has generally restricted viable donor graft storage times to 6 hours in the setting of clinical heart transplantation. Such has been the overall success of modern clinical heart transplantation that the original indications for the procedure have been broadened and as a result, the numbers of patients on the waiting list have been increasing steadily and far exceed the current availability of organs (Macdonald,
2008). This shortage has led to the current practice of referring and increasing number of hearts from “marginal” donors for consideration for transplantation. Unfortunately, up to 60% of the hearts from this group of donors are not transplanted due to poor organ quality (Rosengard et al.,
2002). A recent clinical study has shown that Celsior solution was particularly useful as an arresting and storage preparation for hearts from
“high risk” donors, although the cold ischaemic times for the high risk group was only 190 ± 50 minutes, well below the 6 hour cut-off (De Santo et al., 2006).
It is now well recognised that that the ischaemia reperfusion injury sustained by the donor heart during the obligatory procurement, period of cold storage and reimplantation during the transplant process may adversely affect short and long term outcomes. The recent elucidation of the mechanisms which underpin ischaemia reperfusion injury have
205 suggested new approaches to manage this damage. Such is the complexity of the molecular changes that pre-dispose the heart to ischaemia reperfusion related preservation injury, it is unlikely that any single treatment will provide maximal protection to the donor heart during cold storage and reimplantation. Rather, a combination of approaches is likely to be required.
Three of the most important mechanisms of ischaemia reperfusion injury to cardiac myocytes and endothelium introduced in chapter 1 were intracellular calcium overload (Dong et al., 2006), a decrease in bioavailability of nitric oxide (Loscalzo et al., 1995) and a massive increase in the activity of poly(ADP-ribose) polymerase activity leading to significant loss of high energy phosphates and cell death (Szabo et al., 2004a).
Recruitment of pro-survival signalling kinases of the RISK pathway by ischaemic or pharmacological pre- and post-conditioning provide a paradigm shift in designing adaptive approaches to protect the heart against ischaemia reperfusion injury. A recent review summarising the results of clinical trials using ischaemic pre or post-conditioning in cardiac surgery have shown beneficial effects of these adaptive processes on the ability of the heart to withstand ischaemia reperfusion damage (Hausenloy et al., 2008a). Although there are no published data on the use of pre- or postconditioning in clinical heart transplantation, recent reports of its
206 application to clinical liver transplantation are promising (Amador et al.,
2007, Cescon et al., 2006, Net et al., 2005).
The previous chapters have identified pharmacological approaches for minimising intracellular calcium overload (cariporide), increasing nitric oxide bioavailability (GTN) and inhibiting PARP (INO-1153). Importantly, each agent activates major components of the RISK pathway.
Supplementation of Celsior with the PARP inhibitor, INO-1153 improved post reperfusion cardiac function after 6 hours hypothermic storage, whilst activating the PI 3-k/Akt pathway (chapter 3). The functional improvement in hearts arrested and stored in Celsior supplemented with GTN and
Cariporide after 6 hours hypothermic storage was associated with ERK 1/2 activation (Chapter 4).
The hypothesis to be tested in the study in the present chapter was that pharmacological activation of both the Akt pathway and the ERK 1/2 pathways simultaneously may further enhance cardiac protection and allow viable functional recovery after an extended (10 hr) period of hypothermic storage. The specific aims of the study were i) to assess the recovery of post-storage function of hearts arrested and stored in Celsior supplemented with GTN, cariporide and INO-1153 alone and in all combinations compared to unsupplemented Celsior; ii) to ascertain the extent of phosphorylation (activation) of the major proximal RISK pathway elements, Akt and ERK 1/2; iii) to assess the extent of phosphorylation of
207 GSK 3ß, a major downstream target of the ERK and Akt pathways; iv) to assess the extent of phosphorylation of the ezrin / radixin / moesin (ERM) complex, classically involved in cytoskeletal remodelling and recently identified as a major component in the transduction of survival signalling
(including the Akt pathway) (Louvet-Vallee, 2000) and v) to assess the extent of apoptosis.
208 5.2 MATERIALS AND METHODS
Animals
As described in Chapter 2, section 2.3.
Ethical conduct of studies
As described in Chapter 2, section 2.2.
Chemicals and pharmacological agents
As described in Chapter 2, section 2.1.
Preparation of perfusion buffer
As described in Chapter 2, section 2.1.
Isolated working heart preparation
A detailed account of the surgical steps in preparing the isolated heart appears in Chapter 2, section 2.4. Briefly, the rats were anaesthetised with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). After bolus injection of 500 IU heparin into the renal vein, the heart was rapidly excised and arrested by immersion in chilled (2 – 3°C) perfusion buffer. The aorta was cannulated and immediately perfused retrogradely on a Langendorff perfusion apparatus with Krebs buffer at a hydrostatic pressure of 100 cm H2O. During this time, a small incision was made in the left atrial appendage into which another cannula was inserted
209 and tied off. This non-working preparation was stabilised for 10 minutes and then converted to working mode by switching the supply of perfusate from the aorta to the left atrial cannula at a hydrostatic pressure of 20 cm
H2O (pre-load). The working heart ejected perfusate via the aortic valve into the aortic cannula. The hydrostatic pressure in the aortic cannula was maintained at 100 cm H2O (after-load) throughout the working phase for all rat hearts.
Aortic pressure was monitored in a side arm of the aortic cannula with a pressure transducer (Ohmeda, Pty Ltd., Singapore). Aortic flow was measured by an in-line flowmeter (Transonics Instruments Inc. Ithaca,
NY). Aortic pressure and flow were recorded using MacLab/4e
(ADInstruments Pty Ltd, Sydney, Australia) and heart rate was calculated from the flow trace. Coronary flow was measured by timed collection of the effluent draining from the apex of the heart.
Experimental protocol and timeline (Figure 5.1 a)
All hearts remained in working mode for 15 min prior to storage.
Measurements of heart rate (HR), aortic flow (AF), coronary flow (CF), and cardiac output (CO) were made at 10 minutes after conversion to working mode and used as pre-storage baseline.
Exclusion criteria: Any hearts having a baseline aortic flow less than 35 ml/min, heart rate of less than 200 beats/min, or coronary flow less than
10 mL/min were excluded.
210 After collection of baseline hemodynamic data, the heart was arrested by infusion of cold Celsior preservation solution (at 2–3°C) into the coronary circulation for 3 minutes from a reservoir 60 cm above the heart. All hearts were stored in 100 mL of the same solution for 10 hours under hypothermic conditions (2–3°C). Hearts were then remounted on the perfusion apparatus and reperfused in Langendorff mode for 15 minutes.
Hearts were then switched to working mode then stabilized for 30 minutes.
The Indices of cardiac function measured at baseline were remeasured after 10 hours hypothermic storage and 45 minutes reperfusion. Recovery of each parameter was expressed as a percentage of its pre-storage baseline. After the 30 minutes functional observations were taken, the left ventricular free wall of each heart was rapidly frozen by immersion in liquid nitrogen and stored at -80 °C for Western blot analysis.
211 (a) Cardioplegia Reperfusion
Pre-storage Phase Storage phase Post-Storage Phase (2-3°C) Langendorff Langendorff Working Heart Working Heart
10 min 15 min 10 hrs 15 min 30 min
Celsior + Krebs Krebs Single Additive
Krebs Celsior + Double Krebs Additives
Krebs Celsior + Triple Krebs Additives
# (b) Experimental Groups
Groups GTN Cariporide INO1153 (0.1 mg/ml) (10 μM) (1 μM) A √
B √ C √
D √ √
E √ √
F √ √
G √ √ √
Figure 5.1 Timeline and experimental groups. (a) illustrates the experimental timeline for the functional study. Figure 5.1 (b) shows the treatment groups
(pharmacological supplements to Celsior) employed in the study. Rat hearts were randomised to one of 7 groups (A – G). Numbers of hearts in each group were as follows: (A = 5; B = 6; C = 4; D = 6; E = 7; F = 6; G = 6). Arrows indicate times at which cardiac function were assessed. (#) indicates the point where heart tissue was harvested for western immnoblotting analysis.
212 Experimental groups
For consistency and ease of comparison, the concentrations of the various agents in the cardioplegic solution for the 10 hour hypothermic storage study described in here were the same as those used in Chapters 3 and 4
(ie [INO-1153] = 1 µM; [GTN] = 0.1 mg/ml; [Cariporide] = 10 µM). Figure
5.1b illustrates the experimental timeline and treatment groups. Rat hearts were divided into three groups according to the type of pharmacological additives added to the cardioplegic and storage solution, Celsior.
Compositions were as follows:
Group 1, Single additive - 0.1mg/ml glyceryl trinitrate (GTN) (Group A) or
- 10μM cariporide (Group B) or
- 1μM INO-1153 (Group C);
Group 2, Double additives - 0.1mg/ml GTN + 1μM INO-1153 (Group D);
- 1μM INO-1153 + 10μM cariporide (Group E);
- 0.1mg/ml GTN + 10μM cariporide (Group F);
Group 3, Triple additives
- 0.1mg/ml GTN + 10μM cariporide + 1μM INO-1153 (Group G)
Western immunoblotting
Briefly a subgroup of 4 hearts was chosen from each experimental group for Western blot analysis and sixty milligrams of tissue from each heart were homogenized in cold lysis buffer (section 2.7). Protein samples were boiled in sample loading buffer for 5 minutes before loading onto 10%
213 SDS-polyacrylamide gel under reducing conditions (40 µg protein per lane) as described in section 2.8. Membranes were blocked for 1 hour in
Tris buffered saline, (pH 7.4) which contained 1% BSA and 0.1% Tween
20. Membranes were probed with primary rabbit polyclonal antibodies
(1:3000 dilution) raised against total and phospho-p44/42 MAPK (Erk1/2), total and phospho-Akt (Ser 473), total and phospho-GSK3β, total and phospho-Ezrin (Thr567) / Radixin (Thr564) / Moesin (Thr558), Caspase 3 and Cleaved Caspase 3, β-actin (Cell Signaling Technology, Danvers,
MA). The secondary antibodies used were horseradish peroxidase- conjugated anti-rabbit IgG and anti-mouse IgG (Amersham Life Sciences).
The protein bands were visualized using enhanced chemiluminescence
(Pierce Biotechnology) with bands visualised by autoradiography and intensities were digitised with Image J freeware (National Institute of
Mental Health, Bethesda, MA).
Statistical analysis
All values were expressed as mean ± SE unless otherwise specified. Data were analyzed by the StatView 4.5 statistical software package.
Differences among groups were compared by one-way ANOVA analysis followed by Fisher’s PLSD post hoc test, a P value of less than 0.05 was considered significant.
214 5.3 RESULTS
5.3.1 Prestorage baseline measurements of cardiac function
The pre-storage baseline measurements of cardiac function for control and all treatment groups are shown in Table 5.1. The groups were well matched with no significant differences between any of the experimental groups for any of the parameters measured.
Table 5.1 Pre-storage baseline cardiac measurements and values.
AF CF CO Treatment Groups HR ( bpm) n (ml/min) (ml/min) (ml/min)
Single Additive A Celsior + 0.1mg/ml GTN 42 ± 8.1 19 ± 1.0 61 ± 8.7 234 ± 38 5 B Celsior + 10µM Cariporide 55 ± 15.4 17 ± 3.4 72 ± 18.5 243 ± 57 6 C Celsior + 1µM INO-1153 42 ± 5.5 15 ± 1.6 57 ± 6.5 311 ± 39 4 Double Additives
D Celsior + 0.1mg/ml GTN + 48 ± 8.1 19 ± 1.5 67 ± 9.2 265 ± 15 6 1µM INO-1153
E Celsior + 10µM Cariporide + 48 ± 4.9 19 ± 2.5 67 ± 6.2 269 ± 24 7 1µM INO-1153
F Celsior + 0.1mg/ml GTN + 51 ± 10 18 ± 3 68 ± 11 273 ± 16 6 10µM Cariporide
Triple Additives Celsior + 0.1mg/ml GTN + G 10µM Cariporide + 1µM INO- 46 ± 3.9 18 ± 1.2 64 ± 4 274 ± 29 6 1153
AF=Aortic flow, CF=Coronary flow, CO=Cardiac output, HR=Heart rate,
N=number of rat hearts. Data are expressed as means ± SD in each parameter.
215 5.3.2 Effect of cardioplegia / storage solution composition on post-
reperfusion recovery of cardiac function
Figure 5.2 shows the representative trace recordings of pressure and aortic flow for pre-storage baseline and after 10 hours of hypothermic storage for the various pharmacological additives. The example of post- storage recovery of hearts stored in Celsior preservation with a single additive (GTN) in this case alone was poor. However, better post-storage recovery was observed in hearts that were stored in Celsior preservation solution with 2 of the 3 additives (in this case, GTN and Cariporide). The strongest recovery was observed in hearts that were stored in Celsior supplemented with all three additives together (GTN, Cariporide and INO-
1153).
The group data for recovery of function after 10 hours hypothermic storage are plotted in Figures 5.3 – 5.6 (Figure 5.3, Recovery of aortic flow; Figure
5.4, Recovery of coronary flow; Figure 5.5, Recovery of cardiac output;
Figure 5.6, Recovery of heart rate). For convenience, numerical values for functional recoveries are tabulated in Table 5.2.
Hearts arrested and stored in Celsior supplemented with a single agent only (GTN, cariporide or INO-1153) had no recovery of contractile function
(aortic flow and coronary output) with poor recovery of coronary flow.
Heart rate also showed little recovery in the singly supplemented hearts.
216 On the whole, hearts arrested and stored in Celsior supplemented with 2 of the 3 supplements showed better recovery of the indices of cardiac function examined, especially in the groups containing GTN. However, this recovery was still probably classed as “non-viable” recovery.
Hearts arrested and stored in Celsior supplemented with all three agents,
(group G) in Figures 5.3 – 5.6 and Table 5.2, showed robust and viable recovery of post-storage function. After 45 minutes reperfusion, triple supplemented hearts had regained 42% of baseline aortic flows [P < 0.02 vs all other groups] (Figure 5.3), 67% of baseline coronary flows [P < 0.01 vs all other groups] (Figure 5.4), 49% of baseline cardiac output [P < 0.05 vs all other groups] (Figure 5.5) and 86 % of baseline heart rate (Figure
5.6) [P < 0.02 vs all other groups].
217 (Pressure) Pre-storage Baseline
(Aortic Flow) 200 ml/min
Single Additive
GTN (Group A)
40 ml/min
Double Additives
GTN + Cariporide (Group F)
100 ml/min
Triple Additives
GTN + Cariporide + INO1153 (Group G)
200 ml/min
Figure 5.2 Effect of various pharmacological additives composition in cardioplegic / storage solution on indices of post-storage cardiac function.
Examples of representative trace recordings of pressure and aortic flow for pre- storage baseline and during reperfusion subsequent to 10 hours of hypothermic storage for various pharmacological additives.
218 Aortic Flow 60
* 50
40
30
Recovery (% Baseline) of 20
10
0 A B C DEF G
Single Double Triple Additive Additives Additives
Figure 5.3 Aortic flow represented as a percentage of baseline values in hearts after 45 minutes post-storage reperfusion following 10 hours hypothermic storage. Effect of various pharmacological additives composition in cardioplegic / storage solution on cardiac recovery (Groups A – G). Values are expressed as mean ± SE. * P < 0.02 vs all other groups, ANOVA.
Single additive (A) - 0.1mg/ml glyceryl trinitrate (GTN);
(B) - 10μM cariporide;
(C) - 1μM INO-1153;
Double additives (D) - 0.1mg/ml GTN + 1μM INO-1153;
(E) - 1μM INO-1153 + 10μM cariporide;
(F) - 0.1mg/ml GTN + 10μM cariporide;
Triple additives (G) 0.1mg/ml GTN + 10μM cariporide + 1μM INO-1153
219 Coronary Flow 80
* 70
60 *
50
40
Recovery 30 (% ofBaseline)
20
10
0 A B C D E F G Single Double Triple Additive Additives Additives
Figure 5.4 Coronary flow represented as a percentage of baseline values in hearts after 45 minutes post-storage reperfusion following 10 hours hypothermic storage. Effect of various pharmacological additives composition in cardioplegic / storage solution on cardiac recovery (Groups A – G). Values are expressed as mean ± SE. * P < 0.01 vs all other groups, ANOVA.
Single additive (A) - 0.1mg/ml glyceryl trinitrate (GTN);
(B) - 10μM cariporide;
(C) - 1μM INO-1153;
Double additives (D) - 0.1mg/ml GTN + 1μM INO-1153;
(E) - 1μM INO-1153 + 10μM cariporide;
(F) - 0.1mg/ml GTN + 10μM cariporide;
Triple additives (G) 0.1mg/ml GTN + 10μM cariporide + 1μM INO-1153
220 Cardiac Output 60 *
50
40
30
Recovery (% of Baseline) 20
10
0
A B C DGFE Single Double Triple Additive Additives Additives
Figure 5.5 Cardiac output represented as a percentage of baseline values in hearts after 45 minutes post-storage reperfusion following 10 hours hypothermic storage. Effect of various pharmacological additives composition in cardioplegic / storage solution on cardiac recovery (Groups A – G). Values are expressed as mean ± SE. * P < 0.05 vs all other groups, ANOVA.
Single additive (A) - 0.1mg/ml glyceryl trinitrate (GTN);
(B) - 10μM cariporide;
(C) - 1μM INO-1153;
Double additives (D) - 0.1mg/ml GTN + 1μM INO-1153;
(E) - 1μM INO-1153 + 10μM cariporide;
(F) - 0.1mg/ml GTN + 10μM cariporide;
Triple additives (G) 0.1mg/ml GTN + 10μM cariporide + 1μM INO-1153
221 Heart Rate 100 *
80
60
Recovery Recovery 40 (% of Baseline)
20
0 A B C DGFE
Single Double Triple Additive Additives Additives
Figure 5.6 Heart rate represented as a percentage of baseline values in hearts after 45 minutes post-storage reperfusion following 10 hours hypothermic storage. Effect of various pharmacological additives composition in cardioplegic / storage solution on cardiac recovery (Groups A – G). Values are expressed as mean ± SE. * P < 0.02 vs all other groups, ANOVA.
Single additive (A) - 0.1mg/ml glyceryl trinitrate (GTN);
(B) - 10μM cariporide;
(C) - 1μM INO-1153;
Double additives (D) - 0.1mg/ml GTN + 1μM INO-1153;
(E) - 1μM INO-1153 + 10μM cariporide;
(F) - 0.1mg/ml GTN + 10μM cariporide;
Triple additives (G) 0.1mg/ml GTN + 10μM cariporide + 1μM INO-1153
222 Table 5.2 Recovery of indices of cardiac function aftern 10 hours cold ischaemic time (data from Figures 5.3 – 5.6).
Treatment Recovery of Index of Cardiac Function n Group (% of Pre-storage Baseline Level) AF CF CO HR Single Agent 0.8 ± 0.5 8.0 ± 1.7 3.0 ± 0.7 9.9 ± 9.9 5 A 1.4 ± 0.8 21.0 ± 4.3 5.8 ± 1.2 17.4 ± 10.2 6 B 1.1 ± 0.6 7.0 ± 2.9 1.7 ± 0.7 21.5 ± 12.5 4 C Combinations of 2 Agents 12.1 ± 9.5 24.3 ± 14.9 15.3 ± 10.5 40.2 ± 18.7 6 D 3.0 ± 1.5 18.8 ± 8.7 7.3 ± 3.4 28.2 ± 12.2 7 E 17.8 ± 10.3 48.6 ± 12† 25.7 ± 10.6 54.5 ± 14.7 6 F Three Agents 42.2 ± 10.7* 66.9 ± 4.5† 49.2 ± 8.8¶ 86.4 ± 4.4* 6 G
Celsior Supplements contained in each Group -
A: 0.1 mg / ml GTN;
B: 10 µM Cariporide;
C: 1 µM INO-1153;
D: 0.1 mg / ml GTN + 1 µM INO-1153;
E: 1 µM INO-1153 + 10 µM Cariporide;
F: 0.1 mg / ml GTN + 10 µM Cariporide;
G: 0.1 mg / ml GTN + 10 µM Cariporide + 1 µM INO-1153.
Abbreviations: AF: Aortic Flow; CF: Cononary Flow; CO: Cardiac Output; HR:
Heart Rate. * P < 0.02, † P < 0.01, ¶ P < 0.05 vs all other groups.
223 5.3.3 Effect of cardioplegia / storage solution composition on post-
reperfusion phosphorylation status of ERK 1/2 and Akt
Immunoblotting for phosphorylated and total proteins comprising key elements in pro-survival signalling pathways was carried out for all the doubly supplemented combinations and the triply supplemented group.
Hearts that had been arrested and stored for 10 hours in unsupplemented
Celsior were used as the comparator for this part of the study.
5.7 (a) shows the immunoblots of the phospho and total ERK 1/2 and ß actin loading controls from a representative heart from each group, with
Figure 5.7 (b) showing quantitation of the phospho / total ERK ratios for a subgroup of 4 hearts. Total ERK 1/2 was uniformly expressed throughout
(Figure 5.7 (a)), indicating neither the storage conditions nor the various combinations of pharmacological additives had any effect on the amount of ERK detected. Comparatively low levels of phospho ERK 1/2 were detected in the Celsior alone control. Increased levels of phospho-ERK 1/2 were detected in all the doubly supplemented groups with the increases in phospho / total ratios reaching significance for the double supplements which contained GTN (Figure 5.7 (b)). The largest phospho / total ERK
1/2 ratio was observed in the hearts from the group arrested and stored in
Celsior containing all three supplements.
Figure 5.8 (a and b) show the effect of pharmacological supplements present during arrest and storage on the phosphorylation status of Akt in a
224 (a) Phospho- Erk1/2
Total Erk1/2
Actin
(b) 1.2
** * 1.0
0.8
0.6 Total Erk 0.4
Ratio of Phospho-Erk1/2 / 0.2
0
0.1mg/ml GTN - + + - + 10µM Cariporide - + - + +
1µM INO-1153 - - + + +
Figure 5.7 Effect of various pharmacological additives composition in cardioplegic / storage solution on the extent of Erk1/2 phosphorylation 45 minutes post-reperfusion phase following 10 hr hypothermic storage. (a)
Representative western immunoblots from each group showing levels of Erk1/2 phosphorylation (activation), total Erk1/2 and β-actin. (b)Histogram showing ratio of phosphorylated Erk1/2 to total Erk1/2 (n=4 in each group), values are means ±
SE. *P < 0.01 vs control, ANOVA.
225 representative heart from each group (Figure 5.8 (a)) and the phospho / total Akt ratios in a sub-group of 4 hearts (Figure 5.8 (b)). As was the case with ERK 1/2, the levels of total Akt were constant across all experimental groups, indicating that they were unaffected by arresting and storage conditions and pharmacological supplements. There was minimal presence of phospho-Akt in hearts from the unsupplemented Celsior group (Figure 5.8 (a and b)). The extent of Akt phosphorylation was increased in all the doubly supplemented combinations. The largest increase in the extent of Akt phosphorylation was again observed in hearts arrested and stored in Celsior supplemented with all three agents, with an approximately 6 fold increase in the phospho / total Akt ratio compared to
Celsior alone control and around a doubling of the ratio compared to the doubly supplemented combinations (Figure 5.8 (b)).
226 (a)
Phospho - Akt
Total Akt
Actin
(b) 1.2
1.0
0.8 *
0.6
Total Akt * 0.4 *
Ratio of Phospho-Akt / * 0.2
0
0.1mg/ml GTN - + + - +
10µM Cariporide - + - + +
1µM INO-1153 - - + + +
Figure 5.8 Effect of various pharmacological additives composition in cardioplegic / storage solution on the extent of Akt phosphorylation 45 minutes post-reperfusion phase following 10 hr hypothermic storage. (a)
Representative western immunoblots from each group showing levels of Akt phosphorylation (activation), total Akt and β-actin. (b) Histogram showing ratio of phosphorylated Akt to total Akt (n=4 in each group), values are means ± SE. * P
< 0.01 vs control, ANOVA.
227 5.3.4 Effect of cardioplegia / storage solution composition on post-
reperfusion phosphorylation status of GSK3β
Having demonstrated that GTN, Cariporide and INO-1153 supplementation in Celsior improves post-storage cardiac function and activates Erk1/2 and Akt, we further investigate evidence of down-stream signalling of Erk1/2 and Akt pathway. We observed that the pattern of
GSK3β phosphorylation (Figure 5.9a) parallels that of Akt phosphorylation
(Figure 5.8) and that of Erk1/2 phosphorylation (Figure 5.7), with significant increase in the levels of GSK3β phosphorylation observed
(Figue 5.9b) in hearts treated with a combination of double additives (blue coloured bars) with the most significant increase recorded (red coloured bar) with the addition of all 3 additives (GTN, Cariporide and INO-1153) in celsior solution when compared to control hearts.
228 (a)
Phospho - GSK3β
Total GSK3β
Actin
(b)
1.2
/ β 1 * β 0.8 * 0.6
Total GSK3 0.4 * *
Ratio of Ratio of Phospho-GSK3 0.2
0
0.1mg/ml GTN - + + - + 10µM Cariporide - + - + +
1µM INO-1153 - - + + +
Figure 5.9 Effect of various pharmacological additives composition in cardioplegic / storage solution on the extent of GSK3β phosphorylation 45 minutes post-reperfusion phase following 10 hr hypothermic storage. (a)
Representative western immunoblots from each group showing levels of GSK3β phosphorylation, total GSK3β and β-actin. (b) Histogram showing ratio of phosphorylated GSK3β to total GSK3β (n=4 in each group), values are means ±
SE. * P < 0.01 vs control, ANOVA.
229 5.3.5 Effect of cardioplegia / storage solution composition on post-
reperfusion phosphorylation status of ERM
ERM (Ezrin-Radixin-Moesin) has been shown to interact with the PI3-k/Akt signalling molecule and could result in the activation of Akt (Wu et al.,
2004). To further determine the evidence of phosphorylation (activation) of
ERM, we examine the extent of ERM phosphorylation and found that there was a significant increase in the levels of ERM phosphorylation observed across all the treatment groups with the most significant increase recorded in the group with the addition of all 3 additives (GTN, Cariporide and INO-
1153) in celsior solution when compared to control hearts (Figure 5.10).
5.3.6 Effect of cardioplegia / storage solution composition on post-
reperfusion phosphorylation status of BAD and Bcl-2
The expression of Bad and Bcl-2 phosphorylation was used to explore the anti-apoptotic activities of Erk1/2 and Akt phosphorylation induced by pharmacological additives GTN, Cariporide and INO-1153 in Celsior solution (Figure 5.11 a and b). In hearts arrested and stored in unsupplemented Celsior, there was almost an absence of both BAD and
Bcl-2 phosphorylation. However, there were increased phosphorylation of both BAD and Bcl-2 expression in doubly supplemented Celsior as well as with all three pharmacological agents observed as compared to unsupplemented Celsior.
230 (a)
Phospho - ERM
Total ERM Actin
(b) 1.2
1 * * 0.8 * 0.6 Total ERM 0.4 *
Ratio of Phospho-ERM / 0.2
0
0.1mg/ml GTN - + + - +
10µM Cariporide - + - + +
1µM INO-1153 - - + + +
Figure 5.10 Effect of various pharmacological additives composition in cardioplegic / storage solution on the extent of ERM phosphorylation 45 minutes post-reperfusion phase following 10 hr hypothermic storage. (a)
Representative western immunoblots from each group showing levels of ERM phosphorylation, total ERM and β-actin. (b) Histogram showing ratio of phosphorylated ERM to total ERM (n=4 in each group), values are means ± SE. *
P < 0.01 vs control, ANOVA.
231 (a) Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Phospho - BAD
Total BAD
(b) Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6
Phospho - Bcl2
Total Bcl2
Lane identification
Lane 1 - Freshly Excised Heart
Lane 2 - Unsupplemented Celsior
Lane 3 - Celsior + 1 µM INO 1153
Lane 4 - Celsior + 0.1 mg / ml GTN + 1 µM INO 1153
Lane 5 - Celsior + 1 µM INO 1153 + 10 µM Cariporide
Lane 6 - Celsior + GTN + Cariporide + INO 1153
Figure 5.11 Effect of various pharmacological additives composition in cardioplegic / storage solution on the extent of BAD and Bcl-2 phosphorylation 45 minutes post-reperfusion phase following 10 hr hypothermic storage. (a) Representative western immunoblots showing levels of BAD phosphorylation and total BAD. (b) Representative western immunoblots showing levels of Bcl-2 phosphorylation and total Bcl-2.
232 5.4 DISCUSSION
This chapter explores the potential of developing a preservation solution strategy that extends the window of protection for the donor heart by combining all three pharmacological additives (INO-1153 from Chapter 3,
GTN and Cariporide from Chapter 4) together in Celsior preservation solution. The aim of this aspect of the study is to identify some of the mechanistic aspects of cardiac protection involved extending the hypothermic storage time that accompany functional recovery.
The present results showed that: i) Hearts arrested and stored in Celsior supplemented with a single pharmacological agent demonstrated minimal or no recovery of cardiac function, whereas hearts exposed to Celsior supplemented all three pharmacological agents have significant recovery of all the indices of cardiac function assessed. Specifically, functional recovery was as follows:
42% of baseline aortic flows (P < 0.02 vs all other groups) (Figure 5.3),
67% of baseline coronary flows (P < 0.01 vs all other groups) (Figure 5.4),
49% of baseline cardiac output (P < 0.05 vs all other groups) (Figure 5.5) and 86 % of baseline heart rate (Figure 5.6) (P < 0.02 vs all other groups).
ii) Significant increases in Erk1/2, Akt, GSK3ß and ERM phosphorylation were observed in hearts exposed to 2 or 3 pharmacological supplements
(Figures 5.7 – 5.10), however, the largest increase was observed in the triply supplemented group which was associated with a 6.5 fold increase in
233 Erk1/2 phosphorylation, 6 fold increase in Akt phosphorylation, 5.3 fold increase in phosphorylation of GSK3ß and a 5.4 fold increase in ERM phosphorylation respectively (All P<0.01 vs control).
iii) The down-stream anti-apoptotic potential of pro-survival kinase activation (both Erk1/2 and Akt) by the pharmacological agents in Celsior solution, was manifested as increased phosphorylation of BAD and Bcl-2 in doubly and triple supplemented groups (Figure 5.11).
It is evident that the addition of these 3 pharmacological additives would involve different biochemical and ionic processes, all of which play important roles in cell survival and contribute to the preservation of heart during hypothermic storage to enable a functioning heart during reperfusion. GTN acts as an exogenous donor of nitric oxide, that may have a number of protective effects. In the present model it may: i) improve vasodilatory activity and cardiac perfusion; ii) reversibly inhibit elements of the electron transport chain resulting in decreased superoxide and ROS formation; iii) by the S-nitrosation reaction, in may inhibit key pro-apoptotic elements such as caspase 3 (discussed in chapter 1, section
1.5.1).
Cariporide may also have multiple cardioprotective effects arising from its ability to inhibit sodium hydrogen exchange during ischaemia and at immediately post reperfusion (Chapter 1, section 1.5.2). These include: i)
234 minimising sodium infux during ischaemia and especially at reperfusion
(minimising oedema); ii) preventing reverse mode activity of the Na+/Ca+ exchanger thus minimising calcium intracellular influx, secondary to intracellular sodium influx that occurs in the absence of Na+/H+ exchange inhibition (minimising protease activity and mitochondrial damage) and iii) because of the inability to clear proton accumulation due to glycolytic activity during ischaemia and early reperfusion, a slightly acidic intracellular environment will be maintained immediately post reperfusion that will help to minimise the open probability of the mitochondrial transition pore and protect the integrity of the mitochondria for the task of rapidly initiating oxidative phosphorylation and ATP production crucial for the recovery of proper cardiac function.
Pharmacological inhibition of PARP has been documented by many investigators in preclinical models showing significant improvement in the recovery of heart function during myocardial ischaemia and reperfusion.
The importance of inhibition of poly(ADPribose) polymerase in minimising ischaemia reperfusion related injury in the heart and other organs has recently been recognised (Chapter 1, section 1.5.3). Hyperactivation of
PARP following ischaemia and reperfusion leads to profound depletion of cellular NAD(P)H and ATP leading to increases in cell death via apoptosis and necrosis.
235 In addition to the protective effects enumerated above, the preceding chapters have shown that the presence of GTN, cariporide and the PARP inhibitor INO-1153 in the arresting and storage solution are able to activate endogenous elements of prosurvival signalling – GTN and cariporide activating the ERK 1/2 pathway and INO-1153 predominantly the PI 3-k /
Akt pathway. The hypothesis examined here was that combining all three agents in the arresting and storage solution, (Celsior), should allow both arms of the RISK pathway to be recruited and activated after storage and this should result in protection against a longer period of cold ischaemic storage than either agent alone.
A distinct and immediate improvement of cardiac recovery is observed in the trace recordings (Figure 5.2) when all 3 pharmacological addtives were present in Celsior solution and this is strengthened by the group data reflected across all parameters of functional recovery (Figures 5.3 – 5.6).
Despite the significant recovery of cardiac function of using 2 additives
(GTN and Cariporide) together to supplement Celsior solution described in
Chapter 4 for 6 hours of storage, maximal recovery of heart function was only observed in presence of all 3 pharmacological additives when hypothermic storage time is increased to 10 hours (Group F, Figures 5.3-
5.6). Similar observations with INO-1153 in group B (Figures 5.3 – 5.6) reflected an almost non existent recovery of contractility and recovery of the heart when compared to the significant recovery of heart function observed in Chapter 3 when INO-1153 was supplemented in Celsior
236 solution. The present 10 hours functional results suggest that no single pharmacological drug or treatment in clinical preservation solution such as
Celsior will provide optimal protection to the heart under conditions of prolong hypothermia (> 6 hours).
As described previously (Chapter 1, section 1.5.5) both the Erk1/2 and
PI3-k/Akt are major elements of the RISK pathway that play a protective role in cardiac ischaemic reperfusion injury during ischaemic or pharmacologic preconditioning as well as regulating other signalling events, we explored the extent of activation of these pathways. A previous landmark report by Chan and colleagues (Chan et al., 1999) looked at
Ras-MEK-Erk kinase cascade in cells exposed to hypothermia and found
Erk1/2 activation when hypothermic cells were warmed to physiological temperature of 37oC during recovery. In addition, they also confirmed Erk activation in a variety of cultured cells when exposed to prolonged hypothermia which led them to conclude that the Ras-Erk signalling can be manipulated to increase cell survival during cold storage. The present finding during post-storage in our hearts supplemented with either 2 or 3 pharmacological additives in Celsior solution demonstrated a significant increase in Erk1/2 phosphorylation (or activation) when the hearts were brought back to physiological temperature during reperfusion after prolonged (10 hours) of hypothermic storage (Figure 5.7). The most significant increase was observed in hearts stored with all 3 pharmacological additives present in the preservation solution.
237 The extent of Akt phosphorylation at Ser 473 (Figure 5.8) somewhat paralleled the increase in ERK1/2 phosphorylation, with hearts exposed to the 3 additives showing the most robust increase in Akt phosphorylation.
As shown in chapter 3, the presence of a single additive, PARP inhibitor
INO-1153, contributed to a significant increase in the phosphorylation of
Akt when these hearts were stored under hypothermic condition for 6 hours. Our present findings also agree with several other reports confirming the beneficial use of PARP inhibitors during cardiac preservation and reperfusion (Fiorillo et al., 2003, Szabo et al., 2002) as well as the activation of Akt and Erk1/2 (Palfi et al., 2005).
Glycogen synthase kinase-3ß is a common down-stream target of both Akt and ERK. Phosphorylation of GSK3ß at Ser 9 could be catalysed directly by activated Akt (D.A. Cross et al., 1995). Phosphorylation of GSK3ß by
Erk1/2 is an indirect 2 stage process. Phosphorylated ERK first docks with
GSK3ß and phosphorylates it at Thr43, priming it for phosphorylation at
Ser9 by p90rsk-1 (Ding et al., 2005, Stambolic et al., 1994). Phospho-
GSK3ß (Ser9) acts as a “pseudosubstrate”, docking with the “priming phosphate site” of GSK. This process prevents access of the normal substrates of GSK to the active sites, resulting in phosphorylation-induced inactivation of the enzyme. The results of GSK3β phosphorylation (Figure
5.9) follows a similar trend to the previous observation of Akt phosphorylation and showed the most robust phosporylation (or inactivation) of GSK3β when hearts were treated with all 3
238 pharmacological additives; this result agrees with data from other groups
(Juhaszova et al., 2004, Tong et al., 2002) showing GSK3β inhibition or increase phosphorylation initiated by preconditioning resulted in cardioprotection which significantly reduces ischaemia and reperfusion injury.
The phosphorylation of GSK3-ß at Ser9 has been demonstrated to be an integral element in the caridioprotective effects of pharmacological and ischaemic “conditioning”. Juhaszova and colleagues (Juhaszova et al.,
2004) implicated phosphorylated GSK3ß in the inhibition of mitochondrial transition pore opening and showed that cells containing a constitutively active form of GSK3ß were resistant to phosphorylation and resistant to pharmacological cardioprotective interventions.
However the mechanism of GSK3ß associated protection is still unclear, not the least because of the evolving nature of the architecture of the mitochondrial permeability transition pore (Halestrap et al., 2009). These workers posit a biphasic protective effect of conditioning involving: i) prevention of mPTP opening immediately post reperfusion and ii) further sustained protection during reperfusion by preventing a cycle of mPTP- induced ROS formation and further mPTP opening. They suggest that Akt mediated GSK3ß phosphorylation and inhibition may facilitate this latter phase by increasing ROS removal or reducing mitochondrial ROS production. A recent study by Das and colleagues suggest that inhibition
239 of GSK3ß may play an adaptive role in reperfusion injury independent of permeability pore transition (S. Das et al., 2008). Their experiments show that inhibition of GSK3ß may adapt cells an ischaemic insult by dephosphorylation of the voltage dependent anion channel in the outer membrane of the mitochondrion.
Ezrin / radixin / moesin (ERM) are a family of structurally related proteins that concentrate in actin rich cell surface structures and act as molecular linkers between the cytoskeleton and the plasma membrane (Louvet-
Vallee, 2000). The rationale for examining the change in phosphorylation status of ERM during hypothermic ischaemic storage and reperfusion was to assess the possible role of the Rho Kinase – PTEN axis in preventing recruitment of the PI 3-k / Akt pathway in hearts stored in celsior alone and the possible modulation of Rho kinase / PTEN activity by the cardioprotective pharmacological supplements. This rationale arose from recent studies (Hamid et al., 2007). Their premise was that Rho kinase activation would attenuate activation of the Akt pathway via activation of
PTEN, the lipid phosphatase that removes IP3. Using ERM phosphorylation as a surrogate for Rho kinase activation in a normothermic rat heart coronary artery ligation model, they demonstrated that the infarct produced by 35 minutes ligation of the left main coronary artery was accompanied by a significant increase in the extent of ERM phosphorylation (and by inference Rho kinase activation) which was significantly decreased in hearts exposed to the Rho kinase inhibitor,
240 fasudil. These studies inferred that the Rho kinase was activating PTEN and prevented recruitment of the Akt pathway.
The situation in the present model of 10 hours hypothermic ischaemic storage and 45 minutes reperfusion showed a completely different picture
(Figure 5.10). In hearts arrested and stored in unsupplemented celsior, there was virtually no evidence of ERM phosphorylation and in fact the extent of ERM phosphorylation increased in the hearts arrested and stored in doubly supplemented celsior and was maximal in hearts arrested and stored in celsior supplemented with all three pharmacological agents. We used the same strain of animal and the same source of antiphosphoERM antibody as Hamid and colleagues (Hamid et al., 2007), so the present results suggested that ERM phosphorylation was indicative of a possible enhancement of pro-survival signalling rather than a marker of decreased flux through the RISK pathway.
A further investigation of the literature indeed suggested that this may be the case and that ERM proteins have a significant role in recruitment of pro-survival signalling as well as a structural role. There are several studies to suggest that ERM interacts with the cystolic tail of the sodium hydrogen exchange to regulate cell survival and form a signalling complex that includes NHE, ERM, PI3-k and Akt (Gautreau et al., 1999, Khan et al.,
2006, Schelling et al., 2008, Wu et al., 2004). Under cellular stress conditions, the NHE-ERM interaction is able to activate pro-survival
241 pathways to prevent apoptosis in in vivo models and culture cells by forming a signalling complex with PI3-k/Akt which leads to phosphorylation of downstream substrates and inhibition of apoptosis (Wu et al., 2004).
Further a recent study demonstrated that VEGF – dependent endothelial nitric oxide production was enhanced by a signalling cascade consisting of
Ezrin → Calpain (at sub-pathological level of activation) → PI 3-k /Akt →
AMPK → eNOSSer1179 (Youn et al., 2009). Importantly, in the context of organ preservation injury it was recently shown in a model of hypothemic renal preservation, that cytoskeletal degradation was observed in warm renal ischaemia, reperfusion and hypothermia and cytoskeletal disruption during hypothermia contributes to injury upon rewarming (Mangino et al.,
2008). These workers also commented on the likelihood that dephosphorylating and inactivation of ezrin by hypothermia and ischaemia exacerbated preservation injury. A parallel situation may be operational in the present model of cardiac preservation injury. Figure 5.10 shows little ezrin phosphorylation and poorest functional recovery in hearts arrested and stored in celsior alone, whilst the hearts arrested and stored in celsior supplemented with GTN, Cariporide and INO-1153 showed the highest extent of phosphorylation and the best post storage function.
The Bcl-2 family of proteins are intimately involved in the apoptotic process with some such as Bcl-2, Bcl-XL and MCL-1 promoting survival and others such as BAD performing a pro-apoptotic function (Adams et al.,
242 2007). In the present study, hearts arrested and stored in celsior supplemented with all three protective agents, shows an increase in BAD phosphorylation on the Ser 136 residue (Figure 5.11). BAD has previously been shown to be phosphorylated by a number of “conditioning” protocols
(Uchiyama et al., 2004). The serine 136 residue has been shown to be a substrate for phosphorylation by activated Akt (Datta et al., 1997). These workers demonstrated that phosphorylation of Ser 136 facilitates its binding to “14-3-3” protein. Such binding prevents the pro-apoptotic interaction of BAD with mitochondria. Recently it has been shown that activation of the ERK pathway may also lead to phosphorylation of BAD at
Ser 112 another kinase target elucidated by Datta and colleagues which may lead to increased proteasomal turnover of BAD (Balmanno et al.,
2009).
Conclusion
The salient findings from this chapter were – i) Significant post-storage recovery of hearts stored for an extended (10 hour) period was only achieved by arresting and storing them in Celsior supplemented with agents that have previously been shown to recruit both
“arms” of the RISK pro-survival signalling pathway (Akt being activated by
INO-1153 and ERK 1/2 being activated by GTN and Cariporide). Hearts exposed to triply supplemented Celsior during arrest and storage recovered 67% vs 15% of baseline CF (P<0.01 vs any single supplement),
42%vs 1% of AF (P<0.02 vs any single supplement), 49% vs 4%of CO
243 (P<0.05 vs any single supplement) and 86% vs 17% of HR (P<0.02 vs any single supplement).
ii) Post-storage functional recovery in the triple supplemented group was associated with significant activation of both Akt and Erk1/2 pro-survival kinases (6 fold increase and 6.5 fold increase, both P<0.01 vs control hearts) over doubly supplemented groups and control hearts after extended (10 hours) period of hypothermic storage.
iii) GSK3ß, a downstream target of both Akt and Erk1/2 was significantly phosphorylated (inhibited) in the triple supplemented group (5.3 fold increase, P<0.01 vs control hearts), suggesting that the mechanism by which this combination of pharmacological agents operates may involve maintenance of outer mitochondrial membrane integrity post reperfusion.
iv) The robust increase observed similarly in ERM phosphorylation (5.4 fold increase, P<0.01 vs control hearts) in the triple supplemented groups may imply that the cytoskeletal integrity of the myocytes in hearts arrested and stored in all three agents maybe superior.
244 CHAPTER 6
Overall Discussion
245 6.1 PRECIS OF MAJOR FINDINGS – HYPOTHESES AND AIMS
REVISITED
The major findings of this thesis are summarised below in terms of the hypotheses and aims outlined in the Introduction (Chapter 1, section 1.7).
a) “Short” 6 hour periods of hypothermic ischaemic storage
Hypothesis 1: Supplementation of a standard cardioplegic / storage solution (Celsior) with agents that inhibit poly(ADP-ribose) polymerase, optimise bioavailability of nitric oxide or inhibit intracellular calcium efflux can improve post-reperfusion cardiac function.
Findings: Supplementation of Celsior with either the PARP inhibitor INO-
1153, the nitric oxide donor - glyceryl trinitrate or the sodium hydrogen exchange inhibitor cariporide alone significantly improved post-storage cardiac function after 6 hours hypothermic storage. As an illustration, recovery of cardiac output as a percentage of pre-storage baseline was
55% vs 23% for hearts arrested and stored in INO-1153, 50% vs 20% for hearts arrested and stored in GTN and 70% vs 20% for hearts arrested and stored in Cariporide, all P<0.05 vs control hearts arrested and stored in unsupplemented Celsior). The combination of GTN and cariporide was unable to significantly improve post-storage cardiac function after 6 hours hypothermic storage over that achieved by cariporide alone (cardiac output of 66% vs 20% compared to 70% vs 20% respectively, P<0.05 vs control). Unlike ischaemic or pharmacological pre- or postconditioning
246 where the protective stimulus is applied immediately before or after the period of index ischaemia, hearts in the present study see the pharmacological protective stimulus at and during the “index” hypothermic ischaemic period.
Hypothesis 2: Exposure of the heart to these agents at arrest and during index hypothermic storage recruits and activates pro-survival kinase signalling and suppresses cell death pathways post reperfusion.
Findings: Significant recovery of function of hearts arrested and stored in
INO-1153 (CO of 55% vs 23%, p<0.05) was associated with 4.3 fold increase in phosphorylation (activation) of Akt (P<0.05 vs control) and a
2.4 fold increase in ERK 1/2 phosphorylation (P<0.05 vs control).
Functional recovery produced by PARP inhibition could be completely abolished by the PI 3-k inhibitor, wortmannin, highly suggestive of a major role for an active Akt pathway in functional recovery. The increase in Akt phosphorylation was paralleled by 1.2 fold increase in the phosphorylation status of phospholamban (P<0.05 vs control), which leads to activation of the sarcoplasmic reticular Ca-ATPase, normalisation of intracellular calcium levels and cardiac contractility (Chapter 3).
In contrast, recovery of hearts arrested and stored in either GTN or cariporide or a combination of these two agents was associated with 7 fold increase in the phosphorylation status of Erk1/2 (P<0.001 vs control) with
247 no apparent phosphorylation of Akt. In parallel with this increase in ERK
1/2 phosphorylation there was a 2 fold increase in phosphorylation of the anti-apoptotic Bcl-2 (P<0.001 vs control), a substrate for ERK 1/2 phosphorylation as well as a 3 fold decrease in cleaved (activated) caspase 3 (P<0.001 vs control), a key mediator of apoptotic cell death.
Pre-treatment of hearts arrested and stored in GTN and cariporide with the
MEK inhibitor, PD98059 (to block the ERK pathway), produced only partial
(non significant) decrease in recovery of cardiac function, that was accompanied by only a small decrease in ERK 1/2 phosphorylation and a partial increase in the phosphorylation status of Akt.
b) Extended (10 hour) period of hypothermic ischaemic storage
Arising from the findings of these two studies was a supplementary hypothesis.
Hypothesis 3: Arresting and storing hearts in a combination of INO-1153,
GTN and cariporide should activate both arms of the “RISK” pathway (ie the PI 3-k pathway and the ERK 1/2 pathway). Greater pro-survival signalling activity may allow post-storage recovery of hearts stored for an extended (10 hour) period of hypothermic ischaemic storage.
Findings: Hearts arrested and stored in Celsior supplemented with only a single agent (INO-1153, GTN or cariporide) failed to recover any contractile function after 10 hours hypothermic storage. Hearts arrested
248 and stored in the combinations of two of the three additives showed modest recovery, but only hearts arrested and stored in all three agents (ie
INO-1153 + GTN + cariporide) showed significant functional recovery after
10 hours hypothermic storage. As an example, recovery of cardiac output in the triple therapy group was 49% of baseline function (P<0.05 vs all other singly and doubly supplemented experimental groups).
This contractile recovery in the triply supplemented hearts was accompanied a 6 fold increase in Akt phosphorylation (P<0.01 vs hearts stored in unsupplemented celsior), a 6.5 fold increase in Erk1/2 phosphorylation (P<0.01 vs hearts stored in unsupplemented celsior) and a 5.3 fold increase in GSK3ß phosphorylation (P<0.01 vs hearts stored in unsupplemented celsior). An incidental finding was a 5.4 fold increase in the phosphorylation status of the ERM family of proteins (P<0.01 vs hearts stored in unsupplemented celsior), whose phosphorylation is crucial to the maintenance of the integrity of the cytoskeleton.
249 6.2 POTENTIAL STRENGTHS OF PHARMACOLOGICAL
ACTIVATION OF PRO-SURVIVAL SIGNALLING AS AN
APPROACH TO IMPROVING POST-STORAGE DONOR HEART
SURVIVAL
Ischaemic preconditioning has been shown to elicit significant cardioprotection (driven by activation of survival kinases) after a period of potentially damaging or fatal ischaemia in experimental models.
Theoretically, its translation into clinical practice must be limited to specific
(surgical) contexts where the ischaemic period can be exactly predicted (ie contexts that closely mimic experimental models). One such situation would be cardiac or organ procurement for transplantation.
The harnessing of various pharmacological compounds to recruit pro- survival signalling maybe even more appropriate to this clinical situation as the protective effect can be elicited by a single controlled dose of the agent(s) rather than mechanical manipulation of major arteries. This approach may be more appealing to surgeons and perfusionists than repeated clamping and unclamping of a potentially unstable atherosclerotic aorta (that may be the case in older donors) which may result in debris being shed and lodging in small cardiac vessels or the vessels of other transplantable organs. Also, in the context of multi-organ procurement, the time taken for mechanical preconditioning may be incompatible with the requirements of the other organ procurement teams.
250 The present findings show that pharmacological supplementation of the cardiac arresting and storage solution is sufficient to elicit the maximal cardioprotective effect. This is particularly attractive to the logistics of clinical organ procurement as it obviates the need for systemic exposure of the organ donor to the agent and prevents potential unforseen deleterious effects to other transplantable organs. This approach also has advantages over “remote preconditioning” of the donor where a number of cycles of inflation and deflation of a blood pressure cuff produce a pulse of humoral substances that diffuse to the appropriate receptor to transduce the appropriate protective response (Hausenloy et al., 2008b). Instead of exposure of all potential donor organs to the products of remote preconditioning which may or may not be optimal for each organ, pharmacological supplementation of the preservation solution will allow only the organ of choice to be exposed to the most appropriate agent or a combination of agents at the most appropriate dose(s).
The present study provides proof-of-principle evidence for the role of pro- survival kinase activation in improvement of donor heart function using the
PARP inhibitor, INO-1153, the nitric oxide donor, GTN and NHE inhibitor, cariporide. Although the only drug in routine clinical use from this group is
GTN, the ischaemic pre- and postconditioning literature predicts that a number of other agents in routine clinical use or under clinical trials may also be efficacious in this model. Indeed, other recent studies in our laboratory have shown that erythropoietin (activating Stat3) (Watson et al.,
251 2009), the erbB2 receptor agonist neuregulin (activating both Akt and ERK
1/2) (Jabbour et al., 2009) and the third generation NHE inhibitor, zonpioride (activating ERK 1/2) (Tsun et al., 2009) are also cardioprotective as single agents after 6 hours hypothermic storage and in combination after extended (10 hour) periods of hypothermic ischaemic storage.
Another potential advantage of recruitment of endogenous pro-survival signalling pathways is the ability to access many potential targets which may not be necessarily amenable to routine pharmacological treatment.
From the present study, these down-stream targets include sarcoplasmic reticulum proteins that may normalise intracellular calcium and improve cardiac contractility (phospholamban), activation of “anti-apoptotic” markers such as Bcl-2, decrease in markers of apoptosis (cleaved caspase 3), elements that are important in maintenance of mitochondrial integrity (the phosphoylated form of GSK3ß) and the maintenance of normal cellular cytoskeletal architecture (phosphorylated ezrin / radixin / moesin, ERM).
The nature of these novel targets may also suggest completely new therapeutic paradigms. A case in point is the inhibitory effect of the post reperfusion interaction of cyclosporine A with cycophylin D in preventing the opening of the mitochondrial transition pore. Recent reports by Ovize and colleagues have demonstrated a reduction of infarct size (measured
252 by creatine kinase release during the immediate post-operative period) after administration of a single dose of cyclosporine immediately prior to reperfusion to patients with profound blockage of culprit coronary arteries undergoing PCI (Gomez et al., 2009, Piot et al., 2008).
This approach would be particularly easy to translate to clinical transplantation. In many if not most heart transplant recipients, cyclosporine A is already the immunosupressive drug of chioce.
Recipients receive their first dose of cyclosporine A some hours before reperfusion of the donor heart. A further top-up dose could be easily administered to the recipient via the cardiopulmonary bypass reservoir immediately before reperfusion of the donor heart.
6.3 ISSUES TO BE RESOLVED BEFORE PHARMACOLOGICAL
RECRUITMENT OF PRO-SURVIVAL KINASES AS AN
APPROACH TO DONOR HEART PRESERVATION CAN BE
TRANSLATED TO CLINICAL PRACTICE
Apart from the occassional “domino” transplant, where the donor heart comes from a live donor, all donor hearts are procured from brain dead donors. Donor brain death is accompanied by a rapid and profound release of endogenous stores of catecholamines. This and the current donor management practices of maintaining multiorgan donors on
(sometimes escalating) doses of catecholamines such as adrenaline and noradrenaline to maintain adequate systemic blood pressure and maintain
253 organ perfusion may often result in proinflammatory changes and are a clinically important source of potentially irreversible damage to the donor heart (Hicks et al., 2006). The “donor” rats used in the present study were live and anaesthetised when hearts were procured for mounting on the perfusion apparatus. Whether pro-survival signalling can be successfully recruited under after brain death has yet to be systematically tested.
However, there is some emerging positive evidence. Our laboratory uses a porcine orthotopic cardiac transplantation model incorporating donor brain death to test the positive findings obtained from the proof-of-principle rat heart model. A recent study in our laboratory has employed Celsior supplemented with GTN and cariporide (identical with that used in Chapter
4) as the arresting and storage solution in this translational model (Hing et al., 2009). Donor animals were rendered brain dead and managed for 6 hours before hearts were arrested and stored in either Celsior supplemented with a combination of 0.1mg/ml GTN and 10 µM cariporide,
Celsior + GTN, Celsior + cariporide or unsupplemented celsior. Hearts were then stored for 14 hours at 4°C, then reimplanted in a recipient animal. The double supplemented Celsior was shown to produce viable recovery of the donor heart with 5 of 6 hearts from the GTN + cariporide group successfully weaned from cardiopulmonary bypass but only 1 of 5 of each of the other groups successfully weaned (P = 0.001). Although the extent of pro-survival kinase activation was not measured in this study, the significant improvement in post-storage cardiac function after such a long
254 cold ischaemic period is encouraging. The study also demonstrates that positive results from the isolated working rat heart model can be replicated in a more clinically relevant model.
An alternative approach to managing the donor after brain death, especially in the “marginal donor” has been the use of a “hormonal resuscitation” cocktail of vasopressin, methylprednisolone ± triiodothyronine to maintain blood pressure allowing the donor to be weaned off catecholamines. Clinical trials have shown a significant increase in donor hearts from marginal donors after hormonal resuscitation (Rosendale et al., 2003). At least two of the elements of hormonal resuscitation protocol can recruit pro-survival signalling pathways. Firstly, corticosteroids such as methyl prednisolone, have recently been shown to up-regulate cardiac e-NOS through activation of the PI 3-k / Akt pathway subsequent to binding of the glucocorticoid to the glucocorticoid receptor (Hafezi-Moghadam et al., 2002). Second, stimulation of the V1 receptor by vasopressin activates ERK 1/2 and Akt in a neonatal rat cardiac fibroblast model (He et al., 2008). It is interesting to speculate whether a component of the ability of this protocol to improve the function of marginal hearts before brain death may be due to recruitment of pro-survival signalling as well as the catecholamine-sparing effects of this approach.
255 Other issues that must be addressed in the setting of the current donor population that have already been flagged as potential confounding factors are: i) Recruitment of pro-survival signalling in the elderly donor. ii) Recruitment of pro-survival signalling in donors with pre-existing disease such diabetes and hypertension.
In conclusion, the complexity of the molecular and cellular mechanisms that mediate all facets of ischaemia reperfusion injury in the donor heart is such that it is unlikely that any single treatment will ensure maximal cardiac protection during hypothermic storage and reperfusion.
Pharmacological activation of pro-survival signalling offers an approach whereby many important targets of reperfusion injury can be targeted by minimal pharmacological interventions. If the positive findings described here can be fully replicated in translational and clinically relevant models that reflect the human population, the opportunity may exist to further improve the function of the donor heart post implantation after
“conventional” cold ischaemic times, increase the cold ischaemic times from which a donor heart is viable and make better use of the increasing population of marginal donors.
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