Thesis Proposal - TSpace

The Role of Candidate G-protein Coupled Receptors

in Mediating Remote Myocardial Ischemic

Preconditioning.

by

Harinee Surendra

A thesis submitted in conformity with the requirements for the degree of

Master of Science

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

© Copyright by Harinee Surendra (2009)

i

ACKNOWLEDGEMENTS

I would like to extend my heartfelt gratitude to my family for supporting me through this

rewarding and demanding experience. Their constant encouragement has made this thesis

possible.

Also, my sincere thanks are given to the guidance and support of Dr. Gregory Wilson

(Division of Pathology, Department of Paediatric Laboratory Medicine, Hospital for Sick

Children) and Roberto Diaz (Dr. Wilson‘s Lab, Hospital for Sick Children). Their extensive

knowledge in the field of ischemic preconditioning has provided excellent direction for this

thesis and given me opportunities to progress in my career. Also, tremendous guidance was

provided by members of my graduate committee through Dr. Herman Yeger (Research Institute,

Hospital for Sick Children) and Dr Aleksander Hinek (Research Institute, Hospital for Sick

Children). My genuine appreciation is given to Alina Hinek (Dr. Wilson‘s Lab, Hospital for Sick

Children) and Taneya Hossain (Dr. Wilson‘s Lab, Hospital for Sick Children) for their assistance

in providing training.

I would like to thank Jing Li (Dr. Redington‘s Lab, Research Institute, Hospital for Sick

Children) for providing the dialysate used in this study and Michael Tropak (Dr. Callahan‘s Lab,

Research Institute, Hospital for Sick Children) for measuring substances in the dialysate with

mass spectrometry. Finally, I would like to thank Foundation Leducq, the Heart and Stroke

Foundation of Ontario, and the University of Toronto for providing funding for my research.

ii

ABSTRACT

The Role of Candidate G-protein Coupled Receptors in Mediating Remote Myocardial

Ischemic Preconditioning.

By Harinee Surendra

Degree of Master of Science (2009)

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

This study investigated the role of opioid, adenosine, bradykinin, and calcitonin-gene

related peptide (CGRP) receptors, and potential ‗cross-talk‘ among suspected G-protein coupled

receptors in a humoral model of remote ischemic preconditioning (rIPC) cardioprotection.

Compared to Control dialysate (from non-preconditioned donor rabbit blood), rIPC dialysate

(from remotely preconditioned blood) reduced cell death in rabbit cardiomyocytes following

simulated ischemia and reperfusion. Non-selective, δ-, or κ-opioid receptor blockade and non-

selective adenosine receptor blockade abolished rIPC dialysate protection; whereas, bradykinin

B2 and CGRP receptor blockade had no effect. Non-selective adenosine receptor blockade fully

and partially abolished protection by κ- and δ-opioid receptors, respectively. Multiple reaction

monitoring mass spectrometry detected low levels of adenosine, and other preconditioning

substances, in the dialysate. An increase in extracellular adenosine was not detected during

opioid-induced preconditioning to explain this cross-talk. These results suggest that δ-opioid, κ-

opioid, adenosine receptors, and opioid-adenosine cross-talk are involved in rIPC of freshly

isolated cardiomyocytes.

iii

TABLE OF CONTENTS

ABSTRACT ................................................................................................................................... ii

GLOSSARY................................................................................................................................ viii

1. INTRODUCTION......................................................................................................................1

ISCHEMIC PRECONDITIONING (IPC) 1.1 .........................................................................................1

Background in IPC 1.1.a ...........................................................................................................1

Signalling Mechanisms in IPC 1.1.b .........................................................................................2

REMOTE ISCHEMIC PRECONDITIONING (RIPC) 1.2 .........................................................................5

Background in rIPC 1.2.a .........................................................................................................5

Signalling Mechanisms in rIPC 1.2.b .......................................................................................5

INVOLVEMENT OF OPIOID RECEPTORS 1.3 .....................................................................................8

Opioids and Their Receptors 1.3.a ...........................................................................................8

Evidence of Opioid Involvement in IPC1.3.b ............................................................................9

Evidence of Opioid Involvement in rIPC1.3.c.........................................................................10

INVOLVEMENT OF BRADYKININ B2 RECEPTORS 1.4 .....................................................................11

Bradykinin and Their Receptors 1.4.a ....................................................................................11

Evidence of Bradykinin Involvement in IPC 1.4.b ..................................................................12

Evidence of Bradykinin Involvement in rIPC 1.4.c .................................................................13

INVOLVEMENT OF CGRP RECEPTORS 1.5 ....................................................................................14

CGRP and Their Receptors 1.5.a ............................................................................................14

Evidence of CGRP Involvement in IPC 1.5.b..........................................................................15

Evidence of CGRP Involvement in rIPC 1.5.c ........................................................................16

INVOLVEMENT OF ADENOSINE RECEPTORS 1.6 ...........................................................................17

iv

Adenosine and Their Receptors 1.6.a......................................................................................17

Evidence of Adenosine Involvement in IPC 1.6.c....................................................................18

Evidence of Adenosine Involvement in rIPC 1.6.b ..................................................................19

OTHER POTENTIAL PRECONDITIONING SUBSTANCES 1.7 .............................................................20

OPIOID-ADENOSINE RECEPTOR CROSS-TALK 1.8 ........................................................................22

Background 1.8.a ....................................................................................................................22

Adenosine Levels in the Blood 1.8.b .......................................................................................23

Potential Mechanisms of Cross-talk 1.8.c ...............................................................................23

2. RATIONALE ...........................................................................................................................26

3. OBJECTIVES ..........................................................................................................................27

HYPOTHESIS 3.1 ..........................................................................................................................27

SPECIFIC AIMS 3.2 .......................................................................................................................27

4. RESEARCH DESIGN & METHODS ...................................................................................28

ANIMALS AND HUMAN SUBJECTS 4.1 ..........................................................................................28

ISOLATION OF ADULT CARDIOMYOCYTES 4.2 .............................................................................28

Operative Procedure 4.2.a ......................................................................................................28

Digestion Protocol 4.2.b .........................................................................................................29

Comparison of Digestion Protocols 4.2.c ...............................................................................32

DIALYSATE PREPARATION 4.3 .....................................................................................................34

FRESH-CELL EXPERIMENTAL MODEL 4.4 ....................................................................................37

General Protocol 4.4.a ............................................................................................................37

AIM (1) Protocol 4.4.b ............................................................................................................39

v

AIM (2) Protocol 4.4.c ............................................................................................................40

AIM (3) Protocol 4.4.d ............................................................................................................41

TRYPAN BLUE EXCLUSION ASSAY 4.5 .........................................................................................42

Inter-Observer Error Data 4.5.a .............................................................................................44

DRUGS 4.6 ...................................................................................................................................46

OTHER METHODS 4.7 ..................................................................................................................47

Western Blotting 4.7.a .............................................................................................................47

Multiple Reaction Monitoring Mass Spectrometry 4.7.b ........................................................48

Statistics 4.7.c ..........................................................................................................................50

5. CHARACTERIZATION OF THE PROTECTION INDUCED BY RIPC .......................51

RABBIT AND HUMAN PRECONDITIONED DIALYSATE INDUCES PROTECTION 5.1 .........................51

Dialysate Characterization 5.1.a ............................................................................................53

Discussion 5.1.b ......................................................................................................................53

6. THE ROLE OF CELL MEMBRANE RECEPTORS IN RIPC .........................................56

THE ROLE OF OPIOID RECEPTORS 6.1 ..........................................................................................56

Survey of Opioid Receptors in Rabbit Cardiomyocytes 6.1.a .................................................56

Non-selective Opioid Receptor Blockade of Protection 6.1.b .................................................58

δ-Opioid Receptor Blockade of Protection 6.1.c ....................................................................60

κ-Opioid Receptor Blockade of Protection 6.1.d ....................................................................62

Discussion 6.1.e.......................................................................................................................64

THE ROLE OF BRADYKININ B2 RECEPTORS 6.2 ............................................................................66

Bradykinin B2 Receptor Blockage Conserves Protection 6.2.a ..............................................66

Discussion 6.2.b ......................................................................................................................68

vi

THE ROLE OF CGRP RECEPTORS 6.3 ...........................................................................................70

Existence of Calcitonin-like Receptors in Rabbit Cardiomyocytes 6.3.a ................................70

CGRP Receptor Blockage Conserves Protection 6.3.b...........................................................71

Discussion 6.3.c.......................................................................................................................73

THE ROLE OF ADENOSINE RECEPTORS 6.4 ..................................................................................74

Non-selective Adenosine Receptor Blockade of Protection 6.4.a ...........................................74

Discussion 6.4.b ......................................................................................................................76

7. RIPC MEDIATES OPIOID-ADENOSINE CROSS-TALK ................................................78

OPIOID-ADENOSINE CROSS-TALK 7.1 ..........................................................................................78

Adenosine Deamination of the Dialysate Conserves Protection 7.1.a ...................................78

Partial Adenosine Blockade of δ-Opioid Receptor-Induced Protection 7.1.b ........................80

Compete Adenosine Blockade of κ-Opioid Receptor-Induced Protection 7.1.c .....................82

Discussion 7.1.d ......................................................................................................................84

THE INHIBITION OF ADENOSINE KINASE HYPOTHESIS 7.2 ...........................................................86

Exposure to Dynorphin B Does Not Accumulate Extracellular Adenosine 7.2.a ...................86

Discussion 7.2.b ......................................................................................................................89

8. SUMMARY OF FINDINGS ...................................................................................................92

9. GENERAL DISCUSSION ......................................................................................................93

OVERALL PERSPECTIVE 9.1 .........................................................................................................93

OTHER MECHANISMS OF OPIOID-ADENOSINE CROSS-TALK 9.2 ..................................................95

Dimerization of G-protein Coupled Receptors 9.2.a ..............................................................95

FUTURE DIRECTIONS 9.3 .............................................................................................................96

vii

Specific Adenosine Receptor Subtypes in Cross-talk & rIPC 9.3.a ........................................96

Heterodimerization of Opioid and Adenosine Receptors 9.3.b ...............................................98

Cell Signalling in rIPC 9.3.c ...................................................................................................98

LIMITATIONS 9.4 .......................................................................................................................103

CONCLUSIONS 9.5 ......................................................................................................................104

APPENDIX .................................................................................................................................105

APPENDIX I: LIST OF FIGURES ..................................................................................................105

APPENDIX II: LIST OF TABLES ..................................................................................................107

APPENDIX III: RIPC SUMMARY: MYOCARDIUM AS THE TARGET ORGAN .................................108

APPENDIX IV: RIPC SUMMARY: SKELETAL MUSCLE AS THE PRECONDITIONING ORGAN ..........109

APPENDIX V: MRM MASS SPECTROMETRY PLOTS OF STANDARD CONCENTRATIONS ..............110

REFERENCE LIST ...................................................................................................................113

viii

GLOSSARY

TERM DEFINITION

8-SPT 8-(p-sulfophenyl)theophylline

ADA Adenosine deaminase

ADO Adenosine

BNTX 7-benzylidenealtrexone

cAMP cyclic adenosine monophosphate

CAO Coronary artery occlusion

CCPA 2-chloro-N6-cyclopentyladenosine

CGRP (8-37) Human fragment of the calcitonin-gene related peptide

DynB Dynorphin B

FAO Femoral artery occlusion

GNTI 5‘-guanidinyl-17-(cyclopropylmethyl)-6,7-dehyrdo-4,5α-epoxy-3,14-

dihydroxy-6,7-2‘,3‘-indolomorphinan

HOE140 Hoechst 140

IPC Ischemic preconditioning

IR Ischemia-reperfusion

KH Krebs-Henseleit

MAO Mesenteric artery occlusion

ME Met-enkephalin

mKATP Mitochondrial ATP sensitive potassium channels

ix

MPG N-2-mercaptopropionyl glycine

mPTP Mitochondrial permeability transition pore

MRM MS Multiple reaction monitoring mass spectrometry

Nal Naloxone

NO Nitric oxide

NOS nitric oxide synthase

NTI Naltrindole

PGI2 Prostacyclin

PI3 Kinase Phosphatidylinositol 3-kinase

PKC Protein kinase C

RAO Renal artery occlusion

rIPC Remote ischemic preconditioning

ROS Reactive oxygen species

SI Simulated ischemia

sKATP Sarcolemmal ATP sensitive potassium channels

SR Simulated reperfusion

TNF-α Tumour necrosis factor –α

WB Western blotting

Wort Wortmannin

1

- Chapter 1 -

INTRODUCTION

1.1 Ischemic Preconditioning (IPC)

1.1.a Background in IPC:

Ischemic preconditioning (IPC) is one of the most potent strategies, under experimental

conditions, to reduce myocardial infarction to date. One or more periods of brief ischemia (~5

min) and reperfusion (~5 min) render the myocardium protected from a much longer damaging

ischemic stress. This phenomenon, first discovered by Murry et al.1 in 1986, exhibits two distinct

windows of protection. This thesis will focus on the first window of protection (called acute

preconditioning), which occurs within 2 hours of the IPC stimulus, whereas the second window

(called delayed preconditioning) occurs 24-72 hours later and is thought to be gene transcription-

dependent.

Since the discovery of IPC to induce protection in a wide range of tissues and species, the

triggers involved in this phenomenon have been of great interest as a therapeutic intervention for

myocardial infarction. Thornton et al.2 determined that G-protein coupled receptors were vital to

preconditioning when pertussis toxin (an inhibiter of G-protein coupled receptors) administered

in vivo to rabbits blocked myocardial preconditioning. Since then, the major extracellular

molecules involved in IPC at the trigger phase were found to be adenosine, bradykinin and

opioid peptides. Goto et al.3 postulated that receptors for these three peptides exert an additive

effect to reach a hypothetical threshold that is required to induce cardioprotection. Thus,

blockade of any one of these G-protein coupled receptors is enough to abolish protection by IPC.

2

1.1.b Signalling Mechanisms in IPC:

The downstream signalling pathways with respect to classical IPC have been well

established. According to Downey et al. 4

, cell signalling exhibits three distinct phases: the

‗trigger‘ phase, the ‗mediator‘ and the ―end effector‖ phase.

Mocanu et al.5 demonstrated that cardioprotection was abolished by the

phosphatidylinositol 3-kinase (PI3 kinase) inhibitor, wortmannin and LY 294002. The activation

of PI3 kinase results in translocation of Akt to the plasma membrane6 and its subsequent

phosphorylation7. Akt then stimulates nitric oxide synthase (NOS) to generate nitric oxide (NO)

8,

an important molecule that also acts as a trigger in IPC9 as suggested by Lochner et al.

10.

Oldenburg et al.11

also proposed the involvement of guanylyl cyclase (GC) in IPC by abrogating

protection with the GC inhibitor, ODQ. GC then produces cGMP and activates protein kinase G

(PKG).

PKG then induces the opening of mitochondrial potassium channels (mKATP) which

results in swelling of the mitochondria due to potassium influx. This leads to reactive oxygen

species (ROS) generation as shown by Forbes et al.12

when protection from diazoxide (an mKATP

channel opener) was abolished by ROS scavengers. Finally, the target of this downstream

signalling pathway converges on protein kinase C (PKC)13

.

To summarize, the trigger phase involves the release of endogenous substances, such as

opioid peptides and bradykinin, that activate a complex pathway which includes, in the following

3

order: phosphatidylinositol 3-kinase (PI3-kinase), Akt, nitric oxide synthase (NOS), nitric oxide

(NO), guanylyl cyclase, protein kinase G, opening of mitochondrial KATP channels, which in turn

generates reactive oxygen species (ROS) and actives protein kinase C (PKC). In addition, opioid

peptides undergo an additional step of activating an epidermal growth factor receptor (EGFR)

before activating PI3-kinase and Akt. Adenosine, another important trigger in IPC, is thought to

activate PKC directly through adenosine A1 and A3 receptors.4

The ‗mediator‘ phase occurs either during the long ischemia (called the index ischemia)

or early during reperfusion and is characterized by adenosine-dependent activation of adenosine

A2b receptors. PKC can also modulate the activity of this adenosine receptor directly. Adenosine

A2b receptors, in turn, lead to ERK and PI3-kinase/Akt activation. These kinases phosphorylate

GSK-3β, which is thought to inhibit formation of mitochondrial permeability transition pores

(mPTP) (see Figure 1). The mPTP has been proposed to be an end effector of IPC but this a

subject of intense debate. 4 (See Figure 1 for an overall summary)

4

14

This schematic diagram is identical to Figure 1 from the review by Cohen & Downey in 2008.14

Figure 1. IPC Signalling Mechanisms in the Target Organ.

Preconditioning exhibits three distinct phases: the ‗trigger‘ phase (prior to index ischemia), the

‗mediator‘ phase (during index ischemia and reperfusion) and the ―end effector‖ phase.

During the trigger phase, endogenously released substances, such as opioid peptides and

bradykinin, activate in the following order: phosphatidylinositol 3-kinase (PI3 kinase), Akt, nitric

oxide synthase (NOS), produce nitric oxide (NO), guanylyl cyclase, protein kinase G, opening of

mitochondrial KATP channels, generation of oxygen radicals (ROS), finally activating protein

kinase C (PKC). Opioids undergo an additional step of activating an epidermal growth factor

receptor (EGFR, or HB-EGF in this figure) before activating PI3 kinase and Akt. Adenosine

activates PKC directly through adenosine A1 and A3 receptors.

The ‗mediator‘ phase is characterized by adenosine-dependent activation of adenosine A2b

receptors. PKC can also modulate the activity of this adenosine receptor directly. Adenosine A2b

receptors activate in the following order: ERK and PI3-kinase/Akt, phosphorylation of GSK-3β,

which inhibits formation of mitochondrial permeability transition pores (mPTP). The mPTP has

been proposed to be an end effector of IPC.

5

1.2 Remote Preconditioning (rIPC):

1.2.a Background in rIPC:

In 1993, Przyklenk et al.15

first conceived the idea of remote ischemic preconditioning

(rIPC) wherein repeated brief episodes of ischemia and reperfusion in a non-local tissue/distant

organ has the ability to render the myocardium protected against ischemia/reperfusion injury.

Remote preconditioning of the myocardium has can be induced by other organs such as the liver,

small intestine, kidneys, and also from skeletal muscle ischemia (see Appendix III & IV).

Similarly, two windows of protection have been confirmed for rIPC16

. However, the focus of my

thesis will be on the first window of protection (called acute preconditioning/rIPC) which occurs

within 2 hours of the preconditioning stimulus and not the second window (called delayed

preconditioning/rIPC).

1.2.b Signalling Mechanisms in rIPC:

There is much controversy over the involvement of either a humoral (via blood) or

neurogenic (via nerves) pathway, or both, as a mechanism of transferring protection from the

preconditioned tissue/organ to the myocardium16

. Coronary effluent from preconditioned donor

rabbit hearts elicited protection in untreated hearts in the study by Dickson et al.17

, suggesting

the involvement of humoral protective factors. However, Gho et al.18

demonstrated that

protection by mesenteric artery occlusion (MAO) was abolished by ganglion blockade, providing

support for a neurogenic pathway in rIPC.

The actual trigger mechanisms involved to induce protection in the myocardium are

unclear. Multiple triggers have been suggested such as opioid peptides, adenosine, bradykinin,

6

and calcitonin-gene related peptide (CGRP). Once G-protein coupled receptors are triggered on

the cardiomyocyte cell surface, there is translocation of PKCε from the cytosol to the

mitochondrial membrane fraction, an event thought to be dependent on mitochondrial ROS

generation. However, it is yet to be determined whether activation of survival kinases during

reperfusion or inhibition of mPTP occurs. 19

(See Figure 2 for an overall summary)

7

This schematic diagram is identical to Figure 2 from the review by Hausenloy & Yellon in

2008.19

Figure 2. rIPC Signalling Mechanisms of the Myocardium. Either humoral or neurogenic pathways, or both, act as a mechanism of transferring protection

from the preconditioned tissue/organ to the myocardium. Once this signal triggers G-protein

coupled receptors on the cardiomyocyte cell surface, kinases such as ERK or Akt may be

activated, and there is translocation of protein kinase C-epsilon (PKCε) from the cytosol to the

mitochondrial membrane fraction, an event thought to be dependent on mitochondrial reactive

oxygen species (ROS) generation. Finally, inhibition of mitochondrial permeability transition

pore (mPTP) may occur, resulting in preconditioning of the myocardium.

8

1.3 Involvement of Opioid Receptors:

1.3.a Opioids and Their Receptors:

Opioids exhibit various functions in the heart such as cardiac arrhythmogenesis and

modulation of vasculature20

. Opioid peptides consist of enkephalins, endorphins, dynorphins, and

endomorphins as well as other non-opioid substances, such as nociceptin, that exhibit similar

pharmacological properties20

. Enkephalins are most abundant in cardiac ventricles compared to

any other organ in the body aside from the central nervous system21

, suggesting the important

role of opioids in cardiomyocyte regulation and maintenance during stress. Each opioid class is

derived from a corresponding gene which produces a distinct hormone precursor. Enkephalins

are produced from pro-enkephalin, endorphins from pro-opiomelanocortin (POMC), dynorphins

from pro-dynorphin, and finally the endomorphin precursor has yet to be identified20

. In

particular, opioids in the myocardium exhibit unique features such as larger molecular weighted

peptides (e.g. Met5-enkephalin-Arg

6-Phe

7 or MERF and dynorphin A)

22. Regardless, the

myocardium contains all necessary machinery to generate all peptide forms and receptor types. 20

Minimum concentrations reported to protect rabbit cardiomyocytes from

ischemia/reperfusion damage by common endogenous opioids such as dynorphin B is 10µM152

and 1µM148

for Met-enkephalin. However, this does not include all known and unknown opioids

which may induce protection at lower concentrations.

The above mentioned peptide classes bind with varying affinities to three subtypes of Gi-

protein coupled opioid receptors, which are: delta (δ), kappa (κ), and mu (μ). Endorphins and

enkephalins are known to associate mostly with δ receptors (and to some extent μ), while

9

dynorphins are highly selective for κ opioid receptors. Endomorphins are most potent at μ

receptors. The κ receptor can be divided into κ1 and κ2, and the δ receptor into δ1 and δ2, and all

of these have been identified on rat and human cardiomyocytes20

. However, there is evidence

that the μ subtype (which exists as three isoforms, µ1, µ2, µ3 23

) is not expressed in adult

cardiomyocytes24,25

. The μ receptor is highly abundant in the central nervous system, suggesting

this subtype may be present at nerve ends in the myocardium; however, this requires further

investigation. Nonetheless, the tissue specificity of these opioids and their receptors as well as

their pharmacological functions within the heart is still a subject of study.

1.3.b Evidence of Opioid Involvement in IPC:

During episodes of myocardial ischemic stress, opioid levels were found to be elevated in

humans26

, suggesting an involvement of opioid peptides in preconditioning. A number of studies

have investigated the role of opioids in IPC. Mesenteric artery occlusion (MAO) in rats has

shown that morphine (a non-selective opioid agonist) could mimic IPC and this protection was

abolished by naloxone (a non-selective opioid antagonist)27

. In addition, coronary effluent that

was collected following IPC measured increased levels of endogenous opioid peptides.

There is controversy regarding which opioid receptor subtype is involved in ischemic

preconditioning. The involvement of δ receptors was proposed by Schultz et al.28

when µ and κ

receptor antagonists failed to block protection from 3 cycles of 5 min ischemia-5min reperfusion

in in vivo rat hearts. In addition, the detrimental effects of κ receptors to aggravate ischemia-

reperfusion injury have been reported in κ-opioid perfused isolated rat hearts29

and found to be

proarrhythmic for in vivo κ opioid-induced preconditioning in swine30

. Interestingly, κ receptors

10

have also been reportedly involved in the beneficial effects of IPC in rats by Wang et al. 31

.

Though δ and κ agonists reduced infarct size in isolated perfused hearts, Wang and colleagues

suggested that only the κ opioid agonist mimicked the antiarrhythmic effects of IPC in this study.

Very few studies have investigated the role of opioids during reperfusion. Gross et al.32

in

2004 determined that activation of opioid receptors during the reperfusion phase by morphine

(non-selective opioid agonist) and BW373U86 (selective δ agonist) reduced infarct size in rats.

However, this is an aspect of IPC that requires further exploration but is not the focus of my

thesis.

1.3.c Evidence of Opioid Involvement in rIPC:

The involvement of opioids in rIPC was proposed in a study by Dickson et al.33

in which

pre-treatment with naloxone (a non-selective opioid antagonist) blocked protection from

transferred coronary effluent from one isolated rabbit heart to another. The coronary effluent was

analyzed and found to contain Met- and Leu-enkephalin (endogenous agonists of δ and µ opioid

receptors)33,34

. Patel et al.35

had found that MAO in rats could reduce myocardial infarction, and

this effect was blocked by naloxone. Preconditioning by femoral artery occlusion (FAO) in rats

also reduced infarct size and lactate dehydrogenase levels (a marker of oxidative stress)36

. In a

separate study in isolated rat hearts, Weinbrenner et al.37

found that protection was abolished by

pre-treatment with naloxone and the free radical scavenger N-2-mercaptopropionyl glycine

(MPG), thus suggesting a relationship between opioid signalling in rIPC and ROS generation.

The same study demonstrated the involvement of the δ1 opioid receptor subtype in protection

11

induced by limb ischemia. However, a recent study by Zhang et al.36

has proposed the idea that it

is κ receptors, not δ, that mediate rIPC through femoral artery occlusion in rats.

The exact mechanism of opioid involvement is unclear. Skeletal muscle ischemia with

ganglion blockade by hexamethonium did not affect preconditioning in pigs38

. Thus, it is thought

that opioid peptides enter the blood stream through a humoral pathway and mediate their effects

through receptor binding at the target organ20

. In addition, similarities in signalling pathways

between rIPC and IPC are even less clear. However, there are implications of ROS generation

and inhibition of the mPTP with regards to opioids and rIPC37

.

1.4 Involvement of Bradykinin B2 Receptors:

1.4.a Bradykinin and Their Receptors:

Bradykinin (BK), a major player of the kinin family, is a potent vasodilatory protein that

is released into the blood stream to lower blood pressure following stimuli such as ischemia and

tissue damage. BK is produced when high-molecular-weight kininogen is released into the blood

stream following proteolytic cleavage by serine proteases called kallikreins39,40

. Bradykinin is

also degraded in the blood by angiotensin converting enzyme (ACE) (in this role, ACE is called

kininase II), carboxypeptidase N, and neutral endopeptidase 1.Kinins generally reduce vascular

resistance and increase vascular permeability41

through the release of nitric oxide (NO) and

prostacyclin (PGI2) from the endothelium42

.

The kinin receptors, B1 and B2 were first cloned in the 1990‘s from humans43,44

. The

constitutively active rabbit bradykinin B2 receptor (a Gi- and Gq- protein coupled receptor) was

12

cloned in 199545

and is the target of endogenous kinins such as bradykinin (BK) and Lys-

bradykinin (aka: kallidin). This receptor is also highly expressed in rabbit kidney and duodenum

but mRNA is present in rabbit heart45

. Bradykinin B2 receptors were reported on rat

cardiomyocytes in 199446

. The cloned rabbit B2 receptor is 92% homologous with humans,

indicating the homogeneity of this receptor across species. The inducible B1 receptor is thought

to activate as a result of tissue injury47

. Also, bradykinin is highly selective for the B2 receptor

over B1 in rabbit smooth muscle cells48

. The potent antagonist, Hoechst 140 (HOE140) is highly

selective for B2 receptors such that it is considered a non-competitive antagonist for this

receptor49,50

.

There is also some controversy over the existence of other kinin receptor subtypes such

as B3, B4, and B5 which are species specific51

. In addition, these receptor subtypes may be

expressed as varying homologs in different species52

.

The source of bradykinin in the heart is cardiac endothelium53

. BK levels are ten-fold

higher in tissues, such as the kidneys, heart, and brain, compared to plasma in rats53

and levels of

circulating BK in the blood are low in humans54

at basal conditions. However during ischemia,

kinin levels dramatically increase five-fold in plasma55,56

.

1.4.b Evidence of Bradykinin Involvement in IPC:

The first studies of bradykinin in IPC illustrated the beneficial effects of exogenous BK

to recover cardiac function through increased coronary flow and an improved metabolic profile

following ischemia57

. However, this protection in rats was abolished by the B2 receptor

13

antagonist, HOE140. This finding was recapitulated in dogs58

and rabbits59

when investigating

cardiac necrosis following ischemia. In vivo canine studies in which BK was injected into the

coronary artery showed a decrease in infarct size following coronary artery occlusion (CAO)60

,

which was subsequently abolished by HOE140. With respect to rabbits, ischemic

preconditioning was blocked by the administration of HOE140 and exogenous bradykinin has

been shown to reduce infarct size59

. Studies have also revealed increased kinin release following

local and global ischemia in rats, dogs, and humans61,62,63

.

Ebrahim et al.64

determined that exogenous bradykinin limits infarct size through a

concentration-dependent manner. Male rats were preconditioned on a Langendorff apparatus

with increasing concentrations of bradykinin. Concentrations >0.1µM were able to induce

protection from ischemia-reperfusion injury in rat hearts.

Kinin activation is thought to increase intracellular endothelial cyclic guanine

monophosphate (cGMP) through NO and increase cyclic adenosine monophosphate (cAMP)

through activation of prostacyclin (PGI2)65

. Also, B2 receptors from neonatal rats show G-protein

coupled activation of PI3 kinase66

.

1.4.c Evidence of Bradykinin Involvement in rIPC:

Bradykinin has been implicated in a combined neuronal and humoral pathway in rIPC

through the G-protein coupled B2 receptor. Schoemaker et al.67

abolished rIPC protection with

mesenteric artery occlusion (MAO) in early reperfusion through administration of the bradykinin

B2 receptor antagonist, HOE140. The authors suggested that bradykinin exerts its effects in rIPC

14

by activating the neuronal pathway via stimulatory sensory nerves. In a later finding by Wolfrum

et al.68

, the involvement of PKCε downstream of bradykinin was also implicated.

1.5 Involvement of CGRP Receptors:

1.5.a CGRP and Their Receptors:

Calcitonin gene-related peptide (CGRP) is a vasodilatory neuropeptide released by vagal

and capsaicin-sensitive sensory nerves. CGRP is widely recognized as the most potent

vasodilator in the cardiovascular system69

through its actions on vascular smooth muscle70

. In

addition to these roles, CGRP is thought to be involved in neurogenic inflammation,

nociception71,72

, and vascular hypertrophy73

. Recently, this peptide has been implicated in

protection from myocardial infarction70,74

.

CGRP is a 37 amino acid peptide present in cardiac C-fibres (unmyelinated free nerve

fibres)75

and cardiomyocytes76

, and is highly abundant in central and peripheral neurons77

. Of the

two isoforms, α- and β-CGRP, α-CGRP is known to bind to the CGRP receptor. α-CGRP is a

neuropeptide derived from the calcitonin gene in neural tissues78

. β-CGRP is highly homologous

to the α isoform but is derived from another gene located near the calcitonin gene on

chromosome 1179

. Since the cloning of human CGRP in the early 1980‘s, CGRP is found to be

highly homologous among mammalian species80

.

The binding of CGRP to its receptor depends on the association of the calcitonin-like

receptor with two accessory proteins. Calcitonin-like receptor (CLR) is a rhodopsin-like Gs-

protein couple receptor first discovered in the 1990s81,82

exhibiting an unknown function83

. The

15

association of the CLR to specific receptor activity-modifying proteins (RAMPs) determine

which ligands can bind to the receptor84,85

. RAMPs are single-pass transmembrane proteins

which are 148 amino acids in length. CLR coupled to RAMP1 allows binding of CGRP to the

receptor (now a CGRP receptor). CLR coupled to RAMP2 allows binding of another vasodilator,

adrenomedullin (a peptide associated with the adrenal medulla tumour, pheochromocytoma).

Finally, CLR coupled with RAMP3 produces a dual CGRP and adrenomedullin receptor. When

comparing the different accessory proteins, RAMP2 and RAMP3 are 30% homologous to

RAMP185

. The second accessory protein associated with CLR is the receptor component protein

(RCP) which forms a component of the CLR-RAMP1 complex86

. Both rat and human CLRs

have been cloned and expression of CLR in rat aortic smooth muscle cells has been reported87,88

,

however, no such investigations have been conducted in rabbits.

1.5.b Evidence of CGRP Involvement in IPC

CGRP, adrenomedullin89

, and intermedin90

(another protein that binds to CLR in

association to all three RAMPs) are considered cardioprotective 91,92,93

. With respect to

cardioprotection, coronary effluent from isolated rat hearts displayed elevated CGRP levels

during preconditioning94

. Several clinical studies have also shown elevated CGRP during early

reperfusion following acute myocardial infarction in human patients74

suggesting that CGRP is

an endogenous substance produced by the myocardium95

. In isolated rat hearts, preconditioning

by global ischemia was abolished by CGRP (8-37), a CGRP receptor antagonist91,94

. In vivo

studies with a CGRP antibody also abolished ischemic preconditioning in rats96

(only the

abstract is available, since the article is in Chinese). CGRP has also been implicated in delayed

preconditioning and has been extensively studied in gastrointestinal preconditioning97,98,99

.

16

Chai et al.92

examined the role of CGRP in isolated rat hearts to induce IPC. The authors

measured CGRP release in coronary effluent and determined that CGRP improved left

ventricular pressure and coronary flow in a heart subjected to ischemia and reperfusion. A

concentration of 1µM improved coronary flow and left ventricular pressure, suggesting that

concentrations >1µM will protect isolated rat hearts from ischemia-reperfusion injury.

CGRP downstream of receptors is known to activate protein kinase C, but not ATP-

sensitive K+ (KATP) channels, and inhibits tumour necrosis factor –α (TNF-α)

100,101. Also,

suggestions have been made that CGRP-induced cardioprotection in rats is activated by nitric

oxide release102

.

1.5.c Evidence of CGRP Involvement in rIPC:

A number of remote intestinal preconditioning studies of the myocardium suggest that

CGRP is released by capsaicin sensory nerves into the blood stream97,98

and activates PKCε in

the myocardium103

. Wolfrum et al.103

found that administration of the CGRP receptor antagonist,

CGRP (8-37) abolished this protection. The experiments by Wolfrum et al. demonstrated

increased CGRP plasma levels following preconditioning, thus suggesting a humoral pathway in

rIPC. However, this CGRP protective effect was abolished by ganglion blockade, yet CGRP

levels in the plasma remained unaffected. Also, a clinical study by Li et al.104

has shown that

cardiac ischemic preconditioning improved lung preservation during value replacement

operations through CGRP receptor activation.

17

1.6 Involvement of Adenosine Receptors:

1.6.a Adenosine and Their Receptors:

Adenosine plays several important biological roles, such as energy transfer, an anti-

inflammatory agent, and can act as an inhibitory neurotransmitter. This purine nucleoside is

formed both intracellularly and extracellularly in cardiomyocytes, endothelial cells, and vascular

cells by dephosphorylation of AMP by 5‘-nucleotidase or by hydrolysis of S-adenosyl-

homocysteine105,106

. Degradation of adenosine occurs when extracellular concentrations of

adenosine increase, facilitating diffusion of adenosine into the cell. Adenosine is then broken

down to AMP by adenosine kinase or into inosine by adenosine deaminase (ADA) which is

found in all mammalian tissues107

. Though extracellular adenosine is mostly broken down by

ADA via erythrocytes in the blood stream105

, ventricular cardiomyocytes do not posses

extracellular adenosine deaminase108

.

There are four known G-protein coupled adenosine receptors: A1, A3 (both inhibitory;

coupled to Gi, thus resulting in a decrease in cAMP), A2a and A2b (both stimulatory, coupled to

Gs which increases cAMP; A2b is also coupled to Gq, which mediates phosphoinositide

metabolism)109

. A1 and A3 receptors are involved in the trigger phase of classical IPC, whereas

the A2b subtype has been implicated in the mediator phase14

. The binding affinity of adenosine to

rat A1 receptors is 3-30nM and is >1µM for A3 receptors110

. A2b is considered a low affinity

receptor as adenosine concentrations required to stimulate cAMP levels in the brain are >10µM

compared to A2a (requires 0.1-1µM of adenosine)111

. Only A1, A2b and A3 receptors have been

shown to be definitively expressed in cardiomyocytes112

, while A2a has been found in coronary

18

arterioles113

. With respect to cell signalling, A1 receptors are known to activate phospholipase C,

while A3 receptors activate phospholipase D109

.

1.6.b Evidence of Adenosine Involvement in IPC:

Studies have shown that in vivo infusion of adenosine deaminase through coronary

arteries in swine throughout the preconditioning event and index ischemia abolished IPC

protection114

. Also, in vivo rabbit studies by Thornton et al.115

demonstrate that A1 adenosine

agonists, PIA and 2-chloro-N6-cyclopentyladenosine (CCPA), triggered protection before the

index ischemia (i.e. longer, damaging ischemia), but not activation via A2a receptors by

CGS21680. This finding was later recapitulated in other species such as dogs116

, swine114

,

rabbits117

, and humans118

. In vivo studies with rabbits indicated that 8-(p-

sulfophenyl)theophylline (8-SPT) and PD115,199, non-selective and A2 adenosine blockers

respectively, blocked protection from ischemia by coronary artery occlusion (CAO)119

.

In addition, rabbit hearts perfused with adenosine and the A1 agonist, N6-1-(phenyl-2R-

isopropyl) adenosine (abbreviated: PIA), conferred protection on a level similar to IPC 119

. The

above study determined that saturation of adenosine receptors was required to induce

cardioprotection and short pulses or low concentrations of receptor activation was insufficient to

confer protection. Armstrong et al.120

states that the minimum adenosine concentration required

to protect rabbit cardiomyocyte is 10µM. This is also supported by Peart et al.121

in which 10µM

of adenosine reduced cell necrosis but did not limit contractile dysfunction in isolated perfused

rat hearts. However, 50µM of adenosine was able to reduced cell death and improve contractile

dysfunction.

19

Lui et al.122

demonstrated the involvement of A3 receptors by agonism with N6-2-(4-

aminophenyl)ethyl-adenosine (APNEA) and A3 antagonism with BW-A1433 in isolated rabbit

hearts. Later in 1997, Auchampach et al.123

illustrated that activation of A3 receptors by N6-(3-

iodobenzyl) adenosine-5‘-N-methyluronamide (IB-MECA) mimicked IPC in rabbits as well.

Murry et al.1 discovered that IPC protection could be abolished when 8-SPT, a non-

selective adenosine blocker was administered during reperfusion following the index ischemia.

In addition, the A1/A2 receptor agonist 5‘-(N-ethylcarboxamido) adenosine (NECA) given at

reperfusion induced protection, but was blocked by the A2b blocker MRS1754 in rabbits124

.

MRS1754 administered at reperfusion in classical IPC also abolished protection125

. Recently in

2007, the novel agonist BAY60-6583, that is highly selective for A2b receptors, induced

protection at reperfusion126

. However, this protection was abolished by MRS1754. Thus, the

above studies demonstrate the role of A2b receptors in the mediator phase (i.e. during

reperfusion) of IPC.

1.6.c Evidence of Adenosine Involvement in rIPC:

The mechanism of adenosine activation in rIPC was proposed by Liem et al.127

to act via

a non-humoral pathway that differed from classical IPC. In this study, 4 cycles of 15 min CAO

followed by 2 cycles of 15 min adenosine-dependent CAO induced preconditioning tolerance in

rats. Also, interstitial adenosine initially increased, but rapidly decreased to basal levels.

However, 4 cycles of 15 min CAO followed by 3 cycles of adenosine-independent 3 min

mesenteric artery occlusion (MAO) maintained cardioprotection. Even though tolerance of

20

adenosine-dependent preconditioning can occur in IPC (i.e. repeated cycles of ischemia-

reperfusion can no longer induce protection), rIPC may employ alternate pathways to maintain

this protection. To further support the role of adenosine in rIPC, Pell et al.128

in 1998

demonstrated that protection from renal artery occlusion (RAO) in rabbits was abolished by

treatment with 8-(p-sulfophenyl)theophylline (8-SPT). The same study determined that rIPC

blockade could also be achieved when 8-SPT was administered during reperfusion, suggesting

the role of adenosine in both the trigger and mediator phase.

A study by Pang et al.129

demonstrated that plasma adenosine concentrations increased

with skeletal muscle rIPC, and this effect was partially abolished by reserpine, an inhibitor of the

vesicular monoamine transporter (VMT, a transporter of catecholamines at synaptic nerve

endings). In addition, Addison et al.38

has shown that blockade by 8-SPT and the free radical

scavenger, mercaptopropionyl glycine (MPG) completely abolished skeletal muscle rIPC

cardioprotection in pigs.

1.7 Other Potential Preconditioning Substances:

A number of G-protein coupled and non-G-protein coupled receptors have been

confirmed to play a role in classical IPC and their involvement in rIPC has been suggested.

However, the focus of my thesis will be on G-protein coupled receptors, and in particular, the

major receptors that have been implicated: δ and κ opioid receptors, adenosine receptors, and

bradykinin B2 receptors; as well as the recently proposed CGRP receptor. Unfortunately,

investigating the claims of other receptors (both G-protein and non-G protein coupled) is beyond

21

the scope of this thesis. Nonetheless, this section will bring to light other receptors that may be

involved in rIPC.

With respect to other GPCRs, α1 adrenergic receptors which bind both norepinephrine

and epinephrine have been implicated in myocardial rIPC; however, the results in this area are

conflicting130,131

. Losartin (an angiotensin I receptor blocker) was found to block renal artery

occlusion (RAO) preconditioning in rat myocardium132

. Prostaglandin E2 (PGE2) has also been

implicated in rIPC gastric preconditioning by Brzozowski et al.133

. However, there is yet to be a

study on the role of PGE2 in myocardial preconditioning. The endocannabinoid receptor CB2 has

also been implicated in a humoral role in rIPC. Intestinal ischemia studies have shown that

blockage of the CB2 receptor abolished myocardial protection in mice134

and rats135

. Finally,

studies have demonstrated that carbachol (a muscarinic M2 receptor agonist) mimicked

preconditioning in isolated rabbit whole hearts, suggesting the role of acetylcholine in IPC2. To

date, no studies have been conducted in rIPC involving muscarinic M2 receptors.

A number of substances have been implicated to trigger rIPC. These include nitric oxide

(NO) and the following cytokines: tumour necrosis factor-α (TNF-α), nuclear factor- κB (NF-

κB), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Also, free radicals and heat shock proteins

such as hemoxygenase-1 (HO-1) have been suggested. However, these substances do not activate

G-protein coupled receptors and are beyond the scope of this thesis. That being said, the findings

presented in this section are not conclusive and requires exploration of the mechanisms involved

in rIPC. Consequently, many of the above studies are preliminary and require further testing.19

22

1.8 Opioid-Adenosine Receptor Cross-talk:

1.8.a Background:

A number of non-preconditioning studies have implicated opioid-adenosine cross-talk in

the central nervous system. Interestingly, adenosine A1 and A2 blockers antagonize the

synergistic signalling actions between opioid and dopamine D2 receptors in the rat CNS136

. In

this study, adenosine deaminase and the adenosine blocker, BW A1434U, prevented increased

activation of cAMP downstream of colocalized δ opioid and dopamine D2 receptors in

transfected NG108-15/D2 cells. Also, Eisenach et al.137

conducted a clinical trial in which

patients were administered intrathecal opioid (morphine and fentanyl), which resulted in

increased adenosine release in cerebrospinal fluid. This study provided support for an opioid-

adenosine role in analgesia in humans.

The novel idea of ‗cross-talk‘ between opioid and adenosine receptors in ischemic

preconditioning was first proposed by Peart et al.138

in 2003. Peart et al. found that myocardial

protection induced by either morphine (non-selective opioid receptor agonist) or CCPA (A1

adenosine receptor agonist) was blocked, for both morphine and CCPA, by either δ opioid or A1

adenosine specific blockers. This study was conducted in an in vivo coronary artery occlusion

(CAO) model in rats. Later, Peart et al.139

in 2005, they claimed that adenosine kinase inhibition

reduced infarct size in the same in vivo CAO model in rats. A1 adenosine, A3 adenosine, and δ

opioid receptor antagonists abolished this protection, suggesting that stimulation of these

receptors resulted in adenosine kinase inhibition. Currently, there are no studies of opioid-

adenosine receptor cross-talk in rIPC.

23

1.8.b Adenosine Levels in the Blood:

Concentrations of extracellular adenosine are 30-300nM in tissues and can increase

during ischemia to 10µM when ATP is converted to adenosine111

. An increase in adenosine

during ischemia suggests that adenosine could enter the blood stream to reach the target organ.

However, the half-life of adenosine in the blood stream is 0.6-1.5 seconds due to constant

deamination by adenosine deaminase from erythrocytes, pericytes, and endothelial cells140

. Since

there are no studies demonstrating an inhibition of nucleoside transportation that prevents

adenosine degradation in the blood, it is postulated that adenosine either works entirely through

the neurogenic pathway, or partially activates this pathway to induce the release of humoral

mediators4. Liem et al.

141 provided evidence of a neurogenic pathway in which exogenous

adenosine mimicked MAO rIPC, however this protection was abolished by prior ganglion

blockade.

1.8.c Potential Mechanisms of Cross-talk:

Two mechanisms have been proposed by Peart et al.139

to mediate ‗cross-talk‘ between

opioid and adenosine receptors. One possibility for cross-talk is via adenosine kinase inhibition

in cardiomyocytes (see Figure 3). Adenosine kinase continually converts adenosine to AMP,

effectively maintaining very low concentrations of adenosine within cardiomyocytes. In turn,

adenosine continually diffuses into cardiomyocytes to replenish adenosine levels. However,

Peart et al. postulates that opioid receptor stimulation inactivates adenosine kinase, resulting in a

build-up of adenosine within cardiomyocytes. Since the uptake of adenosine by cells is

dependent on a concentration gradient, there is an accumulation of extracellular adenosine. The

amassing of adenosine around cardiomyocytes activates adenosine receptors on the cell

24

membrane and initiates downstream signalling that ultimately results in ischemic

preconditioning. In support of this mechanism, Deussen et al.142

provides evidence that

inhibition of adenosines deaminase doubles cardiac adenosine release, whereas inhibition of

adenosine kinase causes a 30-fold increase in adenosine. In addition, the role of adenosine kinase

is thought to be important in IPC since a low pH due to hypoxia inhibits adenosine kinase, which

may contribute to increases in adenosine during ischemia142

.

The second mechanism of cross-talk proposed by Peart et al.139

is heterodimerization of

opioid and adenosine receptors. This hypothesis is addressed in detail in the General Discussion

chapter of this thesis (see section 9.2.a Dimerization of G-protein Coupled Receptors).

25

(Peart et al., 2005)139

A

Figure 3. The Adenosine Kinase Inhibition Hypothesis.

(A) Adenosine kinase continually converts adenosine to AMP, effectively maintaining very low

concentrations of adenosine within cardiomyocytes. In turn, adenosine continually diffuses into

cardiomyocytes to replenish adenosine levels.

(B) Opioid receptor stimulation inactivates adenosine kinase, resulting in a build-up of adenosine

within cardiomyocytes. Since the uptake of adenosine by cells is dependent on a concentration

gradient, there is an accumulation of extracellular adenosine. The amassing of adenosine around

cardiomyocytes activates adenosine receptors on the cell membrane and initiates downstream

signalling that ultimately results in ischemic preconditioning.

B

26

- Chapter 2 -

RATIONALE

The mechanisms involved in IPC have been studied extensively; however, the exact

triggering molecules involved in rIPC are a subject of controversy. Possible candidates for rIPC

include opioid peptides, bradykinin, adenosine, and CGRP. Even less is known about possible

cross-talk between the G-protein coupled receptors for these molecules in IPC or rIPC. Thus, a

comprehensive study is needed to evaluate the similarities between classical IPC and rIPC in

regard to receptor activation and explore the possibility of receptor cross-talk, a novel concept in

myocardial preconditioning.

The most appealing aspect of rIPC is its clinical applicability. Unlike classical IPC and

the recent phenomenon of post-conditioning (i.e. short periods of re-occlusion during long

reperfusion), which is restricted to specific settings such as cardiac surgery, rIPC does not

require invasive treatment that can initiate harmful side effects. Cheung et al.143

have already

demonstrated the use of rIPC in a clinical setting wherein four cycles of 5 min limb ischemia and

reperfusion was able to reduce myocardial injury in children undergoing cardiac surgery. The

clinical use of rIPC in acute myocardial infarction may prove to be a major medical advance.

Given the practical value of rIPC alone, the elucidation of mechanisms involved in rIPC is of

significant importance.

27

- Chapter 3 -

OBJECTIVES

3.1 Hypothesis

I hypothesize that one or more humoral protective factor(s) released by hind limb rIPC

activate two G-protein coupled receptors on the cardiomyocyte cell surface, adenosine and

opioid receptors, and involves a cross-talk mechanism between these two receptors to induce

protection against cardiomyocyte cell death from ischemia-reperfusion injury.

3.2 Specific Aims

1. To characterize the protection induced by rIPC in freshly isolated rabbit ventricular

cardiomyocytes.

2. To determine the role of G-protein coupled cell membrane receptors in rIPC in

cardiomyocytes.

3. To determine whether rIPC involves cross-talk between adenosine and opioid receptors.

28

- Chapter 4 -

RESEARCH DESIGN & METHODS

4.1 Animals and Human Subjects

Male New Zealand rabbits were used to obtain both preconditioned and control dialysate.

Isolated cardiomyocytes for fresh cell experiments were obtained from either male or female

New Zealand adult rabbits. All rabbits were ~12-14 weeks old, weighed ~3.2-3.5 kg, and had

been designated specific pathogen free. All animals were maintained and used in accordance

with recommendations from the Department of Laboratory Animal Services at the Hospital for

Sick Children.

Human dialysate was obtained from male human volunteers. Prior to obtaining blood,

subjects were instructed not to exercise or consume caffeine and remain calm during the

procedure.

4.2 Isolation of Adult Cardiomyocytes

4.2.a Operative Procedure:

Male or female New Zealand White rabbits were treated with a mild topical anaesthetic

(xylocaine) prior to being anesthetised via the ear vein with pentobarbital (30mg/kg) and heparin

(100U/kg) to prevent coagulation. Once rabbits reached a surgical level of anaesthesia, hearts

were rapidly excised and hung by the aortic root onto a Langendorff apparatus.

29

4.2.b Digestion Protocol:

Buffers were made in 1L of distilled water from a Millipore filtration system according to

specifications in Table 1and Figure 4. This Krebs-Henseleit buffer (contains the following in

mM: 0.5 MgSO4, 4.7KCl, 10.0 NaHCO3, 1.2 KH2PO4, 10.0 Dextrose, 20.0 HEPES, and 128.3

NaCl) was filtered and adjusted to pH 3.7. All buffers were oxygenated at 37°C with 95% O2 and

5% CO2. Rabbit hearts were mounted on a pressure-controlled Langendorff apparatus via the

aorta and immediately perfused with Ca+ Buffer (with 0.99mM CaCl2) for 2 min in order to

expel blood from the heart. Hearts were then perfused with Ca- Buffer containing 9.99mM

ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) for 7 min to chelate

calcium and suppress heart contraction. Enzymatic digestion was conducted via recirculation of

the Collagenase Buffer (with 200U/ml crude collagenase) until the heart was soft. Samples were

taken throughout the digestion process to ensure sufficient perfusion of the heart. Once digestion

was complete, the heart was agitated in the Mincing Buffer to separate cardiomyocytes from

connective tissue.

The isolate then underwent subsequent filtration and wash steps in Wash Buffer (2%

albumin). Dead cells were removed by low centrifugation (at 500g for 2 min, all subsequent

spins occur at 500g for 1min) and removal of the supernatant. Cardiomyocytes in Wash Buffer

were gradually re-introduced to calcium in a step-wise manner until concentrations reached

0.9mM CaCl2. Finally, calcium tolerant cardiomyocytes were re-suspended in Reactive Buffer

(0.1% albumin, 0.99mM CaCl2) to remove excess calcium from the re-introduction mentioned

earlier. Only calcium tolerant cardiomyocyte isolates with <30% cell necrosis were used in fresh

cell experiments.

30

Table 1. Krebs-Henseleit Buffer Composition.

Reagent Concentration (mM) Osmolarity (mOsm)

MgSO4 0.5 1.0

KCl 4.7 9.4

NaHCO3 10.0 20.0

KH2PO4 1.2 4.8

Dextrose 10.0 10.0

HEPES 20.0 20.0

NaCl 128.3 256.6

31

Figure 4. Preparation of Buffers Derived from Krebs-Henseleit Buffer.

Krebs-Henseleit (KH) buffer was filtered and adjusted to pH 3.7. Rabbit hearts were mounted on

a Langendorff apparatus and perfused with Ca+ Buffer (with 0.99mM CaCl2) then with Ca

-

Buffer (9.99mM EGTA, 0.1% albumin). Enzymatic digestion was conducted via recirculation of

the Collagenase Buffer (with 200U/ml crude collagenase, 0.1% albumin, 9.99mM EGTA). Once

digestion was complete, the heart was agitated in the Mincing Buffer (from the Collagenase

Buffer). The isolate then underwent subsequent filtration and wash steps in Wash Buffer (2%

albumin) to remove dead cells. Cardiomyocytes in Wash Buffer were gradually re-introduced to

calcium in a step-wise manner until concentrations reached 0.9mM CaCl2. Finally, calcium

tolerant cardiomyocytes were re-suspended in Reactive Buffer (0.1% albumin, 0.99mM CaCl2).

All buffers were oxygenated at 37°C with 95% O2 and 5% CO2.

0.1% albumin

9.99mM EGTA

200U/ml collagenase

0.1% albumin

Ca+ Buffer

0.99 mM CaCl2

Wash Buffer

2% albumin

Ca- Buffer

Reactive Buffer

Mincing Buffer

Collagenase

Buffer

Krebs- Henseleit

Buffer

400ml 400ml 100ml

150ml

100ml

40ml

1L

32

4.2.c Comparison of Digestion Protocols:

A number of modifications to the digestion protocol were studied in an effort to improve

isolate yields. Digestions were modified in two protocols, each supplemented with DNase I

(degrades DNA, 0.02mg/ml; Worthington)144

, hyaluronidase (degrades hyaluronic acid,

chondroitin, and chondroitin sulphates, 0.6mg/ml; Sigma)145

, ovamucoid trypsin inhibitor

(inhibits trypsin, derived from ovamucoid extracts, 0.1mg/ml; Worthington)145

, or soybean

trypsin inhibitor (derived from soybean extracts, 1mg/ml; Worthington)144

and compared to

digestions with only collagenase (200U/ml crude collagenase; Worthington).

In the first protocol, hearts were initially digested with collagenase for 10 min, removed

from the Langendorff system, minced, and re-suspended in collagenase buffer supplemented

with a reagent for a further 10 min (n=2). This protocol was designed to ensure that

cardiomyocytes from the same heart were used in order to avoid heart-to-heart variability. Also,

these were preliminary experiments to confirm the supplemented reagents were non-toxic. The

second protocol involved re-circulation of collagenase with the supplemented reagent in a

Langendorff until digestion was complete (n=1-2). All isolates underwent similar wash and

filtration steps as in a non-supplemented digestion. The results of these modifications indicate

that re-circulation with only collagenase is the most optimal method of digestion. (See Figure 5)

33

Figure 5. Comparison of Digestion Protocols.

Digestions with collagenase were modified in two protocols that were supplemented with DNase

I (degrades DNA, 0.02mg/ml), hyaluronidase (degrades hyaluronic acid, chondroitin, and

chondroitin sulphates, 0.6mg/ml), ovamucoid trypsin inhibitor (inhibits trypsin, derived from

ovamucoid extracts, 0.1mg/ml), or soybean trypsin inhibitor (derived from soybean extracts,

1mg/ml) and compared to digestions with only collagenase (200U/ml crude collagenase).

In the first protocol (Minced Heart), hearts were initially digested with collagenase for 10 min,

removed from the Langendorff system, minced, and re-suspended in collagenase buffer

supplemented with a reagent for a further 10 min. The second protocol (Perfusion) involved re-

circulation of collagenase with the supplemented reagent in a Langendorff until digestion was

complete. All isolates underwent similar wash and filtration steps for a non-supplemented

digestion. The results of these modifications indicate that re-circulation with only collagenase is

the most optimal method of digestion.

34

4.3 Dialysate Preparation

Male New Zealand White rabbits were anaesthetized with akmezine (0.25mg/kg) (a pre-

anaesthetic containing ketamine, acepromazine, and atropine) followed by pentobarbital

(30mg/kg) with heparin (100U/kg) via the ear vein then placed on a ventilator. rIPC rabbits were

preconditioned by 4 cycles of 5 min hind limb ischemia and 5 min reperfusion using a blood

pressure cuff. Control rabbits were only anaesthetized and ventilated for the same time period as

rIPC rabbits. About 100-150ml of blood was quickly drawn from the left carotid artery

immediately after the preconditioning or control protocol. Rabbits were monitored throughout

the procedure to detect any hemodynamic variability. The whole blood obtained was centrifuged

(3000g for 20 min) to obtain 50 or 100ml of plasma which was then dialysed using a 12-14kDa

cut-off membrane against a 10 fold greater volume of water. Thus, the dialysate contents were

limited to <14 kDa. Dialysate was divided into 900µl aliquots and immediately stored at -80°C.

Prior to use, the frozen aliquots were thawed only once and reconstituted with 10µl of 10 fold

concentrated Krebs-Henseleit buffer. Since dialysate was created against water, a concentrated

salt solution was supplemented in order to achieve the same physiological salt levels as in 1x

Krebs-Henseleit buffer. Osmolarity and pH was measured in the dialysate before exposing the

solution to cardiomyocytes.

For human subjects, 100 ml of blood was withdrawn before (Control Human Dialysate)

and after (rIPC Human Dialysate) 4 cycles of 5 min ischemia (>20mmHg above systolic

pressure) and 5 min reperfusion to the upper arm by a blood pressure cuff. Dialysate was

obtained using the identical procedure as described above for rabbits. The protocol for preparing

35

both rabbit and human dialysate is shown on Figure 6 and was previously described by Shimizu

et al.146

. All dialysate used in these studies were prepared by an external laboratory*.

* Dialysate prepared by Jing Li (Dr. Andrew Redington‘s Lab; Research Institute, Hospital for Sick Children).

36

(Shimizu et al., 2009)146

Figure 6. Rabbit and Human Dialysate Preparation.

rIPC rabbits were preconditioned by 4 cycles of 5 min hind limb ischemia and 5 min reperfusion

using a blood pressure cuff. Control rabbits were only anaesthetized and ventilated for the same

time period as rIPC rabbits. About 100-150ml of blood was quickly drawn from the left carotid

artery immediately after the preconditioning or control protocol.

For human subjects, 100 ml of blood was withdrawn before (Control Human Dialysate) and after

(rIPC Human Dialysate) 4 cycles of 5 min ischemia (>20mmHg above systolic pressure) and 5

min reperfusion to the upper arm by a blood pressure cuff. Dialysate was obtained using the

identical procedure as described above for rabbits.

The whole blood obtained was centrifuged (3000g for 20 min) to obtain 50 or 100ml of plasma

which was then dialysed using a 12-14kDa cut-off membrane against a 10 fold greater volume of

water. Thus, the dialysate contents were limited to <14 kDa. Dialysate was divided into 900µl

aliquots and immediately stored at -80°C. Prior to use, the frozen aliquots were thawed only once

and reconstituted with 10µl of 10 fold concentrated Krebs-Henseleit buffer. Osmolarity and pH

was measured in dialysate before exposing the solution to cardiomyocytes.

37

4.4 Fresh-Cell Experimental Model

4.4.a General Protocol:

Cardiomyocyte isolates were evenly divided into Eppendorff tubes such that each group

contained >200µl per group. Cardiomyocytes were re-suspended in 1ml Reactive Buffer and

placed in 12-well plates (2ml total volume) at 37°C and 100% O2. Samples from the Baseline

group were taken after stabilization and at the end of the experiment to ensure that

cardiomyocyte viability remained stable. All cardiomyocytes underwent a 20 min stabilization

period in suspension. Cardiomyocytes were subjected to 20 min of a particular treatment,

depending on the protocol. This was followed by another 20 min wash period in suspension with

fresh Reactive Buffer such that any residual drug(s) in the supernatant was/were removed. All

treatment groups subsequently underwent 45 min damaging simulated ischemia (SI) and 60 min

simulated reperfusion (SR). SI was conducted by low speed centrifugation (500g for 1 min) of

cardiomyocytes into a pellet and leaving only a thin layer of supernatant with an oil layer on top

of cells. Cardiomyocytes then underwent SR through suspension in fresh Reactive Buffer.

Samples from the treatment groups were taken before SI and after SR. The purpose of these fresh

cell protocols was to observe the effect of a treatment on cell survival following the simulation of

normally damaging ischemia and reperfusion.147

(See Figure 7)

38

Figure 7. General Protocol: Isolated Rabbit Cardiomyocytes.

All cardiomyocytes underwent a 20 min stabilization period in suspension. Cardiomyocytes were

subjected to 20 min of a particular treatment, depending on the protocol. This was followed by

another 20 min wash period in suspension with fresh Reactive Buffer such that any residual

drug(s) in the supernatant was/were removed. All treatment groups subsequently underwent 45

min damaging simulated ischemia (SI) and 60 min simulated reperfusion (SR). SI was conducted

by low speed centrifugation (500g for 1 min) of cardiomyocytes into a pellet, leaving only a thin

layer of supernatant with an oil layer on top of cells. Cardiomyocytes then underwent SR through

suspension in Reactive Buffer. Samples from the treatment groups were taken before SI and after

SR (see arrows).

39

4.4.b AIM (1) Protocol:

AIM (1): To characterize the protection induced by rIPC in freshly isolated rabbit

ventricular cardiomyocytes.

Cardiomyocytes freshly isolated from non-preconditioned donor rabbit hearts were

preconditioned by exposing them to10 min ischemic pelleting (IPC treatment group) or rIPC

dialysate, human or rabbit derived (rIPC Dialysate group). Cardiomyocyte groups that were not

preconditioned were subjected to 10 min re-suspension in buffer (IR group) or control human or

rabbit dialysate (Control Dialysate group). Treatment groups underwent a wash period in fresh

buffer for 20 min. All groups were then subjecting to a long period of 45 min simulated ischemia

(SI) and 60 min simulated reperfusion (SR). Samples were taken before and after SI/SR. (See

Figure 8)

Figure 8. AIM (1) Protocol: To Characterize Dialysate Protection.

Cardiomyocytes freshly isolated from non-preconditioned donor rabbit hearts were

preconditioned by exposing them to10 min ischemic pelleting (IPC treatment group) or rIPC

dialysate, human or rabbit derived (rIPC Dialysate group). Cardiomyocyte groups that were not

preconditioned were subjected to 10 min re-suspension in buffer (IR group) or control human or

rabbit dialysate (Control Dialysate group). Treatment groups underwent a wash period in fresh

buffer for 20 min. All groups were then subjecting to a long period of 45 min simulated ischemia

(SI) and 60 min simulated reperfusion (SR). Samples were taken before and after SI/SR.

40

4.4.c AIM (2) Protocol:

AIM (2): To determine the role of G-protein coupled cell membrane receptors in rIPC in

cardiomyocytes.

Freshly isolated cardiomyocytes were either subjected to 10 min IR or rIPC (using

dialysate) in the presence of an antagonist (or its vehicle) for 20 min to prevent specific

activation of each receptor being studied (adenosine, bradykinin B2, and κ opioid receptors,

and CGRP receptors). Treatment groups underwent a wash period in fresh buffer for 20 min. All

groups were then subjecting to a long period of 45 min simulated ischemia (SI) and 60 min

simulated reperfusion (SR). Samples were taken before and after SI/SR. (See Figure 9)

Figure 9. AIM (2) Protocol: The Role of Cell Membrane Receptors.

Freshly isolated cardiomyocytes were either subjected to 10 min IR or rIPC (using dialysate)

in the presence of an antagonist (or its vehicle) for 20 min to prevent specific activation of

each receptor being studied (adenosine, bradykinin B2, and κ opioid receptors, and CGRP

receptors). Treatment groups underwent a wash period in fresh buffer for 20 min. All groups

were then subjecting to a long period of 45 min simulated ischemia (SI) and 60 min

simulated reperfusion (SR). Samples were taken before and after SI/SR.

41

4.4.d AIM (3) Protocol:

AIM (3): To determine whether rIPC involves cross-talk between adenosine and opioid

receptors.

Cardiomyocytes were subjected to 10 min classic IPC, specific opioid receptor agonists

(Ag), such as Met-enkephalin (δ-specific agonist) or dynorphin B (κ-specific agonist) in the

presence of adenosine receptor blockers, such as 8-SPT (non-selective adenosine blocker) for 20

min. Treatment groups underwent a wash period in fresh buffer for 20 min. All groups were then

subjecting to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion

(SR). Samples were taken before and after SI/SR. (See Figure 10)

Figure 10. AIM (3) Protocol: Investigate Receptor Cross-talk.

Cardiomyocytes were subjected to 10 min classic IPC, specific opioid receptor agonists, such as

Met-enkephalin (δ-specific agonist) or dynorphin B (κ-specific agonist) in the presence of

adenosine receptor blockers, such as 8-SPT (non-selective adenosine blocker) for 20 min.

Treatment groups underwent a wash period in fresh buffer for 20 min. All groups were then

subjecting to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion

(SR). Samples were taken before and after SI/SR.

42

4.5 Trypan Blue Exclusion Assay

Cardiomyocyte samples were taken before and after simulated ischemia (SI) and

reperfusion (SR) to assess cell death by a trypan blue exclusion assay. 10µL of cardiomyocytes

were suspended in 13µl of 85mOsm hypotonic Tyrode Buffer (contains the following in mM:

10.7 KCl, 1.5 NaH2PO4, 14.7 NaHCO3, 5.6 glucose, 1.6 MgSO4, 7.2 CaCl2, 3.0 amylobarbitone,

0.5% glutaraldehyde, 0.5% trypan blue; see Table 2 for buffer composition) and 13µl of Reactive

Buffer for 1 min in order to allow dead cardiomyocytes to take up the trypan dye. Samples were

place on a hemocytometer and images were taken with a light microscope at 20x magnification.

Cardiomyocytes unable to exclude the trypan blue dye due to the loss of membrane integrity

were counted as dead cells, whereas live cells that maintained membrane integrity were able to

exclude the dye and were seen as clear (see Figure 11). The percent cell death was counted from

>300 cells per sample. Images were taken by the Micro-Cap software (Spectrum) and ImageJ

software (NIH) was used to count sample images.

Table 2. Tyrode Buffer Composition.

Reagent Concentration (mM) Osmolarity (mOsm)

KCl 10.7 21.4

NaH2PO4 1.5 3.0

NaHCO3 14.7 29.4

Glucose 5.6 5.6

MgSO4 1.6 3.2

CaCl2 7.2 21.6

Total Osmolarity ~85 mOsm

Amylobarbitone 3.0

Glutaraldehyde 0.5%

Trypan Blue 0.5%

43

Figure 11. Trypan Blue Exclusion Assay.

Cardiomyocyte samples were taken before and after ischemia (SI) and reperfusion (SR) to

assess cell death by a trypan blue exclusion assay. Cardiomyocytes unable to exclude the

trypan blue dye due to the loss of membrane integrity were counted as dead cells (Nonviable

Cells), whereas live cells (Viable Cells) that maintained membrane integrity were able to

exclude the dye and were seen as clear

44

4.5.a Inter-Observer Error Data:

Inter-observer measurements were collected to ensure consistency in cell counting from

various observers and experiments. Three experiments (Sept. 17, 2007; Sept 19, 2007; Sept 25,

2007) were conducted using the rabbit dialysate protocol described in Figure 8. Treatment

groups included a Baseline, ischemia-reperfusion (IR), ischemic preconditioning (IPC), control

rabbit dialysate (Control Dialysate), and preconditioned rabbit dialysate (rIPC Dialysate).

Sample pictures were taken before and after simulated ischemia and simulated reperfusion.

Cardiomyocytes were exposed to hypotonic trypan blue and images were counted. Four different

observers (Harinee Surendra, Elena Kuzmin, Sue Omar, and Mohammad Escandarian) counted

cell necrosis from the same images in all three experiments (a total of 45 images). The mean was

calculated for the following parameters: live, dead, and total cardiomyocytes counted and the

percent necrosis for reach observer. Statistical analysis was conducted via correlation

coefficients and correlation p-values. The results indicated that all counts correlated among

observers for a given parameter (coefficients approached 1) and the probability of incorrectly

concluding this correlation was very small among the observers (p<0.0001). Thus, the method of

counting cell necrosis through trypan blue staining showed little variation among observers. (See

Table 3 for results)

45

Table 3. Inter-Observer Error Data.

Elena Harinee Sue Mohammad

Mean of

Counts

Live 175.3 228.3 253.3 160.1

Dead 83.58 88.53 87.84 59.53

Total 258.9 316.8 341.1 219.6

% Necrosis 32.51 28.84 26.60 27.37

Elena Harinee Sue Mohammad

Elena

Live 0.82 0.71 0.82

Dead 0.97 0.93 0.91

Total 0.86 0.74 0.87

% Necrosis 0.93 0.91 0.88

Harinee

Live <0.0001 0.90 0.90

Dead <0.0001 0.93 0.87

Total <0.0001 0.90 0.88

% Necrosis <0.0001 0.94 0.91

Sue

Live <0.0001 <0.0001 0.85

Dead <0.0001 <0.0001 0.93

Total <0.0001 <0.0001 0.85

% Necrosis <0.0001 <0.0001 0.92

Mohammad

Live <0.0001 <0.0001 <0.0001

Dead <0.0001 <0.0001 <0.0001

Total <0.0001 <0.0001 <0.0001

% Necrosis <0.0001 <0.0001 <0.0001

Correla

tion

Co

efficient

Correlation p-value

46

4.6 Drugs

All drugs used as agonists or antagonists for these studies competitively bound to cell-

membrane receptors and the concentrations used have previously been published in peer-

reviewed preconditioning literature to pharmacologically precondition (agonist) or inhibit the

protection of classical or remote IPC (antagonists).

Antagonists used for opioid receptors were naloxone (Nal), naltrindole (NTI), and GNTI.

Naloxone (chemical name: (5a)- 4,5-Epoxy-3,14-dihydro-17-(2-propenyl)morphinan-6-one

hydrochloride; from Sigma) is a highly potent, non-selective, competitive inhibitor of opioid

receptors used at 100µM148,149,150,151

. Naltrindole (chemical name: 17-(cyclopropylmethyl)-6,7-

dehydro-4,5a-epoxy-3,14-dihyd roxy-6,7-2',3'-indolomorphinan hydrochloride; Sigma) is a δ

opioid blocker used at 10nM152

with a 223- and 346-fold greater selectivity for δ over µ and κ

opioid receptors, respectively153,154,155

. GNTI (chemical name: 5'-Guanidinyl-17-

(cyclopropylmethyl)-6,7-dehydro-4,5a-ep oxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan

dihydrochloride; Tocris Biosciences) is a κ-opioid blocker used at 1nM152

with a 208- and 799-

fold selectively for κ receptors over µ and δ opioid receptors, respectively156,157,158

.

Agonists used for opioid receptors were Met-enkephalin (ME) and dynorphin B (DynB).

Met-enkephalin is an endogenous ligand of δ opioid receptors used at 100µM152

that is highly

selective for δ receptors, and to a lesser extent µ, over κ receptors20

. The minimum concentration

of Met-enkephalin required to protect rabbit cardiomyocytes from ischemia/reperfusion damage

is 1µM148

. Dynorphin B (aka: rimorphin; Phoenix Pharmaceuticals) is an endogenous peptide

that is highly selective for κ opioid receptors over µ and δ receptors20

. DynB was used at 100µM

47

and the minimum concentration reported to protect rabbit cardiomyocytes from

ischemia/reperfusion damage is 10µM152

.

This study used Hoechst 140 (HOE140) as a blocker of bradykinin B2 receptors and

calcitonin gene-related peptide 8-37 (CGRP (8-37)) as a blocker for CGRP receptors. Hoechst

140 (aka: icatibant; Sigma) is a highly selective inhibitor of bradykinin B2 receptors and is

negligibly active against B1 receptors49,49 ,159

when used at 5µM160

. CGRP (8-37) (calcitonin

gene-related peptide, fragment 8-37; Sigma) is a fragment of the endogenous α-CGRP human

peptide and inhibits CGRP receptors and not calcitonin receptors at 5nM103,161,162

.

Finally, the antagonist used for adenosine receptors was 8-SPT. 8-SPT (chemical name:

8-(p-sulfophenyl)theophylline; Sigma) is non-selective blocker of adenosine receptors used at

100µM120,163

.

4.7 Other Methods

4.7.a Western Blotting:

Western blots were performed to determine the presence of cell-membrane receptors on

rabbit cardiomyocytes. For µ opioid receptors, a synthetic rabbit monoclonal antibody

corresponding to amino acid residues 220–250 of the human µ opioid receptor sequence was

used (Santa Cruz Biotechnology). This human epitope is 100% homologous with both the cloned

rat and mouse µ-opioid receptor sequence over residues 220-250. The δ opioid receptor was

detected with a rabbit polyclonal synthetic antibody which corresponded to amino acids 3-17 of

both the mouse and rat δ opioid receptor (Abcam). The antibody used for κ opioid receptors was

48

a goat polyclonal antibody corresponding to amino acids 330 – 380 of the human κ-opioid

receptor (Santa Cruz Biotechnology). This fragment is 88% homologous to the rat and mouse κ-

opioid receptor sequence over these residues.

The calcitonin-like receptor in rabbits was detected using a mouse polyclonal antibody of

human origin, corresponding to amino acids 23-133 (Abnova). This immunogen has a 92%

homology to the mouse calcitonin-like receptor over these residues.

4.7.b Multiple Reaction Monitoring Mass Spectrometry:

Multiple reaction monitoring (MRM) mass spectrometry was used to identify molecules

in the rabbit dialysate. MRM mass spectrometry is a form of tandem mass spectrometry which

ionizes chemical compounds into multiple fragment ions. Once the particular fragments are

detected by a magnetic and/or electric field, the mass-to-charge ratio is calculated and data

analysis allows identification of the unknown compound. The purpose of utilizing MRM mass

spectrometry, compared to other methods such as chromatography, is the high degree of

sensitivity (can detect substances at concentrations as low as 1-3nM) and selectivity (is able to

distinguish adenosine from other purines, such as inosine which differs by 1Da). In order to

measure accurate concentrations, all substances were compared to known standard

concentrations prepared in water (see Figure 28 in Appendix V for a plot of the standard

concentrations).

In order to investigate the role of adenosine kinase inhibition in cross-talk, MRM mass

spectrometry was also used to measure levels of adenosine in supernatant samples. Supernatant

49

was obtained from freshly isolated rabbit cardiomyocytes after 20 min stabilization, after 10 min

of exposure to dynorphin B (κ-opioid receptor agonist) in fresh Reactive Buffer, and after a 20

min wash period in which cardiomyocytes were suspended in fresh Reactive Buffer (see Figure

12). Measured adenosine concentrations were compared to known standard concentrations

prepared in Krebs-Henseleit buffer (see Figure 29 in Appendix V for a plot of the standard

concentrations).

All mass spectrometry measurements were conducted on an API 4000 Triple Quadrupole

with Agilent LC by an external laboratory†.

† MRM mass spectrometry was conducted by Michelle Young (AIMS Lab; Department of Chemistry, University of

Toronto) and Michael Tropak (Dr. Callahan‘s Lab, Hospital for Sick Children).

Figure 12. Adenosine Kinase Inhibition Protocol.

Supernatant was obtained from freshly isolated rabbit cardiomyocytes after 20 min stabilization,

after 10 min of exposure to dynorphin B (κ-opioid receptor agonist) in fresh Reactive Buffer, and

after a 20 min wash period in which cardiomyocytes were suspended in fresh Reactive Buffer.

MRM mass spectrometry was used to measure levels of adenosine in supernatant samples in

order to investigate the role of adenosine kinase inhibition in cross-talk.

50

4.7.c Statistics:

Averages and standard error of the mean were reported for all results. For all experiment

sets, the N (number of different rabbit dialysates used) and n (number of times experiments were

replicated for a particular dialysate) were stated. All treatment groups were statistically assessed

by an Analysis of Variance (ANOVA) Scheffé post hoc test for multiple comparisons. For

groups that were not distributed normally, an ANOVA Kruskal-Wallis post hoc test was

conducted. Statistics was conducted with Statview (Abacus Corporation).

51

- Chapter 5 -

CHARACTERIZATION OF THE PROTECTION INDUCED BY RIPC

5.1 Rabbit and Human Preconditioned Dialysate Induces Protection

Cardiomyocytes were subjected to 10 min. exposure to either preconditioned rIPC

dialysate or non-preconditioned control dialysate. Results indicate that remotely preconditioned

dialysate, whether rabbit (31.9% ±3.7 vs. 48.0%±2.7 IR, p=0.0002; N=6, n=1 each) or human

derived (32.4%±2.9 vs. 48.0%±2.7 IR, p=0.0001; N=1, n=6), reduces SI/SR induced cell death

similar to classical IPC (30.5%±3.1 vs. 48.0%±2.7 IR, p=0.0006; n=6). (See Figure 13 and Table

4).

52

Table 4. Rabbit and Human Dialysate Results (Mean ± SEM).

Stabilization End

Baseline 26.8 ± 1.0 32.3 ± 2.3

Before SI After SR

IR 33.1 ± 2.9 48.0 ± 2.7

IPC 30.1 ± 1.6 30.5 ± 3.1

Control Rabbit Dialysate 29.5 ± 2.0 49.1 ± 4.8

rIPC Rabbit Dialysate 29.5 ± 1.4 31.9 ± 3.7

Control Human Dialysate 31.0 ± 1.7 47.4 ± 1.9

rIPC Human Dialysate 28.9 ± 1.5 32.4 ± 2.9

Figure 13. Rabbit and Human Dialysate Administered Prior to Ischemia-Reperfusion.

Cardiomyocytes were subjected to 10 min. exposure to either preconditioned rIPC dialysate or

non-preconditioned control dialysate. Results indicate that remotely preconditioned dialysate,

whether rabbit (31.9% ±3.7 vs. 48.0%±2.7 IR, p=0.0002; N=6, n=1 each) or human derived

(32.4%±2.9 vs. 48.0%±2.7 IR, p=0.0001; N=1, n=6), reduces SI/SR induced cell death similar to

classical IPC (30.5%±3.1 vs. 48.0%±2.7 IR, p=0.0006; n=6).

53

5.1.a Dialysate Characterization:

Substances in rabbit control and rIPC dialysate were identified using MRM mass

spectrometry. Opioids such as Met-enkephalin and dynorphin B had no detectable peaks and

Met5-enkephalin-Arg

6-Phe

7 (MEAP) had a concentration of <0.033 µM in control dialysate and

<0.031 µM in rIPC dialysate. Similarly, acetylcholine and angiotensin II were undetectable in

the dialysate. Adenosine in control dialysate was <0.035µM and <0.80 µM in rIPC dialysate.

Inosine was detected at maximum concentrations of 0.583µM in control dialysate and 0.456µM

in rIPC. Bradykinin was <0.0034 µM in control dialysate and was at levels below measurement

in rIPC dialysate. Norepinephrine was <0.021 µM in control dialysate and <0.019 µM in rIPC

dialysate. The above substances were at either undetectable or at minuscule levels that remained

unaffected in either the control or rIPC dialysate (with the exception of adenosine). (See Table 5

for results; see Figure 28 in Appendix V for a plot of the standard concentrations).

Table 5. Characterization of Rabbit Dialysate Using MRM Mass Spectrometry.

Control Rabbit Dialysate (µM) rIPC Rabbit Dialysate (µM)

Met-Enkephalin No peak No peak

Dynorphin B No peak No peak

MEAP No peak – 0.033 No peak – 0.031

Adenosine 0.001-0.035 0.003-0.80

Inosine 0.103-0.583 0.056-0.456

Bradykinin No peak-0.0034 <0

Norepinephrine 0.0087-0.021 0.0080-0.019

Acetylcholine No peak No peak

Angiotensin II No peak No peak

5.1.b Discussion:

Remotely preconditioned (rIPC) rabbit dialysate conferred protection to rabbit

cardiomyocytes on a level similar to IPC. Non-preconditioned (Control) dialysate and

cardiomyocytes subjected to ischemia-reperfusion alone did not provide protection (see Figure

54

13). Thus, there is a protective factor in rIPC dialysate that travels to distant organs through a

humoral route.

Literature on rIPC suggests two mechanisms of transferring protection from one organ to

another. The first is a neuronal pathway, in which substances are released from the stimulus

organ and activate local nerves to release protective factors at the target organ. As evidence for

this pathway, Gho et al.18

demonstrated that ganglion blockade could abolish protection from

mesenteric artery occlusion (MAO). Another method of transferring protection is the humoral

pathway and is the focus of this thesis. Coronary effluent from preconditioned donor hearts

provided protection to untreated virgin hearts in a study by Dickson et al17

. For many substances

implicated in rIPC, there is evidence for a dual neurogenic and humoral pathway which may

work simultaneously in concert or independent of each other16

. However, our model does not

involve the role of the neurogenic pathway, since there is no neuronal involvement in isolated

rabbit cardiomyocytes, and the dialysate used to elicit protection is derived from blood plasma.

Therefore, the protection from rabbit rIPC dialysate indicates that a humoral mechanism alone

can confer protection to distant organs.

Human dialysate can also protect rabbit cardiomyocytes, indicating cross-species

reactivity of the preconditioned dialysate. Though preconditioning has been shown in many

species, very few papers have investigated if protection from one species can elicit protection in

another, i.e. inter-species rIPC, except Shimizu et al. 146

(our data regarding human and rabbit

dialysate to induced protection was presented in this paper) . Since rIPC mechanisms in rabbits

may also apply to humans, cross-species reactivity of preconditioned dialysate is clinically

55

relevant for humans. However, human dialysate from different volunteers are needed to

determine variability in cardioprotection, since control conditions are difficult to enforce in

humans compared to rabbits.

MRM-MS analysis has shown various G-protein coupled receptor ligands are either

undetectable (Met-enkephalin, dynorphin B, acetylcholine, and angiotensin II) or well below

levels that protect in tissues (MEAP, adenosine, bradykinin, and norepinephrine) (see Table 5).

However, this list is a sample survey of molecules that have been suggested in rIPC. For

example, there are a diverse number of opioids and opioid-like molecules that bind to opioid

receptors. To further complicate the matter, Pugsley20

points out that many opioid peptides have

yet to be discovered. Similarly, a number of molecules can bind to a single GPCR.

Unfortunately, an exhaustive survey of substances that bind to G-protein coupled receptors is

beyond the scope of this thesis. However, we did investigate the most common agonist for a

particular receptor through MRM-MS. Considering the above mentioned limitations in

characterizing the dialysate, a better approach would be to investigate the particular receptors

involved in rIPC by blocking their activation.

56

- Chapter 6 -

THE ROLE OF CELL MEMBRANE RECEPTORS IN RIPC

6.1 The Role of Opioid Receptors

6.1.a Survey of Opioid Receptors in Rabbit Cardiomyocytes:

In order to determine the existence of the δ, κ, and µ opioid receptors in cardiomyocytes,

western blotting (n=2) revealed the presence of all three receptors in rabbit whole heart tissues.

The δ and κ subtype were found in rabbit cardiomyocytes, δ was also located in rabbit and rat

brain. The µ subtype was in rabbit brain tissue but absent in rabbit cardiomyocytes, suggesting

the µ subtype is present in neuronal tissue within rabbit whole hearts. (See Figure 14).

57

Figure 14. Western Blot Analysis of Opioid Receptors in Rabbit Tissues.

In order to determine the existence of the δ, κ, and µ opioid receptors in cardiomyocytes, western

blotting (n=2) revealed the presence of all three receptors in rabbit whole heart tissues. The δ and

κ subtype were found in rabbit cardiomyocytes, δ was also located in rabbit and rat brain. The µ

subtype was in rabbit brain tissue but absent in rabbit cardiomyocytes, suggesting the µ subtype

is present in neuronal tissue within rabbit whole hearts.

Survey of Opioid Receptors in Rabbit Cardiomyocytes

58

6.1.b Non-selective Opioid Receptor Blockade of Protection:

The effect of naloxone (a non-selective opioid blocker; 100µM concentration was used;

Takasaki, 1999148

) in conjunction with rabbit dialysate was studied. Blockers of opioid receptor

activity abolished the protection induced by the preconditioned dialysate (43.5%±1.5 vs. 31.3%

± 2.0, p=0.001; N=2, n=4,1). (See Figure 15 and Table 6).

59

Table 6. Naloxone Results (Mean ± SEM).

Group Stabilization End

Baseline 27.4 ± 1.4 28.2 ± 1.5

Baseline + Nal 26.8 ± 0.9 31.0 ± 1.7

Before SI After SR

IR 27.9 ± 2.3 43.0 ± 1.1

IR + Nal 26.7 ± 2.8 45.0 ± 1.6

rIPC Rabbit Dialysate 28.1 ± 1.9 31.3 ± 2.0

rIPC Rabbit Dialysate + Nal 28.7 ± 1.6 43.5 ± 1.5

Figure 15. Naloxone Administered Prior to Rabbit Dialysate.

The effect of naloxone (a non-selective opioid blocker; 100µM concentration was used) in

conjunction with rabbit dialysate was studied. Blockers of opioid receptor activity abolished the

protection induced by the preconditioned dialysate (43.5%±1.5 vs. 31.3% ± 2.0, p=0.001; N=2,

n=4,1).

60

6.1.c δ-Opioid Receptor Blockade of Protection:

The effect of naltrindole (NTI; a δ opioid blocker with a 350 fold selectivity for δ

receptors over κ receptors; 10nM concentration was used on cardiomyocytes; Cao, 2003152

) in

conjunction with rabbit dialysate was studied. Blockers of δ opioid receptor activity completely

abolished the protection induced by the preconditioned dialysate (43.2%±2.0 vs. 29.6%±1.6,

p=0.0003; N=2, n=3,2). (See Figure 16 and Table 7).

61

Table 7. Naltrindole Results (Mean ± SEM).

Group Stabilization End

Baseline 27.4 ± 1.4 29.2 ± 2.4

Baseline + NTI 28.1 ± 1.6 29.3 ± 1.9

Before SI After SR

IR 28.0 ± 1.5 42.4 ± 0.8

IR + NTI 27.6 ± 1.2 45.2 ± 2.1

rIPC Rabbit Dialysate 28.5 ± 1.4 29.6 ± 1.6

rIPC Rabbit Dialysate + NTI 28.8 ± 1.5 43.2 ± 2.0

Figure 16. Naltrindole Administered Prior to Rabbit Dialysate.

The effect of naltrindole (NTI; a δ opioid blocker with a 350 fold selectivity for δ receptors over

κ receptors; 10nM concentration was used on cardiomyocytes) in conjunction with rabbit

dialysate was studied. Blockers of δ opioid receptor activity completely abolished the protection

induced by the preconditioned dialysate (43.2%±2.0 vs. 29.6%±1.6, p=0.0003; N=2, n=3,2).

62

6.1.d κ-Opioid Receptor Blockade of Protection:

The effect of GNTI (a κ selective opioid blocker with an 800 fold selectivity for κ

receptors over δ receptors; 1nM concentration was used; Cao, 2003152

) in conjunction with rabbit

dialysate was studied. Blockers of κ-opioid receptor activity completely abolished the protection

induced by the preconditioned dialysate (46.8%±2.7 vs. 29.6%±1.6, p=0. 0002; N=2, n=3, 2).

(See Figure 17 and Table 8).

63

Table 8. GNTI Results (Mean ± SEM).

Group Stabilization End

Baseline 27.4 ± 1.4 29.2 ± 2.4

Baseline + GNTI 30.9 ± 0.4 32.0 ± 1.3

Before SI After SR

IR 28.0 ± 1.5 42.4 ± 0.8

IR + GNTI 26.5 ± 0.9 45.0 ± 2.3

rIPC Rabbit Dialysate 28.0 ± 1.5 29.6 ± 1.6

rIPC Rabbit Dialysate + GNTI 29.3 ± 1.3 46.8 ± 2.7

Figure 17. GNTI Administered Prior to Rabbit Dialysate.

The effect of GNTI (a κ selective opioid blocker with an 800 fold selectivity for κ

receptors over δ receptors; 1nM concentration was used) in conjunction with rabbit

dialysate was studied. Blockers of κ-opioid receptor activity completely abolished the

protection induced by the preconditioned dialysate (46.8%±2.7 vs. 29.6%±1.6, p=0.0002;

N=2, n=3, 2).

64

6.1.e Discussion:

Literature regarding µ opioid receptors confirms that this receptor subtype is not present

in rabbit cardiomyocytes24,25

. Also, there is indirect evidence that denies the involvement of µ

receptors in preconditioning. Fentanyl (a selective µ agonist) administered during

preconditioning was unable to abolish protection in Langendorff perfused rabbit hearts164

.

There is little surprise regarding the involvement of opioids in rIPC. Multiple studies

have suggested that naloxone (a non-selective opioid antagonist) can abolish protection across

species and in different organs. Dickson et al. 33

used naloxone to blocked protection from

preconditioned coronary effluent in untreated rabbit hearts. Patel et al.35

determined that MAO-

induced cardioprotection was blocked by naloxone in rats. Weinbrenner et al.37

attests to a

similar findings when failing to precondition isolated rat hearts in the presence of naloxone.

However, controversy lies in which receptor subtype is involved in cardioprotection. In

our isolated rabbit cardiomyocyte model, both δ and κ opioid receptors are involved in a humoral

mechanism of rIPC (see Figure 16 and Figure 17). Interestingly, Cao et al.152

has also shown that

both Met-enkephalin (δ opioid agonist) and dynorphin B (κ opioid agonist) administered before

the index ischemia can protect isolated rabbit cardiomyocytes. Naltrindole (δ receptor blocker)

and GNTI (κ receptor blocker) administered during classical IPC abolished this protection. Also,

Weinbrenner et al.37

has observed the involvement of δ1 in isolated rat hearts. More recently,

Zhang et al.36

has reported the involvement of only κ receptors in rIPC when δ receptor blockers

failed to abolish protection from femoral artery occlusion (FAO). However, the authors

65

attributed the controversy of their findings to variations with the experimental models and

species used when compared to previous studies.

Another reason for the discrepancies regarding opioid receptor subtype involvement is

promiscuity of opioid peptides and heterodimerization of receptors. Opioids have the ability to

bind to multiple receptor subtypes20

. Also, many endogenous peptides that are subtype-specific

can operate as non-selective agonists at higher concentrations. The δ-specific Met-enkephalin

and the µ-specific morphine are such examples. The versatility of endogenous opioid peptides to

bind to multiple receptors can be explained by the high degree of homology among receptor

subtypes. The µ subtype is 58% and 67% homologous to δ and κ receptively, whereas δ and κ

share 61% homology165

. Another explanation for the involvement of both δ and κ opioid

receptors is the evidence of dimerization across and within different subtypes. There is evidence

of hybrid δ and µ receptors that have unique structures, pharmacology, and functions upon

activation by either δ or µ-specific agonists166

.

The results so far suggest the involvement of δ and κ opioid receptors in mediating a

humoral mechanism of rIPC protection. However, dialysate characterization using MRM-MS

was unable to detect Met-enkephalin, dynorphin B, or significant levels of the more common

endogenous opioid peptide in coronary effluent, MEAP. Therefore, at least one of the trigger

molecule(s) in dialysate is/are opioid-like in nature. However, since the factor(s) is/are not these

common endogenous peptides, this provides room for discovering a novel/unfamiliar opioid

involved in both δ and κ receptor activation in rIPC.

66

6.2 The Role of Bradykinin B2 Receptors

6.2.a Bradykinin B2 Receptor Blockage Conserves Protection:

The effect of Hoechst (HOE140) (a bradykinin B2 receptor blocker; 5µM concentration

was used; Schoemaker, 200067

) in conjunction with rabbit dialysate was studied. Blockers of

bradykinin B2 receptor activity conserved the protection induced by preconditioned rIPC

dialysate (31.2%±3.0 vs. 26.9%±2.2, p=0.60; N=2, n=4, 1). (See Figure 18 and Table 9).

67

Table 9. HOE140 Results (Mean ± SEM).

Group Stabilization End

Baseline 28.9 ± 3.1 26.5 ± 1.7

Baseline + HOE140 26.9 ± 2.0 28.3 ± 2.1

Before SI After SR

Control Rabbit Dialysate 24.5 ± 1.3 41.3 ± 1.1

Control Rabbit Dialysate + HOE140 26.1 ± 2.2 44.6 ± 1.9

rIPC Rabbit Dialysate 25.1 ± 1.5 26.9 ± 2.2

rIPC Rabbit Dialysate + HOE140 26.2 ± 1.6 31.2 ± 3.0

Figure 18. HOE140 Administered Prior to Rabbit Dialysate.

The effect of Hoechst (HOE140) (a bradykinin B2 receptor blocker; 5µM concentration was

used) in conjunction with rabbit dialysate was studied. Blockers of bradykinin B2 receptor

activity conserved the protection induced by preconditioned rIPC dialysate (31.2%±3.0 vs.

26.9%±2.2, p=0.60; N=2, n=4, 1).

68

6.2.b Discussion:

Blockage with HOE140 did not abolish rIPC dialysate protection, suggesting that

bradykinin B2 receptors are not involved (see Figure 18). To reiterate this finding, levels of

bradykinin were detected at minuscule levels using MRM-MS (see Table 5).

According to Schoemaker et al.67

, MAO-induced rIPC of the myocardium was abolished

when HOE140 (bradykinin B2 receptor blocker) was administered during early reperfusion in in

vivo rats. Also, infusion of bradykinin into the mesenteric artery resulted in cardioprotection, but

was subsequently abolished by the ganglion blocker, hexamethonium. Thus, bradykinin is

thought to exert its effects on distant organs through a dual neurogenic and humoral (since

plasma bradykinin levels increase following ischemia55

) pathway. Once the neurogenic pathway

is blocked, there may be insufficient bradykinin from the humoral pathway to evoke protection at

the target organ. Knocking out one pathway may be enough to abolish protection entirely, as

protection from ischemia-reperfusion injury is commonly an all-or-none phenomenon that

requires a threshold to be met in order to induce protection according to Goto et al.3. To confirm

that dialyzing blood plasma did not dilute bradykinin concentrations (dialysate is 10x diluted

compared to plasma), dialysate was lyophilized into a powder and reconstituted with 1x Krebs-

Henseleit buffer such that dialysate concentrations were comparable to rabbit plasma. This

reconstituted dialysate still protected cardiomyocytes from ischemia-reperfusion damage and

HOE140 did not abolish this protection (N=1, n=1; see Table 10). Thus, in isolated rabbit

cardiomyocytes, the bradykinin B2 receptor and bradykinin molecule do not play a role in

humorally-mediated rIPC nor cross-talk with other receptors.

69

Table 10. 1x Dialysate with HOE140 Results (% Cardiomyocyte Death).

Group Stabilization End

Baseline 24.3 26.4

Before SI After SR

1x Control Rabbit Dialysate 24.9 40.8

1x Control Rabbit Dialysate + HOE140 25.3 34.5

1x rIPC Rabbit Dialysate 25.5 24.4

1x rIPC Rabbit Dialysate + HOE140 24.7 27.3

70

6.3 The Role of CGRP Receptors

6.3.a Existence of Calcitonin-like Receptors in Rabbit Cardiomyocytes:

The presence of calcitonin-like receptors (CLRs) in rabbit cardiomyocytes was

investigated using western blotting (n=1). CLRs were found in rabbit whole hearts, isolated

cardiomyocytes, and cardiomyocyte particulate fractions (i.e. the mitochondrial membrane was

excluded). (See Figure 19).

Figure 19. Western Blot Analysis of Calcitonin-Like Receptors in Rabbit Tissues.

The presence of calcitonin-like receptors (CLRs) in rabbit cardiomyocytes was investigated

using western blotting (n=1). CLRs were found in rabbit whole hearts, isolated cardiomyocytes,

and cardiomyocyte particulate fractions (i.e. the mitochondrial membrane was excluded).

Existence of Calcitonin-Like Receptors in Rabbit Cardiomyocytes

71

6.3.b CGRP Receptor Blockage Conserves Protection:

The effect of CGRP (8-37) (a CGRP receptor blocker; 5nM concentration was used;

Wolfrum, 2005103

) in conjunction with rabbit dialysate was studied. Blockers of CGRP receptor

activity conserved the protection induced by the preconditioned dialysate (24.8%±1.9 vs.

26.0%±3.3, p=0.96; N=2, n=2, 2). (See Figure 20 and Table 11).

72

Table 11. CGRP (8-37) Results (Mean ± SEM).

Group Stabilization End

Baseline 23.4 ± 2.2 23.3 ± 2.6

Baseline + CGRP (8-37) 22.6 ± 7.7 21.8 ± 4.0

Before SI After SR

IR 21.7 ± 2.3 41.6 ± 2.0

rIPC Rabbit Dialysate 22.4 ± 3.3 26.0 ± 3.3

rIPC Rabbit Dialysate + CGRP (8-37) 23.4 ± 2.7 24.8 ± 1.9

Figure 20. CGRP (8-37) Administered Prior to Rabbit Dialysate.

The effect of CGRP (8-37) (a CGRP receptor blocker; 5nM concentration was used) in

conjunction with rabbit dialysate was studied. Blockers of CGRP receptor activity conserved the

protection induced by the preconditioned dialysate (24.8%±1.9 vs. 26.0%±3.3, p=0.96; N=2,

n=2, 2).

73

6.3.c Discussion:

Calcitonin-like receptors (CLRs) have been identified in humans and rat smooth muscle

cells87,

88

. However, calcitonin-like receptors in rabbit tissues and in particular, rabbit

cardiomyocytes have not been studied. Western blotting indicates that CLRs are present in rabbit

whole heart, cardiomyocyte lysate, and the particulate fraction (with mitochondria excluded) (see

Figure 19). Even though this is a significant finding, there are limitations to concluding that

CGRP receptors exist on rabbit cardiomyocytes. The CGRP receptor is a receptor complex that

consists of the CLR and associated receptor activity-modifying proteins (RAMPs). CLR

association with RAMP1 and RAMP3 allows binding of CGRP to the receptor, resulting in CLR

becoming a CGRP receptor85

. CLR association with RAMP2 permits the binding of another

cardioprotective factor, adrenomedullin84

. Thus, in order to conclusively determine the existence

of CGRP receptors on rabbit cardiomyocytes, it is important to establish that RAMP1 and

RAMP3 associate with this CLR.

CGRP (8-37) blocked CGRP receptors and did not affect rIPC dialysate protection,

suggesting that the CGRP receptor is not involved in humoral rIPC (see Figure 20). In addition,

CGRP (8-37) has been used to abolish the protective effects of adrenomedullin167

in rat

cardiomyocytes. Therefore, the results presented in Figure 20 used CGRP (8-37) to block all

CLRs on rabbit cardiomyocytes. CGRP (8-37) was also administered to isolated rabbit

cardiomyocytes under basal conditions (i.e. the Baseline + CGRP (8-37) treatment group). Since

the CGRP blocker did not increase cell necrosis, this antagonist was deemed as non-toxic in

rabbit cardiomyocytes.

74

The claim by Wolfrum et al.103

regarding CGRP and rIPC cardioprotection suggests the

involvement of a dual neurogenic and humoral pathway. The authors determined that infusion of

CGRP could mimic MAO-preconditioned cardioprotection in rats. MAO preconditioning with

CGRP (8-37) abolished protection. Also, CGRP-induced protection was abolished by

hexamethonium (a ganglion blocker).Throughout MAO preconditioning, the level of CGRP was

found to be elevated by radioimmunoassay regardless of ganglion blockade. However, Wolfrum

et al. did not investigate the role of CGRP in a humoral model alone. Since CGRP is released by

caspasin sensitive sensory nerves, most likely this peptide‘s mechanism of action occurs through

a neurogenic pathway. Thus, there may be insufficient CGRP circulating in the blood following

preconditioning to induce cardioprotection. In addition, the work regarding CGRP is in remote

preconditioning of the intestine97,103

in rats to induced cardioprotection, suggesting that CGRP

may play a unique role when the intestine as the preconditioning organ but not in skeletal muscle

ischemia.

6.4 The Role of Adenosine Receptors

6.4.a Non-selective Adenosine Receptor Blockade of Protection:

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration

was used; Armstrong, 1995120

) in conjunction with rabbit dialysate was studied. Blockers of

adenosine receptor activity abolished the protection induced by the preconditioned dialysate

(41.6%±2.2 vs. 28.1%±1.7, p=0 0002; N=2, n=5,1). (See Figure 21 and Table 12).

75

Table 12. 8-SPT Results (Mean ± SEM).

Group Stabilization End

Baseline 26.9 ± 2.3 27.3 ± 0.9

Baseline + SPT 25.2 ± 1.3 26.7 ± 2.2

Before SI After SR

Control Rabbit Dialysate 25.6 ± 1.2 43.7 ± 1.4

Control Rabbit Dialysate + SPT 24.5 ± 1.4 42.3 ± 0.8

rIPC Rabbit Dialysate 25.3 ± 1.4 28.1 ± 1.7

rIPC Rabbit Dialysate + SPT 26.5 ± 1.5 41.6 ± 2.2

Figure 21. 8-SPT Administered Prior to Rabbit Dialysate.

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration

was used) in conjunction with rabbit dialysate was studied. Blockers of adenosine

receptor activity abolished the protection induced by the preconditioned dialysate

(41.6%±2.2 vs. 28.1%±1.7, p=0.0002; N=2, n=5,1).

76

6.4.b Discussion:

My results indicate the involvement of adenosine in rIPC since 8-SPT (a non-selective

adenosine blocker) was able to abolish protection from rIPC dialysate (see Figure 21). To

support this finding, Pell et al.128

suggested the involvement of adenosine in rabbit renal artery

occlusion (RAO). Also, Liem et al.127

confirmed this in rat mesenteric artery occlusion (MAO)

to induce cardioprotection. However, these studies have suggested a neurogenic model for rIPC

in renal and mesenteric preconditioning. Pang et al.129

demonstrated that skeletal muscle

ischemia increased plasma adenosine concentrations and inhibition of vesicular monoamine

transport (inhibition of catecholamines at synaptic nerve endings) partially abolished

preconditioning. Thus, it is possible that skeletal muscle ischemia releases adenosine that

activates local nerves, which in turn affect distant organs like the myocardium through a dual

neurogenic/humoral pathway. However, the protective factor in rIPC dialysate is humoral in

nature and this alone can protect isolated cardiomyocytes without the need of neuronal

intervention (see Figure 13).

According to Pang et al.129

, levels of adenosine in the plasma were elevated during

skeletal muscle ischemia, hinting at this purine‘s involvement in a humoral pathway. However,

the half-life of adenosine is very short (0.6-1.5 sec in plasma) due to continual breakdown by

circulating adenosine deaminase140

. To support this finding, the level of adenosine was

determined to be <0.80µM by MRM-MS in rIPC dialysate (see Table 5). However, adenosine

concentrations are required to be >10uM to protect isolated rabbit cardiomyocytes according to

Armstrong et al.120

. Based on the above evidence, the involvement of the adenosine molecule in

77

a humoral route is unlikely. Therefore, the blockage of dialysate protection with 8-SPT (Figure

21) suggests the involvement of the adenosine receptor and not the adenosine molecule.

A number of controversies exist with the role of adenosine in IPC and rIPC. Auchampach

et al.168

demonstrated that administration of three A1 antagonists (DPCPX, BG9719, BG9928) in

dogs throughout preconditioning until before index ischemia did not block protection. Mice with

knockouts for adenosine receptors also show contradictory results. Lankford et al.169

could not

precondition A1 knockouts, however Guo et al.170

demonstrated that A3 knockout mice are still

capable of preconditioning, thus illustrating the importance of A1 receptors in IPC. However, a

study conducted by Eckle et al.171

demonstrated that A1, A3, and A2a knockouts had preserved

protection following ischemia. It was A2b receptors alone that could not be preconditioned

through IPC. To explain these controversies, a review by Cohen and Downey14

suggests the

involvement of multiple receptor agonists acting in parallel during IPC. This also suggests the

involvement of other receptors in the form of cross-talk.

78

- Chapter 7 -

RIPC MEDIATES OPIOID-ADENOSINE CROSS-TALK

7.1 Opioid-Adenosine Cross-talk

7.1.a Adenosine Deamination of the Dialysate Conserves Protection:

The effect of adenosine deamination of dialysate was studied. Rabbit dialysate was

passed through a column containing adenosine deaminase (ADA) coupled to beads‡. Levels of

adenosine and inosine before and after deamination were measured using MRM mass

spectrometry to ensure complete elimination of adenosine from the dialysate. The adenosine

deamination of the dialysate conserved the protection induced by rIPC dialysate§ (34.9% vs.

33.3% and 30.1% vs. 31.8%, n=2). (See Table 13).

NOTE: These experiments were originally conducted with an n=3.However, adenosine in

rIPC dialysate from one experiment was not deaminated, thus this experiment was excluded.

‡ Dialysate deamination was conducted by Amy Yeung (Dr. John Callahan‘s Lab; Department of Paediatric

Laboratory Medicine, Hospital for Sick Children). § These experiments were conducted by Alina Hinek (Dr. Wilson‘s Lab, Hospital for Sick Children).

79

Table 13. ADA Results (% Cardiomyocyte Death).

Experiment A Stabilization End

Baseline 24.1 35.1

Before SI After SR

Control Rabbit Dialysate 24.3 42.6

rIPC Rabbit Dialysate 28.4 34.9

rIPC Rabbit Dialysate + ADA 31.1 33.3

Pre-ADA (µM) Post-ADA (µM)

Adenosine in rIPC Rabbit Dialysate 0.01 No peak

Inosine in rIPC Rabbit Dialysate 0.22 0.39

Experiment B Stabilization End

Baseline 26.6 33.6

Before SI After SR

Control Rabbit Dialysate 24.0 45.0

rIPC Rabbit Dialysate 29.5 30.1

rIPC Rabbit Dialysate + ADA 30.1 31.8

Pre-ADA (µM) Post-ADA (µM)

Adenosine in rIPC Rabbit Dialysate 0.01 No peak

Inosine in rIPC Rabbit Dialysate 0.26 0.20

80

7.1.b Partial Adenosine Blockade of δ-Opioid Receptor-Induced Protection:

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration

was used; Armstrong, 1995120

) in conjunction with Met-enkephalin (a δ-opioid receptor agonist,

100µM; Cao, 2003152

) was studied. The adenosine receptor blocker partially abolished the

protection induced by δ -opioid receptor activation (41.8%±1.5 vs. 32.8%±1.0 compared to ME,

p=0.0007, and vs. 50.7%±1.1 compared to IR, p=0.0008; n=5). (See Figure 22 and Table 14).

81

Table 14. Met-Enkephalin with 8-SPT Results (Mean ± SEM).

Group Stabilization End

Baseline 29.3 ± 0.8 31.2 ± 0.7

Baseline + ME 23.9 ± 0.0 21.7 ± 0.0

Before SI After SR

IR 28.6 ± 1.3 50.7 ± 1.1

ME 30.6 ± 1.9 32.8 ± 1.0

ME + 8-SPT 29.4 ± 0.8 41.8 ± 1.5

ME + NTI 33.4 ± 0.0 51.7 ± 0.0

Figure 22. 8-SPT Administered Prior to Met-Enkephalin.

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration was

used) in conjunction with Met-enkephalin (a δ-opioid receptor agonist, 100µM) was studied. The

adenosine receptor blocker partially abolished the protection induced by δ -opioid receptor

activation (41.8%±1.5 vs. 32.8%±1.0 compared to ME, p=0.0007, and vs. 50.7%±1.1 compared

to IR, p=0.0008; n=5).

82

7.1.c Complete Adenosine Blockade of κ -Opioid Receptor-Induced Protection:

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration

was used; Armstrong, 1995120

) in conjunction with dynorphin B (a κ-opioid receptor agonist,

100µM; Cao, 2003152

) was studied. The adenosine receptor blocker abolished the protection

induced by κ-opioid receptor activation (44.3%±2.6 vs. 31.8%±2.1 compared to DynB, p=0.02,

and vs. 51.3%±5.0 compared to IR, p=0.17; n=5). (See Figure 23 and Table 15).

83

Table 15. Dynorphin B with 8-SPT Results (Mean ± SEM).

Group Stabilization End

Baseline 26.4 ± 1.1 27.2 ± 2.4

Baseline + DynB 19.2 ± 0.0 22.4 ± 0.0

Before SI After SR

IR 29.1 ± 2.1 51.3 ± 5.0

DynB 28.7 ± 1.8 31.8 ± 2.1

DynB + 8-SPT 30.5 ± 1.9 44.3 ± 2.6

DynB + GNTI 29.1 ± 1.6 48.3 ± 3.1

Figure 23. 8-SPT Administered Prior to Dynorphin B.

The effect of 8-SPT (a non-selective adenosine receptor blocker; 100µM concentration was

used) in conjunction with dynorphin B (a κ-opioid receptor agonist, 100µM) was studied. The

adenosine receptor blocker abolished the protection induced by κ-opioid receptor activation

(44.3%±2.6 vs. 31.8%±2.1 compared to DynB, p=0.02, and vs. 51.3%±5.0 compared to IR,

p=0.17; n=5).

84

7.1.d Discussion:

Preconditioned dialysate was exposed to adenosine deaminase (ADA) conjugated beads

in order to remove any trace of adenosine. The results of our study confirm that adenosine

removal from the rIPC dialysate maintains protection, indicating with certainty that the

protective factor(s) is/are not adenosine (see Table 13). ADA-conjugated beads were utilized in

order to remove ADA prior to cardiomyocyte exposure, thus avoiding deamination of vital

adenosine released during the mediator phase of preconditioning. To summarize the results so

far: the half-life of adenosine in plasma is 0.6-1.5sec140

, MRM-MS detected low levels of

adenosine in the dialysate (see Table 5), and rIPC protection was conserved even in the absence

of adenosine through adenosine deamination. However, rIPC dialysate protection was abolished

by the adenosine receptor blocker, 8-SPT (see Figure 21). Based on this accumulating evidence,

there is involvement of adenosine receptors in rIPC but not the adenosine molecule itself.

ADA also increases inosine concentrations during the breakdown of adenosine. Jin et

al.172

has reported that inosine increases mast cell degranulation during ischemia or

inflammation. The same study demonstrated that inosine can bind only to adenosine A3 receptors

and not A1 or A2a. Recently, Shen et al.173

suggested that inosine preconditions rats from cerebral

brain injury via adenosine A3 receptors. Litsky et al.174

found inosine protects mouse neuronal

and glial cell cultures from hypoxia. The authors constructed a concentration response curve for

inosine and determined the minimum concentration to protect neuronal cells is 500µM in rats.

However, the level of inosine in rIPC dialysate is <0.26µM and upon deamination of adenosine,

inosine levels elevated to <0.39µM (see Table 13). Therefore, the protection seen after adenosine

deamination is not due to inosine.

85

The non-selective adenosine blocker 8-SPT partially abolished protection from Met-

enkephalin (δ-specific opioid agonist) (see Figure 22). The activation of κ receptors by

dynorphin B (κ-specific opioid agonist) was fully abolished by 8-SPT since there was no

statistical significance between ischemia-reperfusion alone and administration of dynorphin B

with 8-SPT (see Figure 23). However, increased number of experiments may result in dynorphin

B exhibiting partial protection similar to Met-enkephalin. Regardless, these studies have

confirmed cross-talk between δ and κ opioid receptors with adenosine receptors during the

trigger phase of classical IPC.

Interestingly, Peart et al.138

suggests that cross-talk between opioid and adenosine

receptors work both ways. However, our rIPC dialysate model does not involve adenosine due to

its deamination in the blood. Also, preliminary data from our lab indicates that cross-talk is

unidirectional (n=2, see Table 16). This may be due to species differences or due to experimental

models since Peart et al. utilized an in vivo rat model.

86

Table 16. Adenosine with Naloxone Results (% Dead Cardiomyocytes).

Experiment A Stabilization End

Baseline 19.2 22.1

Baseline + Adenosine 19.3 22.1

Before SI After SR

IR 26.8 52.9

Adenosine 25.1 29.0

Adenosine + Nal 23.3 31.0

Adenosine + SPT 25.3 45.1

Experiment B Stabilization End

Baseline 26.9 29.0

Before SI After SR

IR 26.2 36.2

Adenosine 28.4 36.2

Adenosine + Nal 26.9 37.6

One explanation, suggested by Peart et al.139

, of opioid-adenosine cross-talk is the

adenosine kinase inhibition hypothesis. Adenosine kinase degrades adenosine into AMP within

cardiomyocytes. According to the authors, activation of opioid receptors inhibits this enzyme,

which leads to an accumulation of intracellular adenosine. Since diffusion of adenosine across

the cell membrane is dependent on a concentration gradient, extracellular adenosine remains

outside cardiomyocytes. The build-up of adenosine activates adenosine receptors and results in

ischemic preconditioning. Therefore, a vital component of the adenosine kinase inhibition

hypothesis is the amassing of extracellular adenosine.

7.2 The Inhibition of Adenosine Kinase Hypothesis

7.2.a Exposure to Dynorphin B Does Not Accumulate Extracellular Adenosine:

The inhibition of adenosine kinase to increase levels of extracellular adenosine was

measured using MRM mass spectrometry. Supernatants from freshly isolated rabbit

cardiomyocytes were obtained after stabilization, dynorphin B (a κ-opioid receptor agonist,

87

100µM; Cao, 2003152

) exposure, and after a wash period. Levels of adenosine did not change

significantly (contained the following in µM: 1.54±0.5 after stabilization, 0.96±0.1 after DynB

exposure, 1.14±0.1 after wash; n=5) and were well below 10µM (the minimum concentration of

adenosine required to protect cardiomyocytes; Armstrong, 1995120

). (See Figure 24 and Table 17

for results; see Figure 29 in Appendix V for a plot of the standard concentrations).

88

Table 17. Adenosine Concentrations After Exposure to Dynorphin B Results (Mean ±

SEM).

Group Concentration (µM)

After Stabilization 1.54 ± 0.5

After DynB 0.96 ± 0.1

After Wash 1.14 ± 0.1

Figure 24. Adenosine Concentrations After Exposure to Dynorphin B.

The inhibition of adenosine kinase to increase levels of extracellular adenosine was measured

using MRM mass spectrometry. Supernatants from freshly isolated rabbit cardiomyocytes were

obtained after stabilization, dynorphin B (a κ-opioid receptor agonist, 100µM) exposure, and

after a wash period. Levels of adenosine did not change significantly (contained the following in

µM: 1.54±0.5, 0.96±0.1, 1.14±0.1; n=5) and were below 10µM (the minimum concentration of

adenosine required to protect cardiomyocytes).

89

7.2.b Discussion:

Supernatant was collected before, during and after cardiomyocytes were exposed to

dynorphin B (DynB, κ-selective agonist) and immediately placed on ice to prevent deamination

of adenosine. The same MRM-MS method used to detect adenosine in the dialysate (see Table 5)

was used to measure adenosine concentrations in supernatant samples (see Figure 24). The

results indicate that adenosine concentrations do not change after DynB exposure and are well

below protective levels (<10µM). Therefore, there is no increase in extracellular adenosine as a

result of opioid receptor stimulation during the trigger phase of IPC.

Another method of increasing interstitial adenosine concentrations is through inhibition

of adenosine deaminase which is normally situated outside cardiomyocytes. Silva et al.175

examined the effects of an adenosine deaminase inhibitor (pentostatin) on myocardial infarction

in dogs. Pentostatin increased plasma adenosine levels 3.5-fold above basal conditions prior to

ischemia and sustained these levels until early reperfusion. However, this attenuation of

adenosine had no effect on infarct size and did not precondition myocardium.

Peart et al.138

in 2003 first noted a relationship between opioid and adenosine receptors in

in vivo cardioprotection in rats. Rats were pre-treated with morphine (a non-selective opioid

agonist) and 2-chloro-cyclopentyladenosine (CCPA, adenosine A1 agonist) throughout the

experiment and this mimicked preconditioning by coronary artery occlusion. This protection was

abolished by the δ1 opioid blocker, 7-benzylidenealtrexone (BNTX). Based on these findings,

Peart et al. concluded receptor cross-talk and/or parallel signalling pathways downstream of

opioid and adenosine receptors. Later in 2005, Peart et al.139

postulated that the opioid-adenosine

90

cross-talk seen in 2003 was attributed to inhibition of adenosine kinase in cardiomyocytes (see

Figure 3). In this study, rats were treated in vivo with the adenosine kinase inhibitor, 5-

iodotubercidin which protected myocardium from damaging CAO. 8-Cyclopentyl-1,3-

dipropylxanthine (DPCPX, adenosine A1 blocker) and MRS1523 (adenosine A3 blocker)

abolished this protection. In addition, the δ1 opioid blocker, BNTX, also eliminated protection.

However, my results measuring adenosine in cardiomyocyte supernatant samples do not

support this hypothesis. This may be due to differences in experimental protocols. For example,

our isolated rabbit cardiomyocyte model was not in vivo and therefore, independent of many

factors such as neuronal tissue in the whole heart. Also, Peart et al. administered adenosine

kinase inhibitors throughout the experimental protocol which may have allowed time for

adenosine kinase inhibition and accumulation of extracellular adenosine, whereas my

cardiomyocyte model only treated cells with an opioid agonist during the trigger phase of IPC.

Most importantly, Peart et al.139

determined that δ1 opioid receptor inhibitors abolished

protection induced by adenosine kinase inhibition. However, this finding contradicts their

proposed hypothesis. The model undergoes the following steps: (1) activation of opioid

receptors, (2) which inhibits adenosine kinase, (3) leading to extracellular adenosine

accumulation, (4) resulting in cardioprotection. Inhibition of opioid receptors eliminates step (1),

however, since an adenosine kinase inhibitor is still present throughout the procedure, steps (2) –

(4) are still possible and should result in cardioprotection. Nonetheless, Peart et al. states that this

is only a tentative hypothesis that requires further investigation and proposes other possibilities

91

such as heterodimerization of opioid and adenosine receptor or parallel signalling pathways

downstream of these receptors.

92

- Chapter 8 -

SUMMARY OF FINDINGS

The data presented confirms these major findings:

1. Remotely preconditioned dialysate (both human and rabbit derived) protect

cardiomyocytes against ischemia-reperfusion injury to the same extent as classical IPC.

2. Compounds that precondition in IPC are either undetectable (Met-enkephalin, dynorphin

B, bradykinin, acetylcholine, angiotensin II) or at orders of magnitude below the

threshold to confer protection (MEAP, adenosine, inosine, norepinephrine) in rIPC

dialysate.

3. Cardioprotection induced by rIPC dialysate requires activation of δ or κ opioid receptors

or adenosine receptors.

4. Cardioprotection induced by rIPC dialysate does not require the activation of bradykinin

B2 or CGRP receptors.

5. The adenosine molecule does not play a role in rIPC dialysate protection, but the

adenosine receptor does.

6. Protection induced by κ or δ opioid receptors is fully or partially abolished by non-

selective adenosine blockade. This indicates cross-talk between these receptors.

7. The cross-talk between opioid and adenosine receptors is not due to accumulation of

extracellular endogenous adenosine.

These findings support the hypothesis that δ or κ opioid receptors, through cross-talk with

adenosine receptors, act as possible triggers in remote ischemic preconditioning.

93

- Chapter 9 -

GENERAL DISCUSSION

9.1 Overall Perspective

The investigations conducted in this thesis have determined that rIPC can be mediated

from rabbit or human skeletal muscle to rabbit cardiomyocytes through a humoral pathway (see

Figure 13). Also, there is involvement of δ and κ opioid receptors33

(see Figures 15, 16, & 17) in

the target organ, but this is not mediated by molecules (Met-enkephalin, dynorphin B, MEAP,

adenosine, inosine, bradykinin, acetylcholine, norepinephrine, and angiotensin II) that have been

widely involved in classical IPC (see Table 5). In addition, claims have been made regarding

bradykinin B267

(see Figure 18) and CGRP103

(see Figure 20) receptors, but these do not play a

role in our isolated cardiomyocyte model. Interestingly, adenosine receptors128

have also been

implicated in rIPC (see Figure 21). However, the level of adenosine in rIPC dialysate and

subsequent deamination (Table 13), excludes the involvement of the adenosine molecule to

explain these results. However, I determined there is cross-talk between δ and κ opioid receptors

with adenosine receptors (see Figures 22 & 23) to explain these results. However, this

relationship is not due to the inhibition of adenosine kinase139

increasing extracellular adenosine

concentrations (see Figure 24).Thus, this cross-talk may occur through heterodimerization of

opioid and adenosine receptors (See Figure 25)

94

Figure 25. General Diagram of Opioid-Adenosine Cross-talk.

The involvement of δ and κ opioid and adenosine receptors has been determined to play a role in

humorally-mediated rIPC. However, the level of adenosine in rIPC dialysate and subsequent

deamination excludes the involvement of the adenosine molecule to explain these results. This can

be explained through cross-talk between δ and κ opioid and adenosine receptors. However, this

relationship is not due to the inhibition of adenosine kinase increasing extracellular adenosine

concentrations. Thus, this cross-talk may occur through heterodimerization of opioid and

adenosine receptors.

Cross-talk

Adenosine

Receptor

Opioid

Receptor

Opioid-

Adenosine

Receptor

Opioid Opioid Adenosine

Activate cell signalling pathways

95

9.2 Other Mechanisms of Opioid-Adenosine Cross-talk

9.2.a Dimerization of G-protein Coupled Receptors:

Heterodimerization among G-protein coupled receptors has been implicated in opioid-

adenosine ‗cross-talk‘138

. Heterodimerization between G-protein coupled receptors is a well

documented phenomenon that can lead to unique pharmacological properties such as altered

receptor sensitivity, endogenous adenosine release, ligand binding and signalling. Also, these

uniquely dimerized receptors may bind to orphan peptides that have yet to be identified.166,176,177

In addition, there is evidence that opioid receptors dimerize with β2-adrenergic and

somatostatin receptors. Jordan et al.178

studied oligomerization between these distant GPCR

family members: δ and κ opioid receptors with β2-adrenergic receptors. Human embryonic

kidney (HEK)-293 cells were cotransfected with anti-myc tagged opioid receptors and anti-Flag

tagged β2-adrenergic receptors. Immunoprecipitation determined that these receptors associate

with each other. Though these receptors did not display altered ligand binding, there were

changes in protein trafficking such as endocytosis. Coimmunoprecipitation of sst(2A)

somatostatin and µ1 opioid receptors were found to heterodimerize in HEK-293 cells by Pfeiffer

et al. 179

. Binding of either sst(2A) or µ ligands altered receptor phosphorylation and

desensitization of this hybrid receptor.

Adenosine A1 receptors can form dimers with P2Y1 and dopamine D1 receptors. Gines et

al. 180

cotransfected cultured mouse Ltk- fibroblasts and rat embryonic cortical neurons with

adenosine A1, dopamine D1, and dopamine D2 human cDNA. Coimmunoprecipitation and

colocalization with immunofluorescence determined that A1 heterodimerized with D1 receptors

96

but not D2. The authors determined that heterodimerization of these receptors resulted in receptor

desensitization. Yoshioka et al.181

extensively investigated in vivo heterodimerized P2Y1 and

adenosine A1 receptors in rat brain tissues and cortical neurons using immunofluorescence and

coimmunoprecipitation. In addition, adenosine A2 receptors exist as naturally occurring

oligomers with dopamine D2 receptors182

. Though GPCR heterodimerization has been described

for both adenosine receptors and opioid receptors individually, there is currently no study that

investigates heterodimerization of opioid and adenosine receptors with each other.

9.3 Future Directions

The further directions of these studies will be to further investigate the mechanism of

‗cross-talk‘ between opioid and adenosine receptors. Studies will involve investigating another

mechanism of opioid-adenosine cross-talk, i.e. heterodimerization of G-protein protein receptors.

In addition, cell signalling downstream of G-protein coupled cardiomyocyte cell membrane

receptors in rIPC and opioid-adenosine cross-talk is another logical avenue for further

investigation.

9.3.a Specific Adenosine Receptor Subtypes in Cross-talk & rIPC:

Before determining if opioid and adenosine receptors heterodimerize, it is important to

elucidate which adenosine receptor subtype(s) cross-talk(s) with δ and κ opioid receptors. To

investigate this, cardiomyocytes will be subjected to 10 min classic IPC, specific opioid receptor

agonists, such as Met-enkephalin (δ-specific agonist) or dynorphin B (κ-specific agonist) in the

presence of adenosine receptor subtype blockers, such as DPCPX (A1 adenosine blocker),

MRS1523 (A3 adenosine blocker), or MRS1754 (A2b adenosine blocker) for 20 min. Treatment

97

groups will undergo a wash period in fresh buffer for 20 min. All groups will then be subjected

to a long period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion (SR).

Samples will be taken before and after SI/SR. (See Figure 26).

Similar experiments will then be conducted with rIPC dialysate and the above mentioned

adenosine blockers in order to confirm which adenosine receptor subtype is involved in rIPC.

Figure 26. Protocol of Adenosine Receptor Subtypes in Opioid-Adenosine Cross-talk.

Cardiomyocytes will be subjected to 10 min classic IPC, specific opioid receptor agonists, such

as Met-enkephalin (δ-specific agonist) or dynorphin B (κ-specific agonist) in the presence of

adenosine receptor subtype blockers, such as DPCPX (A1 adenosine blocker), MRS1523 (A3

adenosine blocker), or MRS1754 (A2b adenosine blocker) for 20 min. Treatment groups will

undergo a wash period in fresh buffer for 20 min. All groups will then be subjected to a long

period of 45 min simulated ischemia (SI) and 60 min simulated reperfusion (SR). Samples will

be taken before and after SI/SR.

98

9.3.b Heterodimerization of Opioid and Adenosine Receptors:

Heterodimerization of GPCRs in cardiomyocytes can be investigated using

coimmunoprecipitation of the opioid and adenosine receptors. In addition, immunofluorescence

of the two receptors may also be examined. While these techniques provide information

regarding receptor association, they cannot prove functionally significant heterodimerization.

A technique that can conclusively state the existence of dimerization is fluorescence

energy transfer (FRET) and bioluminescence energy transfer (BRET) to study protein-protein

interactions in living cells. Hebert et al.183

provides a highly detailed overview of these

techniques. FRET involves fluorescence energy transfer between two adjacent proteins, one

being the donor and the other the acceptor. When these proteins are dissociated, only the donor

emission of fluorescence is detected. However, if they are in close proximity, there is significant

energy transfer between the two proteins and the acceptor‘s emission is detected. BRET is

similar to FRET, yet utilizes bioluminescent luciferase. This technique does not require external

illumination to initiate fluorescence transfer like in FRET, but instead uses luciferase which has

less background noise. Currently, these techniques study interactions between receptors and

downstream signalling molecules, however, Gandia et al.184

describes the potential of the FRET

and BRET techniques to study GPCR oligomerization.

9.3.c Cell Signalling in rIPC:

Signalling downstream of receptors has been studied in rIPC. However, the mechanisms

involved are unclear compared to IPC. Similar to IPC, activation of PKC68

, nitric oxide185

,

reactive oxygen species186

, mitochondrial KATP channels128

, and the reperfusion injury salvage

99

kinase (RISK) pathway146

have been implicated in rIPC. However, inhibition of the

mitochondrial permeability transition pore (mPTP) in rIPC is still unclear.

As a reminder, signalling in IPC involves ligand binging to cell membrane receptors that

result in activation of kinases (e.g. PI3 kinase, Akt, and ERK) and nitric oxide (NO) synthesis,

causing mitochondrial (mKATP) channel opening that produces reactive oxygen species (ROS),

which leads to PKC translocation. During the mediator phase, pro-survival kinases187

of the

reperfusion injury salvage kinase (RISK) pathway are activated and ultimately lead to inhibition

of the mitochondrial permeability transition pore (mPTP) from opening188

.

Nitric oxide (NO) is very important in early and delayed classical IPC, however, the role

of NO in rIPC is still unclear. Tokuno et al.185

proposed that cerebral preconditioning could

induce cardioprotection, however, this protection was absent in nitric oxide synthase (NOS)

knockout mice. In addition, NFκβ and NOS have been implicated in delayed limb rIPC in rat

hearts. Li et al.189

showed that rIPC increased translocation of NFκβ to the nucleus and increased

NOS mRNA. Inactivation of the NFκβ protein or the NOS gene abolished rIPC protection.

Phosphatidylinositol 3-kinase (PI3) is known to be involved in classical IPC. However,

there are no current studies that have investigated the role of PI3 in rIPC. To address this

question, I investigated the effect of wortmannin (a PI3 kinase inhibitor; 100nM concentration

was used; Oldenburg, 2003160

) in conjunction with rabbit dialysate. Blockers of PI3 kinase

activity abolished the protection induced by the preconditioned dialysate (41.0%±4.9 vs.

24.3%±4.0, p=0.01; N=2, n=2,2). (See Figure 27 and Table 18).

100

Table 18. Wortmannin Results (Mean ± SEM).

Stabilization End

Baseline 22.1 ± 1.7 22.9 ± 2.2

Baseline + Wort 23.1 ± 2.0 23.7 ± 1.9

Before SI After SR

IR 21.0 ± 1.8 41.0 ± 1.7

rIPC Rabbit Dialysate 21.3 ± 2.5 24.7 ± 3.0

rIPC Rabbit Dialysate + Wort 20.3 ± 0.3 40.2 ± 3.6

Figure 27. Wortmannin Administered Prior to Rabbit Dialysate.

The effect of wortmannin (a PI3 kinase inhibitor; 100nM concentration was used) in conjunction

with rabbit dialysate was studied. Blockers of PI3 kinase activity abolished the protection

induced by the preconditioned dialysate (40.2%±3.6 vs. 24.7%±3.0, p=0.01; N=2, n=2,2).

101

The activation of mitochondrial KATP has been proposed in early and delayed rIPC. KATP

channel opening is thought to reduce calcium overload and result in inhibition of the

mPTP190,191,192

. As evidence, rat cardioprotection by limb ischemia was blocked by

glibenclamide (non-selective KATP blocker) and 5-HD (a mitochondrial KATP blocker) but not by

HMR 1098 (a sarcolemmal KATP blocker)193,194,195

. However, studies in pigs show that both

sKATP and mKATP are involved in early and delayed limb ischemia196,197

. In these studies, BMS

191095, a mKATP channel opener, mimicked rIPC by limb ischemia. However, 5-HD removed

this protection.

Reactive oxygen species (ROS) generation plays a role in both detrimental lethal

reperfusion injury and in the protective effects of preconditioning187

and post-conditioning198

. As

evidence, Weinbrenner et al.37

determined that a free radical scavenger (MPG) was able to

abolish in vivo cardioprotection from renal artery occlusion (RAO) in rats.

In rIPC, the role of protein kinase C (PKC) has been confirmed in infrarenal aortic

occlusion186

and MAO194

, when chelerythrine (a PKC blocker) abolished cardioprotection in rats.

A number of studies investigating other triggers have suggested the role of PKC in rIPC, such as

adenosine128

, bradykinin68

, and opioids16

. In these studies, pharmacological preconditioning with

bradykinin B268

and CGRP103

receptor activation is thought to induce PKCε translocation in a

manner similar to rIPC cardioprotection.

With respect to the RISK pathway, Heidbreder et al.199

suggest that mitogen-activated

protein kinases (MAPKs), p38, and ERK1/2 are activated in the small intestine during

102

mesenteric rIPC to protect the myocardium. In addition, inhibition of these kinases reduced rIPC

protection. Also, Shimizu et al.146

determined that these MAPK kinases from the RISK pathway

were activated in the heart itself during in vivo skeletal muscle rIPC in rabbits.

To further complicate the role of adenosine in rIPC, cardiomyocytes not only utilize

adenosine as a trigger, but also a mediator in IPC with its claimed involvement of adenosine A2b

receptors during reperfusion14

. Thus, it is important to investigate the role of adenosine as a

mediator in rIPC, and potentially, a mediator in opioid-adenosine cross-talk. However, the role

of adenosine receptors in the studies described by this dissertation has only been investigated in

the trigger phase.

The role of mitochondrial permeability transition pore (mPTP) as an end effector in

classical IPC is a subject of great interest, but to date, there are no published studies of the role of

mPTP in rIPC. The mPTP is located on the inner mitochondrial membrane and opened during

the beginning of reperfusion. Activation of the mPTP results in uncoupling of oxidative

phosphorylation, ATP depletion, mitochondrial swelling, and ultimately results in cell death.

However, preconditioning is thought to inhibit mPTP opening200

. Zhang et al.36

has shown that a

κ-opioid agonist (U-50,488H) induced mPTP opening, as seen by fluorescent calcein, in rat

cardiomyocytes. In the same study, the mPTP activator (atractyloside) abolished femoral artery

occlusion (FAO)-induced cardioprotection in rats.

Infiltration, adhesion and activation of neutrophils have also been proposed as an end

effector in early and late rIPC201

. Cardioprotection by limb ischemia in mice has shown down-

103

regulation of proinflammatory genes and up-regulation of genes involved in cytoprotection and

protection from oxidative stress202

. This suggests that rIPC suppresses neutrophils from releasing

proinflammatory cytokines and from expressing adhesion markers. However, the role of

neutrophils in rIPC requires an in vivo model and is not suitable to be studied by our isolated

cardiomyocyte model.

9.4 Limitations of the Study:

There are some limitations to the model described in this dissertation. Unfortunately,

utilizing an isolated cardiomyocyte model excludes the ability to study a neuronal pathway in

protection. Though my results indicate that a humoral mediator is sufficient to induce rIPC

cardioprotection (see Figure 13), this does not exclude a neurogenic involvement in vivo. An in

vivo model is also useful in investigating the involvement of an inflammatory pathway via

neutrophils in rIPC. Finally, my study did not measure apoptosis as an indicator of myocardial

reperfusion injury. Such an investigation is more suitable for cultured cardiomyocytes since

apoptosis takes several hours to develop. Regardless, it is important to note that an isolated

cardiomyocyte model is more efficient (since many treatment groups can be derived from a

single heart) and reduces heart-to-heart variability.

In addition, my studies only investigated rIPC during the trigger phase of acute

preconditioning. A comprehensive study would involve the role of rIPC dialysate to induce

protection during the reperfusion phase and in delayed rIPC. Fortunately, these investigations

can be readily conducted either in isolated or cultured cardiomyocytes.

104

9.5 Conclusions:

This dissertation provides important insights into the role of particular G-protein coupled

receptors in a humoral model of remote preconditioning. Also, there are benefits of using this

model over others in investigating this area. For example, characterization of the dialysate (see

Table 5) using MRM-MS excluded the possibility of certain substances to mediate protection in

the rIPC dialysate. However, this method does not encompass all endogenous ligands that can

bind to a particular GPCR. Considering this, selective inhibition of receptors is a better method

of determining which receptors are involved even if the stimulatory agent is not present.

Also, our model can be used to determine cross-talk between receptors on a functional

level. MRM-MS also did not exclude the possibility of certain receptors involved in cross-talk

with opioid receptors. However, our model is able to determine which receptors can participate

in cross-talk (i.e. opioid with adenosine receptors) and which possibilities can be excluded (i.e.

opioid cross-talk with bradykinin B2 and CGRP receptors).

In conclusion, remote preconditioning is a clinically relevant strategy to reduce

myocardial infarction. However, the mechanistic pathways in remote precondition are still

unclear, with some evidence resulting to conflicting results. Thus, it is important to compare the

pathways in classical and rIPC in order to create beneficial therapeutic interventions.

105

APPENDIX

APPENDIX I: List of Figures

Figure 1. IPC Signalling Mechanisms in the Target Organ. ........................................................... 4

Figure 2. rIPC Signalling Mechanisms of the Myocardium. .......................................................... 7

Figure 3. The Adenosine Kinase Inhibition Hypothesis. .............................................................. 25

Figure 4. Preparation of Buffers Derived from Krebs-Henseleit Buffer. ..................................... 31

Figure 5. Comparison of Digestion Protocols............................................................................... 33

Figure 6. Rabbit and Human Dialysate Preparation. .................................................................... 36

Figure 7. General Protocol: Isolated Rabbit Cardiomyocytes. ..................................................... 38

Figure 8. AIM (1) Protocol: To Characterize Dialysate Protection. ............................................. 39

Figure 9. AIM (2) Protocol: The Role of Cell Membrane Receptors. .......................................... 40

Figure 10. AIM (3) Protocol: Investigate Receptor Cross-talk..................................................... 41

Figure 11. Trypan Blue Exclusion Assay. .................................................................................... 43

Figure 12. Adenosine Kinase Inhibition Protocol......................................................................... 49

Figure 13. Rabbit and Human Dialysate Administered Prior to Ischemia-Reperfusion............... 52

Figure 14. Western Blot Analysis of Opioid Receptors in Rabbit Tissues. .................................. 57

Figure 15. Naloxone Administered Prior to Rabbit Dialysate. ..................................................... 59

Figure 16. Naltrindole Administered Prior to Rabbit Dialysate. .................................................. 61

Figure 17. GNTI Administered Prior to Rabbit Dialysate. ........................................................... 63

Figure 18. HOE140 Administered Prior to Rabbit Dialysate. ...................................................... 67

Figure 19. Western Blot Analysis of Calcitonin-Like Receptors in Rabbit Tissues. ................... 70

Figure 20. CGRP (8-37) Administered Prior to Rabbit Dialysate. ............................................... 72

106

Figure 21. 8-SPT Administered Prior to Rabbit Dialysate. .......................................................... 75

Figure 22. 8-SPT Administered Prior to Met-Enkephalin. ........................................................... 81

Figure 23. 8-SPT Administered Prior to Dynorphin B. ................................................................ 83

Figure 24. Adenosine Concentrations After Exposure to Dynorphin B. ...................................... 88

Figure 25. General Diagram of Opioid-Adenosine Cross-talk. .................................................... 94

Figure 26. Protocol of Adenosine Receptor Subtypes in Opioid-Adenosine Cross-talk. ............. 97

Figure 27. Wortmannin Administered Prior to Rabbit Dialysate. .............................................. 100

Figure 28. Standard Plot of Substances in Water. ...................................................................... 110

Figure 29. Standard Plot of Adenosine in Krebs-Henseleit Buffer. ........................................... 112

107

APPENDIX II: List of Tables

Table 1. Krebs-Henseleit Buffer Composition. ............................................................................ 30

Table 2. Tyrode Buffer Composition. ........................................................................................... 42

Table 3. Inter-Observer Error Data. .............................................................................................. 45

Table 4. Rabbit and Human Dialysate Results (Mean ± SEM). ................................................... 52

Table 5. Characterization of Rabbit Dialysate Using MRM Mass Spectrometry. ........................ 53

Table 6. Naloxone Results (Mean ± SEM). .................................................................................. 59

Table 7. Naltrindole Results (Mean ± SEM). ............................................................................... 61

Table 8. GNTI Results (Mean ± SEM). ........................................................................................ 63

Table 9. HOE140 Results (Mean ± SEM). ................................................................................... 67

Table 10. 1x Dialysate with HOE140 Results (% Cardiomyocyte Death). .................................. 69

Table 11. CGRP (8-37) Results (Mean ± SEM). .......................................................................... 72

Table 12. 8-SPT Results (Mean ± SEM). ..................................................................................... 75

Table 13. ADA Results (% Cardiomyocyte Death). ..................................................................... 79

Table 14. Met-Enkephalin with 8-SPT Results (Mean ± SEM). .................................................. 81

Table 15. Dynorphin B with 8-SPT Results (Mean ± SEM). ....................................................... 83

Table 16. Adenosine with Naloxone Results (% Dead Cardiomyocytes). ................................... 86

Table 17. Adenosine Concentrations After Exposure to Dynorphin B Results (Mean ± SEM). . 88

Table 18. Wortmannin Results (Mean ± SEM). ......................................................................... 100

Table 19. Summary of Literature in which Myocardium was the Target Organ. ....................... 108

Table 20. Summary of Literature in which Skeletal Muscle was the Preconditioning Organ.... 109

Table 21. Results of Standard Concentrations of Substances in Water. ..................................... 111

Table 22. Results of Standard Concentrations of Adenosine in Krebs-Henseleit Buffer. .......... 112

108

APPENDIX III: rIPC Summary: Myocardium as the Target Organ

Table 19. Summary of Literature in which Myocardium was the Target Organ.

This table is identical to Table 1 from the review by Hausenloy & Yellon in 2008.19

109

APPENDIX IV: rIPC Summary: Skeletal Muscle as the Preconditioning Organ

203

Table 20. Summary of Literature in which Skeletal Muscle was the Preconditioning Organ.

This table is identical to Table 5 from the review by Tapuria et al. in 2008.203

110

APPENDIX V: MRM Mass Spectrometry Plots of Standard Concentrations

Figure 28. Standard Plot of Substances in Water.

Known standard concentrations of all substances were prepared in water and measured by MRM

mass spectrometry in order to measure accurate concentrations.

111

Table 21. Results of Standard Concentrations of Substances in Water.

Standard

Concentrations

(nM)

Measured Concentrations (nM)

Met

-en

kep

hali

n

Dyn

orp

hin

B

ME

AP

Ad

enosi

ne

Inosi

ne

Bra

dyk

inin

Nore

pin

eph

rin

e

Ace

tylc

holi

ne

An

gio

ten

sin

II

Water --- --- --- --- --- --- --- --- ---

2.5 2.64 --- --- 2.45 0.068

3.4 3.57

3.5 3.77

5.0 4.46 --- --- 5.08 4.94

5.9 7.20

6.9 6.79

10.0 9.9 --- --- 10.5 10.3

14.2 12.4

14.3 14.4

15.0 17.1

17.0 15.8

25.0 24.8 27.0 27.6 24.7 25.1

50.0 50.7 --- 40.7 48.5 47.2

56.6 57.1

57.4 51.3

59.0 55.6

68.0 74.4

100 104 54.3 96.8 101 99.1

113 114

115 108

150 149 155

250 252 296 233 241 255

296 295

342 329

500 517 680 578 521 493

566 751

574 566

592 593

685 687

1000 981 823 959

NOTE: --- represents ‗No Peak‘.

112

Table 22. Results of Standard Concentrations of Adenosine in Krebs-Henseleit Buffer.

Standard Concentrations (nM) Measured Concentrations (nM)

Water ---

100 96.3

1000 1070

10000 9940

10000 10800

NOTE: --- represents ‗No Peak‘.

Figure 29. Standard Plot of Adenosine in Krebs-Henseleit Buffer.

Known standard concentrations of adenosine was prepared in Krebs-Henseleit buffer and

measured by MRM mass spectrometry in order to measure accurate concentrations.

113

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