METHODS AND MECHANISMS OF ENDOGENOUS AND EXOGENOUS M Y O C A R D I A L PRECONDITIONING

Girlcon Cohen, MD

Institute for Medical Sciences, University o f Toronto. .

Division o f Cardiovascular Surgery, The Toronto Hospital. Toronto, Ontario, Canada.

A thesis submitted in conformity with the requirements for the degree of Master of Science. Gradunte Department o f the Institute of Medicnl Sciences,

University of Toronto

National Library Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibtiographic Services services bibliographiques

395 Weiiington Street 395, rue wdrmgton Ottawa064 K l A W O(tewa0N KlAONI Canada Canada

The author has granted a non- exciusive licence allowing the National Library of Canada to reproduce, loan, distribute or seli copies of this thesis in rnicrofonn, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fiom it may be printed or othewise reproduced without the author's permission.

L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique.

L'auteur consewe la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation,

ACKNOW LEDGEMENTS

First and foremost, 1 wish to thank rny supervisor and mentor, Dr. Richard D. Weisel, for his

support and guidance, without which this work would have not been possible. His tireless efforts in

pursuit of academic excellence and his dedication and cornmitment to his students are unsurpassed,

and 1 am eternally gratefûl to have been granted the opportunity to work under his s u p e ~ s i o n .

1 wish to thank the members o f my thesis committee, Dr. Donald A. G. Mickle and Dr.

Stephen E. Fremes, for their valuable input and guidance. Their expertise in their respective fields

provided an invaluabte resource throughout my academic training and 1 am indebted to them for their

time and effort on my behalf.

The continued commitment of the Division of Cardiovascular Surgery at the University o f

Toronto towards the training of surgical scientists remains a valuable asset and has provided an ideai

backdrop fo r residents such as myself who are interested in pursuing an academic career. 1 am

indebted to the members ofthe division for their continued suppon and 1 hope to achieve a standard

which is worthy o f such cornmitment in the coming years.

My family and fiends have always provided the unending support to necessary to achieve

my personal and career goals. I am forever gratefiil to them for their love and commitment.

Finally, 1 would like to thank the Heart and Stroke Foundation o f Canada for their fellowship

grants in support o f this work.

iii

METHODS AND MECHANISMS O F ENDOGENOUS AND EXOGENOUS MYOCARDIAL PRECONDITIONING

TABLE OF CONTENTS

Title Page ... a

Acknowledgements . .

.*.II

Table of Contents ... ... 111

Abstract ..,VI

List of Abbreviations ... viii

Legends to Figures a.. x

Ch npter One: KNOWLEDGE TO DATE

1 . 1 Introduction -.. 1

1.1 - 1 The Probiem: Low Output Syndrome 1.1.2 The Solution: Myocardial Preconditioning

1.2 Myocardial Preconditioning . . -4

1.2.1 Historical Overview . . -4

1.3 Adenosine . . -6

Historical Overview Adenosine Metabolism Endosenous Adenosine Production Adenosine Transport Adenosine Catabolism Regulation of Interstitial Adenosine Concentrations Adenosine Receptors Signal Transduction Adenosine Effector Mechanisms

1.4 Protein Kinase C (PKC) 1.5 Cardioprotective Properties of Adenosine

1.6 Exoçenous Adenosine Studies ... 21

1.7 Ischemic Preconditioning in Humans . . -25

1.8 Exoçenous Adenosine in Humans . . -28

1.8.1 Physiologie Effects 1.8 -2 Electrophysiologic Effects 1.8.3 Regulation of Coronary Blood Flow 1 -8.4 Hemodynamic and Respiratory Effects

1.9 Adenosine Preconditioninç in Humans .--30

1 -9.1 Adenosine Pretreatment (pre-ischemic treatment) ... 30 1.9.2 Cardioplegic Adenosine Treatment (ischemic treatment) ,. -3 1 1.9.3 Adenosine Post-treatment (reperfùsion treatment) ... 3 1 1.9.4 Continuous Adenosine Treatment ... 32

1.10 Sumrnary of Study Rationale, Hypotheses, and Objectives -. -34

Ch np ter TI vo: ENDOGENOUS PRECONDITIONING STUDIES: Reconditionirtg is mcdiated r ,icr (denosine reïeme in human ventriculnt myocyta

2.1 Summary

2.2 Introduction ... 39

2.3 Materials and Methods ... 39

2.3 1 Isolation and Culture of Human Ventricular Myocytes ... 39 2.32 Experimental Design , . ,39 2.3 3 Experi ment al Pro t oc01 s : (. Graded Precottditior~ing Strrdy ... 40

II. S~fperrratar~r Preconditioning S t lrdy . . -4 1 Ili. Memtrernertt of Endcgenolts ... 41

Adertosirie Corrcerttrations I K Adetrosine Receptor AI? fagonist Sfudies . . -4 1

2.34 Assessment of Cellular Injury ... 42 2.3 5 Biochemical Measurements . . -42

2.36 StatisticaI Analysis . . -43

2.4 1 Graded Preconditioning Study . . -44 2.42 Supernatant Preconditioning Study ... 44 2.43 Measurement of Endogenous Adenosine Concentrations ... 45 2.44 Adenosine Receptor Antagonist Studies . . -45

2.5 Conctusions ... 45

Chapte+ Three: E X O G E N O U S PRECONDITIONING STUDLES: Reproducing the protective effects of ischenric preconditioning using erogenous m/en mine

3 . 1 Summary ... 48

3 -2 Introduction ...50

3.3 Materials and Methods ... 50

3 .3 1 Experimental Protocols: i? Oprimal dose ami timing of adetrosine 3 0 VI. Selecfive adenosine receptor ... 5 1

critrc~gorzisr smdies 3.32 Assessrnent of CeIIular Injury ... 52 3 -3 3 Biochemical Measurements ..S2 3.34 Adenosine Assay ... 53 3 .3 5 Statistical Analysis ... 53

3 .4 Results ... 54

3.4 1 Optimal Dose and Timing of Adenosine 3.42 Selective Adenosine Receptor Antagonist S tudies

3 . 5 Conclusions ... 56

Chnptcr Fouc PROTELN KINASE C STUDIES: Adetrosine preconditions hunrnn ven rriculnr ntyocytcs vin n PKC ntcihted path ~vay

4.1 Summary ... 59

4.2 Introduction .. -60

4.3 MateriaIs and Methods ..,61

4.3 1 ExperimentaI Protocols: Yii. Protein Kinase C Sftrdies ... 62

4.32 Protein Kinase C Analyses ... 63 4.33 . Statistical Analysis . ..63

4.4 Results ... 64

4.4 1 Protein Kinase C Studies ... 64

4.5 Conclusions ... 65

DISCUSSION

Introduction

Hu man Cardiomyocyte Cell Culture Mode1

Endogenous Preconditioning

Exogenous Preconditioninç

Alternate Clinical Applications

5.5 1 Donor Heart Preservation 5 -52 Reduction of Post-bypass Transfusion Requirement 5.53 Off Pump Coronary Bypass Sursery 5.54 Second Window of Protection

Summary of Investigations and Original Contributions

Conclusions

APPENDIX ONE

APPENDIX TWO

APPENDIX THREE

viii

LIST OF ABBREVIATIONS

A D 0 ADP ANOVA ATP ATPase BSA CABG Ca '+ CaCl, Cal-C CK CK-MB CO2 CP OC DAG DCA DMSO DNA et nf

g G protein H+ Hcl HEPES HPLC H2P04 HVM HXN kG I N 0 IP, K+ KCI KH2HP04 K2 H PO, kDa LA D ni- M -

Adenosine Adenosine dip hosp hate Analysis of variance Adenosine triphosphate Adenosine triphosphatase Bovine Semm Albumin Coronary Artery Bypass Grafting Catcium Calcium ChIoide Calphostin C Creatine Kinase MB fraction of creatine kinase Carbon dioxide Creatine phosphate Degees Centigrade 1,2-Diacylglycerol Dichloroacetate Dimethylsulphoxide Deoxyribonucleic acid "and others" grams Guanosine triphosphate binding protein Hydrogen ion Hydrogen chloride N-[2-hydroxyethyl]piperatine-N'-[2-ethanesulfonicJ acid High performance liquid chromatography Sulphuric acid Human ventricular myocytes Hypoxanthine Immunoglobulin G Inosine lnositol 1,4,5-triphosphate Potassium Potassiuni chloride Potassium phosphate (monobasic) Potassium phosphate (dibasic) KilodaIton Lefi enterior descendiog coronary artery MiIli- ( 1 O -3)

Moles per litre

NIARCKS Mg' MgClz min mol mOsm mRNA n-

N2 Na+ NaRCO, NaHSO, Na2C0, NaCI NaOH Na2HP0, Na H, PO, NAD NADH OMA 0 2

'!AB PBS PLA PIP, PH PKC PMA RNA SAS SEM SPT

M ynstolated. alanine-rich, C-kinase substrate Magnesium Magnesium chloride minutes Mole (6.023 x 10" particles) Milliosmoles Messenger RNA Nano- (1 0-4 Nitrogen Sodium Sodium bicarbonate Sodium bisulphite Sodium carbonate Sodium chloride Sodium hydroxide Disodium phosphate Sodium phosphate Dihydronicotinarnide adenine dinucleotide (oxidized) Dihydronicotinamide adenine dinucleotide (reduced) ?'-O-met hyfadenosine Oxygen Percent Phosphate buffered saline R(-)N1-(phenyl-ZR-isopropy1)-adenosine Phosphatidyl 4.5-biphosphate Negative logarithm of hydrogen ion concentration Protein kinase C Phorbol 12-myristate 13-acetate RibonucIeic acid Statistical Analysis Systems Standard error of the mean 8-p-sulphophenyl theophylline Micro- (1 04)

FIGURE LEGENDS

Figure L: A:Schematic structure of adenosine combining a purine base and a ribose moiety. B:Schematic structure of adenosine triphosphate combining adenosine and three phosphate groups.

Figure 2: Adenosine Metnbolism. The cardiac adenosine system is compnsed of three components; ( 1) formation; (2) receptor complex effects; and (3) degradation. I - Adenosine (ADO) can be formed intracellularly via the adenosine triphosphate (ATP) or S-adenosylhomocysteine ( S M ) pathway, or extracellularly via breakdown of adenine nucleotides. 2 - The adenosine receptor (ADO-R) is coupled to ion channels via the guanine binding regdatory proteins (Gi). Theophylline (THEO) derivatives act as cornpetitive antagonists for the adenosine receptors. 3-AD0 can be transported into the ceIl and then degraded via deamination to inosine or phosphorylated to adenosine rnonophosphate (AMP). Dipyridamoie can block the cellular uptake of ADO, t hus prolonging it s effect . ADP=adenosine di phosphate; cAMP=cyciic AMP; GTP=guanosine triphosphate.

Figure 3: Purine Metabolism. Ado=adenosine; Whypoxanthine; lno=inosine; UA=uric acid. a=ATP consuming reactions; b=oxidative phosphorylation; c=myokinase; d=S1- nucleotidase; e=AMP deaminase; f-adenylosuccinate synthase and lyase; radenosine kinase; h=adenosine deaminase; I=purine nucleoside phosphorylase; j=xanthine dehydrogenase; k=guanine phosphoribosyl transferase; kadenine phosphoribosyl transferase.

Figure 4: Summary of the adenosine-protein kinase C mechanism of ischemic preconditioning. Brief ischemia results in the degradation of adenosine triphosphate (ATP) through adenosine diphosphate (ADP) and adenosine monophosphate (AMP) to adenosine. Adenosine k l y difises across the ce11 membrane to interact with surface adenosine receptors.(A I ). Adenosine receptors are believed to be coupled to inhibitory guanosine triphosphate binding proteins (Gi proteins) consisting of a, b, and g subunits. The activated a subunit stimulates membrane bound phospholipase C (PLC) to conven membrane phosphatidylinositol biphosphate (PIP2) to inositol triphosphate (IP3) 2nd diacylglycerol @AG). IP3 induces interna1 mobilization of calcium stores tiom sites such as the sarcoplasmic reticulum (SR). As the intracellular calcium concentration rises, inactive cytosolic protein kinase C (PKCinact) translocates to ceIl membranes and is activated by DAG (PKCact). Activated PKC may now mediate the cardioprotective response through modulation of final effectorls such as ion channels, intermediary metabolic pathways, and gene expression.

Figure 5: Simplified summary of the adenosine-protein kinase C mechanism of ischemic preconditioning. Brief ischemia results in the degradation o f adenosine triphosphate (ATP) to form adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine. Adenosine d i a s e s across the cell membrane t o interact with extracellular adenosine recepton (Al). Through a senes of intermediary steps including G protein activation and hydrolysis o f membrane phospholipids, protein kinase C (PKC) is activated. Activated PKC goes on to phosphorylate intra- o r extracellular final effectors t hereby conferring protection.

Figure 6: Representative photomicrographs o f pnmary cultures of human pediatric (A) and adutt (B) ventncular cardiornyocytes. (200x magnification; reprinted from Li, et al."')

Figure 7: Schematic diagram of simulated "ischemia" and "reperfllsion" model. Culture dishes of human ventricular cardiomyocytes are placed in an air-tight plexiglass chamber. T o ensure anoxic conditions, 100% nitrogen (NJ gas bubbled through two oxygen traps is utilized to continuously flush the sealed chamber thereby displacing any ambient oxygen. Four culture dishes are placed in the chamber which is equipped with a central sampling dish t o enable venfication of anoxic conditions and to allow temperature monitoring with each ischemia/reperfiision experiment. (Reprinted from Tumiati, et al.""')

Figure 8: Light microyraph o f cardiomyocytes stained with Trypan Blue. Lefi Panel: cardiomyocytes stabilized in phosphate-buffered saline for 30 minutes show little evidence o f ceilular injury. Middle Panel: cardiomyocytes preconditioned with 20 minutes o f "ischemia" followed by 20 minutes of ''repefision" reveal relatively few injured cells (denoted by arrows) following prolonged "ischemia" and "reperfùsion". Righi Panel: non-preconditioned cardiomyocytes reveal large numbers of injured celis (denoted by arrows) following prolonged "ischemia" and "reperfùsion". (200x magnitication; scale bar=20pm; Reprinted from ~konornidis~')

Figure 9: Endogenous preconditioning studies: In study 1) cells undenvent either anoxic (PCO) o r hypoxic (PC 16) preconditioning for a period o f 20 minutes prior to prolonged ischemia and reperfusion. In study 2) non-preconditioned cells were preconditioned for a penod of 20 iiiin. using the supernatant of cells which underwent either anoxic (SUPO) or hypoxic (SUP16) preconditioning. In study 4) supernatant from anoxically preconditioned cells was treated with either SPT or adenosine deaminase (ADA) and applied to non-preconditioned cells which were pre-treated with SPT o r adenosine deaminase. All groups were compared to non-ischemic controls @TIC) which underwent 190 min. o f stabilization, and ischemic controls (IC) which underwent 70 min. of stabilization followed by prolonged "ischemia" (90 min.) and "reperfùsion" (30 min.).

xii

Figure 10: Anoxic preconditioning (PCO) reduced cellular injury to a greater extent than did hypoxic preconditioning (PC 16) (+p<0.05). Both forms of preconditioning reduced cellular injury compared to ischemic controls (IC) (*p<0.05 vs. IC). ( N C : Non-ischemic Controls).

Figu re II: Upper panel: Extracellular lactate levels were significantly elevated at 50 minutes in the anoxic preconditioning group (PCO), however not significantly. Extracellular lactate concentrations following both "ischemia" and "repefision" did not differ between groups. Lower panel: Intracellular ATP levels decreased significantly in the anoxic preconditioning group (PCO) in cornparison to ischemic controls OC; pc0.05) ('ATP debt'). However intracellular ATP levels following both "ischemia" and "reperfùsion" did not differ between groups.

Figure 12: Lower Panel: Preconditioning with the supernatant of anoxically preconditioned cells (SUPO) reduced cellular injury to a greater extent than did preconditioning with the supernatant of hypoxically preconditioned celIs (SUP l6)(p<O.OS). Both forms of supernatant preconditioning significantly reduced cellular injury compared to ischemic controls (IC) (p<O.OS) CN[C: Non-ischemic Controls). Upper Panel: HPLC analysis revealed a greater concentration of endogenous adenosine in the supernatant of anoxically preconditioned cells (SUPO)(p=O.O 18, SUPO vs. S U P 16). The supernatant of cells which underwent stabilization only revealed the lowest endogenous adenosine concentrations.

Figure 13: The protective effects of anoxically preconditioned supernatant (SUPO) were abolished when the non-preconditioned cells and the supernatant were first incubated with either SPT or adenosine deaminase (ADA) W C : Non-ischemic wntrols; IC: lschemic controls) (*p<0.05 vs. SPT, ADA, and IC; +p<0.05 vs. SUPO, SPT, A D 4 IC) .

Figure 14: Exogenous preconditioning studies: In study 5) exogenous adenosine was applied to cells either prior to (Pretreat), during (Ischemic treat), or following (Reperksion treat) prolonçed "ischemia" and "reperfùsion", or during al1 three phases (Continuous treat). Compatisons were made with cells which underwent stabilization in normoxic PBS for a total of 190 min. (Non-ischemic controls; NIC) and with cells which underwent stabilization for 70 min. followed by prolonged "ischemia" and "repefision" (Ischemic controls; IC). In study 6) cells treated with adenmine either prior to or durinç ischemia were simultaneously treated with SPT (Pretreat -t SPT and Ischemic Treat + SPT, respectively). Cornparison was made with cells which underwent stabilization in SPT and adenosine only W C + SPT). (A: Adenosine)

xi i i

Figure 15: Upper Panel: Exogenous adenosine was most protective when administered at a dose of 50 umol prior to ischemia (PRE). Application of adenosine during ischemia (1SCl-t) was protective to a significantiy lesser degree. The two protective effects were not found to be additive when adenosine was administered continuously (CONTIN). Adenosine administered dunng reperfusion (REP) was not protective. Al1 groups were compared to both ischemic controls (IC) and non-ischemic controls (MC). Al1 protective effects were abolished when SPT was applied to adenosine treated cells, regardless of timing. Adenosine and SPT had no effect on non-ischemic controls (NLC). Lower Panel: Both PRE and CONTM groups revealed a presemtion of ATP followin~ "ischemia" and "reperfusion" in cornparison to ischemic controls (K). The ISCK group reveated preservation of ATP to a lesser degree. Simultaneous administration of SPT abolished the ATP-preservative effects of adenosine. Adenosine applied duting repefision did not afford ATP-preservative properties.

Figure 16: Extracellular lactate concent rations following "ischemia" and "reperfbsion" (FINAL) were elevated in cells which received adenosine either continuously (CONTIN) or du ring reperfùsion (REP)(*p<O.OS). In evaluating the direct effects of adenosine VOST-ADENOSME), lactate levels were elevated immediately following adenosine administration in al1 groups compared to untreated controls (CONTROL) (+p<0.05 vs. correspondinç CONTROL). SPT blocked the lactate elevating effects of adenosine (ADENOSME+SPT) (pc0.05 vs. corresponding POST-ADENOSINE). (NIC: Non-ischemic controls; PRE: Pretreatment; ISC: Ischemic treatrnent; IC: Ischemic controls)

Figure 17: Protein kinase C studies: Non-preconditioned cells were exposed to PMA for 20 minutes followed by 20 minutes of pre-ischemic reperfusion prior to prolonged "ischemia" and "repertiision". Certain cells which underwent ischemic preconditioning (PCO) or were treated with adenosine (A) or PMA prior to prolonged "ischemia" and "reperfùsion" were also exposed to Calphostin-C (Cal-C) during 30 minutes of stabilization, durinç preconditioninç with ischemia, adenosine (PRE), or PMA, and during pre-ischemic reperfusion. Non-ischemic controls (NIC) were exposed to Calphostin-C for 30 minutes, followed by Calphostin-C with adenosine or PMA for 20 minutes. followed by Calphostin-C for 20 minutes, followed by 120 minutes of stabilization.

Figure 18: The protective effects of preconditioning with either ischemia (PCO), adenosine (PRE), or PMA (PMA) were abolished with the addition of Cal-C (+Cal-C) (*p<0.05 vs. NIC, IC). (Cal-C: Calphostin-C; A: Adenosine; NIC: Non-ischemic controls)

xiv

Figure 19: Representative dot-blot analysis demonstrating isoform-specific translocation of PKC in cells exposed to 50 pmol of adenosine (Pretreatment), 100 pmol ofadenosine, 50 pmol of adenosine with SPT, or 10 nm PMA. Results were compared to those of cells which undenvent stabilization in normoxic PBS only (NIC). Densitornetnc analyses revealed no changes in PKC-a or PKC-E distributions with stabilization. Similarly, PKC-E distributions did not change with either adenosine or the phorbol ester PMA. However, there was a marked cytosolic to membrane translocation of PKC-a in cells exposed to 50 prnol of adenosine (Pretreatment) or PMA. Cells exposed to 100 pmoI of adenosine prior to ischemia revealed a less marked translocation. Exposure of the celis to 50 pmol of adenosine with SPT (non-selective adenosine receptor antagonist) prevented differential translocation.

xv

ABSTRACT

Coronary anery bypass çraft surgery (CABG) is the most comrnonly performed surgery in

Nonh America. Recently changing trends in the population at risk have resulted in increasing

numbers of high risk patients presenting for CABG with an accompanying nse in the rate of

postoperative low output syndrome (LOS). LOS is associated with increased patient rnortality and

places a significant financial burden on the health care system- In the absence of intraoperative

complications, LOS following CABG represents a failure of intraoperative myocardial protection.

As such, improved methods of intraoperative myocardial protection are necessary to prevent

increased morbidity and mortality following CABG.

lschernic preconditioning (PC) is the most powerful endogenously mediated forrn of

myocardial protection known. Unfortunately, the phenornenon is difficult to apply clinically.

Moreover, the ischemic stimulus of PC may entai1 detrimental effects acutely. To avoid such

limitations, we must possess the ability to reproduce preconditioning without the need for ischemia.

A pharmacological mediator which could harness the beneficial eflects of preconditioning would be

ideal in this regard.

Adenosine, believed to be a mediator of ischemic preconditioning, may represent such an

additive. Unfortunately. the benefits of adenosine in hurnan preconditioning are controversial.

Moreover the optimal timing of adenosine administration, and its mechanism of action remain

undetermined.

We propose a series of studies in isolated human ventricular myocytes exposed to simulated

ischemia and repetfusion. Both endogenous and exogenous preconditioning studies will be

undertaken with an eye towards the delineation of the mechanisms of preconditioning, and the

deveIopment of a valid mode1 for tlie clinical appIication ofadenosine.

CaAPTER ONE: KNOWLEDGE TO DATE

INTRODUCTION

Despite tremendous advances in the treatment and prevention of cardiovascular disease,

coronary artery disease remains the single leading cause of death in Canada. According to current

estimates, more than 6 million Canadians s m e r h m coronary artery disease. In Ontario alone,

coronary artery disease accounted for 13% of ail hospital admissions and 18% of al1 inpatient

resource utilization between 1996 and 1997.l Not surprisingi y, coronary artery b ypass graft surgery

(CABG) has become the most comrnonly performed surgical procedure in North America, with

current estimates projecting a doubling in the number of CABGs by the year 2018. In 1991, the

average rate of CABG in the province of Ontario was 75 per 100,000. By 1998, the rate increased

to 99 per 100,000.' A gradually aging population dong with the continued success of surgical

intervention has created a growing deniand for coronary bypass surgery which will likely extend into

the next millennium.

THE PROBLEM

The beneficiai effects of contemporaiy coronary artery bypass surgery (CABG) are well

documented? In patients with lefi main coronary arterial obstruction, double vesse1 dis- or triple

vesse1 disease, coronary bypass surgery has been shown to significantly d u c e mortality when

comparai to medicd management a lone . In addition, surgical intervention has been shown to be

an effective means of relieving symptoms in cases when more conservative measures have been

unsuccessful.2 Recently changing trends in the population at nsk, however, have introduced new

challenges for cardiac surgeons in their attempt to minimize penoperative morbidity and monality.

Longer average Life spans and significant technological advances have made cardiac surgery

accessible to individuals who were previously deemed inoperable, and have enabled previous CABG

patients to return for second and third time revascularization procedures. This trend is likely to

continue owing to a gradually aging population, with a progresively increasing average life-span.

Moreover, patients who would have previously succurnbed to a myocardial infarction are now

surviving due to the success and growing availability of thrombolytic therapy. Since progression of

disease is rarely halted, such surviving patients are iikely to retum for surgicd management with

more advanced and complex disease.

Numemus studies have confirmed the gmwing numbers of such high nsk patients presmting

for CABG. A review by Christakis and colleagues reveaied a higher incidence of patients greater

than 65 years of age, patients undergoing urgent surgery for unstable angina, and patients with

preoperative ejection fractions of Iess than 40%: Although operative mortaiity did not change

significantly, the risk of non-fatal morbidity rose steadily, contnbuting to longer hospital stays and

inaeased resource utiiization. In a similar review by Maharajh and colleagues, elderly patients (>75

years of age) experienced operative moltalities as high as 1496.9 In both studies, the most cornmon

factor contxibuting to death or increased dwation of hospital stay was low output syndrome (LOS -

the requirement in the intensive care unit for inotropic andor mechanical support for greater than

30 minutes to maintain systolic blood pressure above 90 mmHg and a cardiac index greater than 2.1

Uminlm2). which occumd in 494 patients (6.7%). Later studies reveaied that in some high risk

subgroups the risk of LOS approached 70%. and that patients who developed LOS had an operative

mortality of 17%. whereas those who did not, had a mortality of 0.996.~""

In the absence of intraoperative complications, postoperative M S is the direct rcsult of

inadequate intraoperative myocardial protection. Not surprisingly, recent advances in cardiac

surgery have centreci upon improved methods of intraoperative cardioprotection in the hope of

preventing postoperative ventricular dysfunction and improving overall outcome.

To date, strategies aimed at minimizing the nsks associated with comnary bypass surgery

have almost exclusively involved manipulation of ischemia and reperfusion conditions. Parameters

such as cardioplegic composition, temperature, and flow rate have been extensively evaluated in the

hope of optirnizing intraoperative myocardiai protection. In the mid 1980's, a major innovation

involved the conversion from unox ygenated crystalloid cardioplegic solutions to oxygenated blood

cardiopIegia. Clinical studies revealed that blood cardioplegia enhanced aembic metaboiism,

improved postoperative venfcicular function, and reduced anaerobic lactate producti~n.'~ Further

studies demonstrated the benefits of a tenninai infusion of wann blwd cardioplegia in repleting

myocardial ATP levels and increasing postoperative diastolic ~ornpliance.'~ Later, tepid (29' C)

cardioplegia was shown to avoid the potentiai hazards associated with normothermic or hypothemiic

cardioplegia by reducing lactate and acid production during cardioplegic arrest and improving

postoperative ventricular fun~tion.'~ Shidies of combination antegrade and retrograde cardioplegia

demonstrated a reduction in lactate production, a preservation of ATP stores and improved perfusion

of the heart during ~rossclarnp.'~ Recent studies involving variable flows revealed that a cardioplegic

flow rate of 200 mUmin irnproved the washout of deaimental metabolites resulting in improved

ventricular f~nction. '~

Despite such advances, mechanical and metabolic dysfunction of the myocardium following

coronary bypass surgery remains a frequendy encountered complication. Such a rcality has

prompted clinicians and researchers alike to search for yet additional, less traditional methods of

protecting the heart against the effects of ischemia and reperfusion. Moreover, m e r improvements

will likely be required to resuscitate hearts acutely injured pnor to surgery.

THE SOLUTION= Myocdial Preconîütihning

Perhaps most intriguing in the realm of myocardial protection is the advent of myocardial

preconditioning. Unlike previous approaches, the aim of preconditioning at its inception was to

'condition' the heart pnor to an ischemic insult in the hope of affording an increased tolerance to the

effects of subsequent ischemia. hnically, although numerous preconditioning stimuli have been

proposed, none have been as successhil or profond as that of ischemia itself.

MYOCARDU PRECONDITIONING HHcsforical Overview

The effects of ischemia on the heart have been studied for centuries. As early as 1698, a

report by Chirac documented the depressant effects of coronary artes, Ligation on myocardial

function in a canine rn~del.*~ In 1912, Hemck concluded that permanent coronary artenal occlusion

resulted in myocardial infarction." However, despite initial observations. researchers soon came to

realize that myocardial ischemia nsulted in varying outcornes based pnmarily upon duration of the

ischemic insult Blumgm and colleagues revealed that coronary ligation of l a s than 20 minutes

duration in anaesthetized dogs resulted in reversible injwy which could take hours to days to

resolve.lg Further studies would reveal that this reversible injury was associated with loss of tissue

high energy phosphates and contractile d y s f u n c t i ~ n . ~ ~ Braunwald and Kloner would later coin the

term "myocardial stunning" to describe this pend of reversible functional abnomality, which was

thought to be characterized by free radical mediated injury and defects in ionic home os ta sis?^

Moreover, Reimer and colleagues demonstrated that the injurious effects of repeated exposures to

short (10 minutes) episodes of ischemia were non-cumulative. and that ATP levels, although

decreasing by 60% after the first episode, did not decrease with additionai episodes of ischemid6

The finding that an ischemic stimulus need not necessarily be injunous introduced a

significant chapter in the study of ischemia. However, it was not until the mid 1980's that the

possible beneficial effects of ischemîa were described In 1986 Murry, Jennings an Reimer coincd

the term "ischemic preconditioning", to describe what remains by far the most powerful

endogenously mediated f o m of myocardial protection known? In their canine model, the degree

of myocardial infarction produced by a 40 minute circumflex coronary arte'y occlusion was reduced

by 75% when the myocardium at risk had fmt been subjected to four 5 minute coronary artery

occIusions, each separateci by 5 minutes of reperfusion.

Although original accounts of ischernic preconditioning were centered upon the nduction

of myocardial infarction, more recent studies have suggested an effect of preconditioning on

myocardial functional preservation which is independent of such infarct preventative effects.

Various investigators have shown that ischemic preconditioning preserves contractile function as

measured by increased recovery of myocardial segment shortening versus c~n t ro l s .~ Such results

have been observed in rodent,29 rabbit, pig;" and canine m ~ d e l s ~ ~ . In an isolated rat heaxt model,

Mitchell and coileagues demonstrateci that 2 minutes of global ischernia (with 10 minutes of

reperfusion) prior to a more prolonged 10 minute episode of global ischernia resulted in recovery of

84% of left ventricular developed pressure, in comparison to non-preconditioned hearts which

experienced only 54% recovery in developed pressure. Recent studies have also repoaed an effect

of ischemic preconditioning on the prevention of myocardial stunning in donor hearts following

cardiac transplantation in a sheep rn~del .)~

Later studies of preconditioning revealed that the protective attributes of preconditioning may

be indirectiy afforded to myocardial regions adjacent to areas at risk, thus suggesting the presence

of one or more humoral mediators which conferred the protective effects of ischernic preconditioning

and determined its distribution. Rzyklenk and colleagues were able to demonstrate this feature by

revealing a reduction in canine left anterior coronary artery disuibution infarrtion following brief

circumflex territory irherniaY To date, the most commonly implicated mcdiator in this process has

been adenosine: an endogenous nucleoside produced in a variety of organ systems.

ADENOSIN& Historier3 Overview

It is now 83 years since the first recordeci administration of adenosine in h ~ m a n s . ~ ~ Initial

interest in the potential therapeutic uses of adenosine arose in 1929 following a landmark report by

Dniry and Szent-Gyorgyi describing the isolation of crystailine adenosine monophosphate ( A m )

from extracts of ox heart muscle, as well as the observations of the effects of AMP and adenosine

on the heart and circulation of several mammalian species.% The authors determined that the agonist

activity of adenosine depended entirely on both the 6-amino group on the purine base as well as on

the ribose moiety.(Figure 1) The rate of metabolism determined the duration of action. The

eleçtrophysiological effects of adenosine were found to be sinus bradycardia and heart block, and

predisposition to one or both of these effects was entirely species dependent, Intravenous adenosine

injection in dogs terminateci paroxysmal atrial flutter and fibrillation. While adenosine was found

to have negative inotropic effects on the atrium, no effects on ventricdar performance were noted.

Adenosine was also shown to be a powerful coronary and peripheral vasodilator. The concomitant

hypotensive effects were the result of the combined effects of adenosine induced bradycardia and

peripheral vasodilatation. An accompanying defiuise in urine production was the result of a

decrease in glomerular filtration rate. Finally. in umscious animals, adenosine infusion caused initiai

"apprehension" and in larger doses, somnolence. Aithough most of the observations doçumented

were physiological in nature, Drury and Szent-Gyorgyi emphasized the generalizability of their

findings to the basic myocellular elernent, Thus, their innovative contributions formed the

foundation for two Lines of research, the first invesîïgating the d e of adenosine in organ physiology,

and the second examining the biology of adenosine and its associateci purinoreceptors.

Despite the rernarkable insights developed by these investigators. no mention was ma& of

the role of adenosine in cardiovascular physiology. Two years following the initial findings of Dniry

and Szent-Gyorgyi, Lindner and Rigler crystallized adenosine from heart muscle extracts and

confîrmed its potent coronary vasodilatory p p e a i e s in a number of species." Based on adenosine's

existence in the heart and its vasoactive properties, Lidner and Rigler hypothesized that the main

physiological role of adenosine was to regulate coronary blood flow in vivo. Unfortunately, this

concept gained Little support and the advent of adenosine quickly fell from the forefront of clinical

research. With the exception of a smail series of publication~.u-'~ interest in the cardiovascular

effects of adenosine languished for the next three decades.

Modern adenosine research was not revived until the mid 1960's, when two landmark studies

by Berne and Gerlach dexnonstrated the release of adenosine catabolites from ischemic or

hypoxic heart muscle. Modifications of an enzymatic spectrophotometric assay made it possible to

demonstrate that adenasine exists in nomally oxygenated as well as ischemic h m muscle?

Further work attempted to document a relationship between cardiac oxygen consurnption, interstitial

adenosine concentrations and coronary blood flow? Although such studies failed to demonstrate

a consistent relationship between these variables. they yieldeâ much insight into issues of cardiac

purine metabolism, including the existence of a unique intracellular adenosine cornpartment

consisting of adenosine bound to S-adenosylhomocysteine hydrolase (sAH)> the role of the SAH

pathway as a source of adenosine. and the potential importance of the coronary endothelium in

cardiac purine metabolism.

The discovery in 1970 of the adenosine-stimulated accumulation of adenosine 3',5'cyclic

monophosphate (CAMP) in brain slices and the specific antagonism of this effect by theophyllines5

was the first definitive evidence of the existence of specific adenosine receptors. In fact,

theophylline denvatives were found act as competitive antagonists against adenosine

receptors.(Figure 2) Further studies would soon reveal receptor subtypes mediating either the

stimulation or inhibition of acknylate c yclase. the rate lirniting enzyme involved in the formation of

CAMP.% Perhaps most signifiant, however, was the discovery of adenosine receptors which were

coupled to cardiac effectoa other than adenylate cyclase, including G-proteins and potassium

~ h a n n e l s . ~ ~ ~ Adenosine has since reached the forefront of cardiovascular research due largely to

increasing evidence suggesting i ts role in the regulation of normal cellular functions via control of

both intra- and extracellular metaboiic processes.

ADENOSINE METABOUSM

Establishing adenosine's role as a regulator of physiological or metabolic functions requires

identification of: 1) the mechanisms of production and delivery to the target organ; 2) the

mechanisms by which adenosine is degradeci or removed h m its site of action; and 3) the relation

of cellular energy state to the concentration of adenosine at its receptors.

Endogenous Adenosine Production

Adenosine is produceci by the enzymatic hydrolysis of either of two ubiquitous substrates,

adenosine monophosphate (AMP) or S-adenyl homocysteine (SAH).(Figure 2)

1) Adenosine from hydrolysis of AMP. Hydrolysis of S'-AMI? by 5'-nucleotidase accounts

for the vast majority of adenosine production in heart muscle, liver, and blood leukocytes. Five'-

nucleotidase is present in two forms, membrane bound (ecto-5'-nucleotidase) and free in the

cytoplasm (cytosolic-5'-nucleotiàase), both of which are thought to contribute synergistically to

adenosine production during myocardiai ischemia? AMP is deriveci from a number of intracellular

sources, including cytosolic and mitochondriai stores, as well as from extracellular sources of

adenine nucleotides such as platelets and endothelium?'

Although a variety of factors combine to influence adenosine production by the heart, leveis

are primarily increased when myocardial oxygen demand exceeds supply, thus influencing cellular

energy stateO6' The metabolic pathway that generates adenosine h m adenosine triphosphate (ATP)

has two key linkages to cellular energy state? Fit, the consumption of ATP determines the

availability of ADP which subsequently undergoes dismutation by myokinase to form AMP, the

immediate precursor of adenosine. Second, AMP exists at a branch point in the pathway of A T '

degradation.(Figure 3) The cytosolic ATP potential (the chernical potential that drives ATP-

consuming reactions and regulates respiratory rate) mediates the catalytic activities of the two

enzymes which degrade AMP to form adenosine, namely, 5'-nucleotidase and AMP cieaminaseb3

Global oxygen deficit is not a necessary precondition for adenosine production. In fact, a

significant amount of admosine is produced during nonn~xia.~'" Numerous studies have shown

that cyclical flow through the microcirculation of the heart often produces spatial and temporal

heterogeneity of tissue oxygenation despite normoxic conditions- Such small, local imbalances

in oxygen supply and demand wuld collectively act as the stimulus for adenosine production in an

organ which is otherwise well perfused and well oxygenated. Thus, global ischemia may act to

enhance overall adenosine production.

II) Adenosine from hydrolysis of SAH. S-adenosylhomocysteine (SAH) is a byproduct of

transmethylations in which S-rdenosylmethionine (SAM) is the methyl d ~ n o r . ~ ~ S A H is hydrolyzed

by SAH-hydrolase to adenosine and homocysteine. In isolated perfused guinea pig hearts, the overall

adenosine production rate has been reported to be very similar to the hydrolysis rate of S M ,

suggesting that during normoxia, adenosine is mostly produced from S AH? During ischemia and

hypoxia, adenosine release increases approximately 50-fold, while the transrnethylation rate

~~~es only 1.5-fol& Although SAH contributes relatively Iittle to overail adenosine production,

(maximum measurable levels are enough to account for the adenosine found in the cardiac

intentitium 0 n . l ~ ) ~ ~ its additional role as an adenosine binding protein accounts for the large

intracellular pool of adenosine in most tissues? In in-situ dog h e m , the intracellular

cornpartment has been shown to account for 9 0 % of the total adenosine pool? Uniike the case

with AMP, no correlation exists between the catalytic activity of SAH and cellular energy state,

suggesting that SAH derived adenosine has no role in the metabolic regulation of coronary blood

fl0w.(j2

III) Adenosine h m hydrolysis of extracellular adenine nucleotides.

Adenosine Transport

Adenosine crosses ceii membranes by specific nucleoside transport (facilitated diffusion) or

via passive difision into tells?- The carrier which mediates facilitated difision controis bath

the uptake and release of adenosine, and may transport other nucleosides which may in tuni act as

cornpetitive inhibitors of adenosine transpoxtm In dog h m . this c h e r has been shown to be

particularly sensitive to stnicniral modifications in the ribose moiety?' The nbosides which are the

best known inhibitors of adenosine transport include 6-S-(p-nitrobenzy1thio)guanosine (NBTGR)

and 6-S-(pnitrobenzy1thio)inosine (NBMPR)." A number of dnigs, including theophylline and

cafTeine have also bem show to inhibit adenosine eanspon both in vitro and in The

sensitivity of adenosine transport to inhibition varies among ce11 lines," between species;' and

between different ce11 types within the same organ (Le. cardiomyocytes and endothelial celIs within

the heart)."

Although the intracellular catabolism of ATP generates extracellular adenosine, only a small

portion of this intracellular adenosine is exporte4 the remainâer king bound to SAH or recycled

to AMP via either the adenosine kinase or purine saivage pathway~?'~~ Extracellular adenosine,

whether formed by catabolism of extracellular nuclentides or released h m within cells, is efficiently

sequestered by transport into endothelial cells on passage through myocardial capillary bedsW*=

This process is saturatable, and is effectively inhibited by dipyridamoie (Figure 2). Dipyridamole,

cornmoni y utilized for pharmacologie cardiac stress testing, inhibits the transport of adenosine

intracellularly, resulting in a net exiracellular accumulation of adenosine." in s o w species,

sequestration is also accompiished by red blood cells."

Adenosine catabolism

Adenosine is cleared h m tissue via phosphorylation to AMP by adenosine kinase, or via

deamination to inosine by adenosine deaminase. Although extracellular adenosine deaminase does

exist (ecto-adenosine deaminase), both enzymes are primarily located within the ce11 thus

necessitating transport intracellularly prior to degradati~n.~ The purine salvage pathway which can

recycle hypoxanthine to IMP and AMP, ensures that purines are not irretrievably lost with adenosine

deamination.(Figure 3) Although in the heart, exogenous adenosine is pnmarily taken up by vascular

endotheli um and incorporateci into the cellular adenine nucleotide pool, this process is easil y

saturatable, allowing for maintenance of measurable extracellular concentration^.^*^^ SiMlarly,

although endogenous admosine is largely cataboiized intraceliularly (as is evident by the steady-state

release of adenosine degradation products h m the hem) a certain amount is exporteci or maintained

extracellularly pnor to d e g r a d a t i ~ n . ~ ~ ~

In those organs which have been studied to date, much of the available adenosine deaminase

exists within the endothelial ceils of the appropriate vascular bedg3*% Moreover, studies of both rat

and rabbit cardiomyocytes have shown littie or no inuacellular adenosine deaminase.

ReguCtrtion of InterstitUJ Adenosine Concentrations

Adenosine present within the interstitiun of the heart represents the physiologically active

fraction that is available to react with ce11 surface adenosine receptors. Adenosine enters the

interstitium either by release from parenchymal cells or by ecto-phosphatase hydrolysis of

extracellular adenine nucIeotided2 Adenosine is removeci from the interstitial cornpartment by

uptake into parenchymal ceils. by washout into the venous drainage and lymphatics. or via

degradation by cell surface ecto-adenosine deaminase. Studies employing radio-labelled adenosine

in isolated rat hearts have shown that no more than 15% of interstitial adenosine cornes from

endothelial ceils. the remainder k ing released by cardiomyocyte~.~~~

ADENOSIN' RECEPTORS

Studies demonstrating the inhibitory effect of theophylline on adenosine-stimulated

accumulation of CAMP in brain sections provided the first definitive evidence for the existence of

specific adenosine receptors.'' Burnstock et al subdivided adenosine receptors into two types

depending upon the nanual ligands that they recognizcd: P, ceceptors recognize adenosine (and

possibl y AMP), and P, receptors recognize ATP and A D P . % ( T ~ ~ I ~ 1) Van Calker et ai. further

subdivided P, receptors into A, and 4 varieties based upon their effects on aden y late cyclase, A,

being inhibitory and A, being ~tirnulatory.~ Classification of adenosine receptors has since been

accomplished based upon both pharmacologie and biochemical critena, Isolation of the diffenng

receptor subtypes was made possible by the discovery that adipocyte membranes contained only A,

receptors. while ptatelet membranes expressed only A, receptors? A, receptors have been shown

to have a very high affinity for adenosine, requiring agonist concentrations in the nanomolar range

for activation. Although activation of this receptor has no effect on basal adenylate cyclase activity,

activation inhibits the receptor medîated stimulation of this enzyme by altemate agonists.

Conversely, A, receptors are low affinity receptors having an affinity for adenosine approximately

three orders of magnitude lower than that of A, receptors?

In vitro, adenosine has also been found to inhibit adenylate cyclase through a distinctive "P

site". Although the P site has been describecl as a ligand binding peptide , the characteristics of this

site make it quite different from a receptor in that inhibition is only seen under conditions where

adenylate cyclase is fimctionally uncoupled h m its G protein.(see section on signal transduction)62

Whereas A, and 4 receptors are activated by nanomolar and micmmolar concentrations of

adenosine respectively, inhibition of the P site requires concentrations of adenosine in the

rnicromolar to millimoiar range. Selective antagonists to the P site remain unhown. and its

physioIogical role remains undetermined-Çrable 2)

More recently, a newly identified adenosine receptor, the A, subtype. was found to be

expressed on both animai and human ventricular ~a rd io rn~ocy tes .~ '~ ' In a cultured chicken

ventricular m yoc yte model, the protec tive effects secondary to adenosine A, receptor activation were

found to exceed (in duration ) those related to A, or 4 receptor activation.'* The selective 4

receptor antagonist MRS 1 19 1 caused a biphasic inhibition of the protective effects of anoxic

preconditioning. When the A, receptor antagonist DPCPX was applied simultaneously, the biphasic

dose inhibition cwve was converted to a monophasic curve. Thus, activation of both A, and A,

receptors was reqtiired to mediate the cardioprotective effects of anoxic preconditioning. In the sarne

model, cardiac atrial ceils were found to lack native A, receptors, possibly accounting for the shorter

duration of cardioprotection following preconditioning. However, when atrial cells were transfected

with cDNA encoding the human adenosine A, receptor, a prolonged duration of cardioprotection was

demon~trated.'~ In rabbits, selective activation of the adenosine A, receptor reduced infarct size in

a Langendorff model of myocardial iwhemia."' Furthennoce, the degree of A, dependent

cardioprotection was similar to that provideci by A, receptor stimulation or ischemic preconditioning.

Finally, in a mode1 of superfuseci human atrial trabeculae exposed to ischemia and reperfusion,

selective stimulation of both A, and 4 adenosine receptors conferred protection similar to that

observai with ischemic preconditioning, as assessed via recovery of baseline contractile f u n ~ t i o n . ~ ~

Thus, the cardiac adenosine A, receptor is believed to mediate a sustained cardioprotective effect

during prolonged ischemia and reperfusion, and may represent a new cardiac therapeutic target.

SIGNAL TRANSDUCTION

Al1 living cells must possess the ability to interact with their surrounding environment Such

communication between extracellular and intracellular compartments requires an inmcate

transrnembranous system by which cells can respond to or react to extracellular stimuli. One of the

methods by which this can be achieved in eukaryotic organisms is via signai transduction-

Extracellular signals represented by Ligands either penetrate ce11 membranes or activate extemal

membrane receptors. Stimulated receptors, in association with membrane bound tramducers, may

then activate intracellular effector mechanisms either direct1 y or indirect1 y ." Through these

transduction systems, extraceliular ligands may act as cofactors for intracellular enzymes.

The adenylate cyclase system was the fmt plasma membrane signal transduction system to

be ~haracterized.'~ Its specificity and simplicity provided an ideal mechanism for the actions of

adenosine on its receptors. Not surprisingly, initial adenosine nomenclature was based solely upon

the adenylate cyclase system. Further studies, however, would reveal the coupling of adenosine

receptors to a variety of cellular effector systerns based upon diffenng transduction mechanisms-

Among the most highly characterized transduction mechanisms are the G proteins. These

heterotrimeric proteins, so named because they bind guanine nucleotides, consist of a-, f3-, and y-

subunits and play a pivotal role in coupling ce11 surface receptors to one or more effector systems.

This coupling effect cm be characteristically blocked by ribosylation of G proteins with pertussis

toxin. The a-, f%, and y- subunits differ fiam one kind of G protein to another in molecular size and

structure as well as in îùnction? It has been suggested that the diversity of subunits may provide

the means by which one kind of receptor is coupled to more than one kind of effector

mec hanism. '03-'05 Mo st functional differences between G proteins seem to be dependent on the

molecularly heterogeneous and hydrophilic a- subunits. Thus, A, receptors were believed to be

coupled to inhibitory G pmteins (Gï) thereby inhibiting adenylate cyclase activity, whereas A,

receptors were beiïeved to be coupled to stimulatory G proteins (Gs) thereby stimulating adenylate

cyclase activity.'06 Converseiy. the k, and y- subunits are more hydrophobie and tend to remain

associateci as a single @,y- cornplex after the dissociation of the a- subunit during signal transduction.

The B,y- subunit also functions to anchor the a-subunit to the ce11 membrane. Although les diverse

than the a- subunits, recent evidence suggests two types of $-, and two types of y- subunits, thus

implying four possible kinds of f3.y- complexes, each able to influence the selective coupling of a

receptor to its effector. Thus, at the level of G proteins there are a variety of means by which to

confer selectivity on the transduction of a signal from a receptor end effector. W c h , if any of these

factors regulate adenosine receptors is unhown. M m v e r , animal data linking both ischemic and

adenosine preconditioning to G protein stimulation is controversial. Thornton and colleagues

reported thac pretreatment of isolated perfused rabbit hearts with pertussis toxin blocked the

protective effects of ischemic preconditioning. lm However, studies using rat models have been more

variable. Although Lasley et al reported that pertussis toxin blocked adenosine A, mediated

protection of the ischemic rat heart,'" Liu and colleagues demonstrated that preconditioning against

infarction in the rat hem did not involve a pemtssis toxin sensitive G protein.'"

Adenosine Effector Mechanisms

Despite initial beliefs, little confimatory data was available with which to support the

adenylate cyclase hypothesis of preconditioning, and available data was controversial. Although

Szilvassy and colleagues demonstrated a reduction in adenylate cyclase activity in preconditioned

rabbit hearts, both Iwase et al and Fu et ai showed no effect on adenylate cyclase activity with

preconditioning of rabbit and swine h e m , cespectively. ""'* Further rescarch into the mechanisms

of signal transduction suggested that adenosine expressed its biological actions through effectors

other than adenylate cyclase. The ability of G proteins to couple ce11 surface receptorç to more than

one effector mechanism supported such a possibility. The first example of an altemate mechanism.

A, receptors coupled to potassium channels, was discovered in cardiac tis~ue.~*l" Although most

adenosine raceptors acting via altemate effectors are of the A, variety, it is not h o w n whether this

functional diversification reflects the coupling of a single kind of A, receptor to different

transduction mechanisms or whether there is molecular diversity of A, receptors similar to that of

other recept0rs.6z"~

I) A,- POTASSIUM RECEETOR: in the 1970's. electmphysiological studies involving atrial cardiac

tissue revealed that adenosine shortened cardiac action potential duration by facilitating an outward

potassium conductance. Further studies employing patch clamping and protein modification

techniques revealed that G proteins were involved in the signal transduction mechanism for this end

ef fec t~r .~~" '~

Il) A,- GUANYLATE CYCLASE RECEPTOR: Adenosine promotes an accmulation of cGMP in

cultures of aortic smooth muscle cells and stimulates a guanyiate cyclase in partially purifieci plasma

membranes from aortic media.

III) A,- CALCIUM RECEVTOR: Several studies in nerve cells have shown adenosine receptors to

be coupled to calcium channels via specific G prote in^.'^'*'^' To date. however. no such receptor

complexes have been identified in the hem.

IV) A,- GLUCOSE RECEETOR: Adenosine has been shown to stimulate glucose transport in

adipocytes independent of CAMP.^'^ These receptors have also been dernonstrated in myocardium.

where adenosine increases insulin-stimulateci glucose uptake above maximal levels, likely by

increasing the V,, of the glucose tran~porter.'~~*'~' Such an effect suggests an effect of adenosine

which is distal to the insulin receptor. This receptor-mediated effect on glucose transport

overshadows the direct inhibitory effwt (competitive inhibition) of adenosine on the glucose

transporter. In fact, A, receptor bloc ka& prevents insulin-stimulated glucose uptake, suggesting that

insulin's effect on glucose uptake is dependent upon activation of the adenosine receptor."

V) A,- PHOSPHOLIPASE C+ and A, - PHOSPHOLJPASE C- RECEVTORS: Adenosine has an

indirect effect on the histamine Hl receptor-initiated hydrolysis of membrane inositol phospholipids

by phospholipase C, and a direct effect on cellular froe fatty acid production by phospholipase-A2.

62,122

The coupling of adenosine A, nceptor stimulation to the hydrolysis of membrane

phospholipids implicated yet another possible signal transduction mechanism. In 1983. Streb and

colleagues revealed that inositol-1.4,s-triphosphate (IP3). a product of the hydrolysis of membrane

phosphatidyl 4.5-biphosphate (PIPd. was released into the cytoplasm Iikely in response to

extracellular receptor stimulation. The release of IP,, in turn, resulted in the mobilization of Ca2+

from intraceliular stores.'* The other product of PlP2 hydrolysis. diacylglycerol (DAG), was found

to remain within membranes. and to facilitate the activation of a specialized enzyme h o w n as

protein kinase C (PKC).'~

PROTEIN KINASE C

The protein kinase C (PKC) family of enzymes transduces a number of signais which

promote Lipid hydrolysis. The prevalence of PKC in cellular signalling is partially attributable to the

diverse transduction mechanisms that result in the production of protein kinase C's primary

activator, diacylglycerol (Figure 4).lZ Stimulators of G-protein-coupled receptors, tyrosine kinase

receptors or non-receptor tyrosine kinases can promote DAG production either rapidly, by activation

of specific phospholipid Cs o r more slowly, by activation of phospholipase D to yield phosphatidic

acid and then diacylglycerol. PKC activity may be M e r mediateci by phospholipase A, dependent

fatty acid generation. Phorbol esters, also known to be activators of PKC, result in prolonged

activation due to their long half-life in vivo. Regardless of the method of activation, some PKC

isozymes require ionic Ca2+, and all PKC isozymes require the cytoplasmic lipid phosphatidylserine,

for their activation.

Al1 PKC isozymes have in common their single polypeptide structure consisting of an N-

terminal regulatory region and a C-terminal catalytic region. After initial cloning of selective

isozyrnes in the mid-1980's. Coussens and colleagues reported the presence of four conserved

domains, C1-C4.126 The Cl domain forms the diacylglyceroYphorbo1 ester binding site and is

immediately preceded by an autoinhibitory pseudosubstrate ~equence . '~~ The C2 domain forms the

recognition site for acidic Iipids, and in sorne isozymes, the Ca2+ binding site. Calcium increases the

affinity of conventional protein kinase Cs for negatively charged lipids.'" The C3 and C4 domains

represent the ATP and substrate binding sites of the kinase ~ o r e . ' ~ ~ The regulatory and catalytic

halves are separated by a hinge that becomes proteolyticaily labile when the enzyme becomes

membrane bound, thus fieeing the kinase domain h m inhibition by the pseudosubstrate, rendering

the enzyme

To &te, 11 PKC isozymes have been identified and are classifiai into three groups based on

their structure and cofactor reg~lation.''~ The a, $ (variants 1 and II), and y isoymes were the fmt

to be characterized and are distinguishable by their regulatory Ca2+ binding site. The next well

characterized are the novel PKC isozymes which include 6. E. q, 0, and p. These isoymes resemble

the conventional PKCs with the exception of their C2 domain which does not contain a ca2+ binding

site. The third group is comprised of the atypical isozymes and A. which are the least well

charac terized. These isozyrnes di ffer si gnif icantly in structure h m the more typical isozymes and

are insensitive to phorbol esters both in vitro and in vivo.

Protein kinase C typically phosphorylates senne or threonine residues. however. displays fat-

less specificity than other conventional kinases such as protein kinase A.')' Moreover, unlike protein

19

kinase A, PKC has the ability to autophosphorylate in vitro?' In addition to catalyzing

phosp hory lation reactions, PKC possesses both ATPase and phosphatase activities.

Under resting conditions, PKC is present mainly within the cytoplasm in an inactive form.

PKC is rendered catalytically comptent by phosphorylations which correctly align residues for

catalysis. These same phosphorylations localize protein kinase C to the cytoplasrnic compartrnen~'~

Stimulation of PKC is associated with removal of its pseudosubstrate h m the kinase core rendering

the enzyme active? Accompanying this activation is a rapid CaZ* dependent translocation of PKC

to membraks, possibly dong cytoskeletal structures such as rnicrotubuledn This translocation has

been shown to be stirnuiated by DAG and phorbol esters. Both DAG and phorbol esters act as

hydrophobie anchors to attract protein kinase C to the membrane while increasing the enzyme's

membrane affinity."' Thus, PKC is regulated via two distinct mechanisrns: by phosphorylation

which reguIates the active site and subceilular localization of the enzyme, and by second messengers

which promote PKC's membrane association and resulting pseudosubstrate exposure.'"

CARDZOPROTECTWE PROPERTIES OF ADENOSINE

Various mechanisms have been proposed for the cardioprotective properties of adenosine:

1) Coronan, v m ~ : Adenosine release by cardiomyocytes during episodes of ischemia or

hypoxia may facilitate coronary vasodilatation via stimulation of A, receptors. The nsultant increase

in myocardial pemision improves metabolic function and thus, contractility. 139.lo0

2) Antiadrener-: Adenosine may be released to counteract the stimulatory effects of

catecholamines on cardiac function via A, receptor stimulation and by inhibiting the reiease of

noradrenaline from sympathetic newes. The resultant decrease in myocardid oxygen demand may

thereby confer prote~tion."~*~"

3) Protection qf e-: Adenosine inhibits neutrophil adherence to endothelial cells and

prevents neutrophil release of oxygenderived free radicals from neutrophils via A, receptor

stirn~lation."~*~~

4) Prevention 4fmicrov&r o b ~ c t & : Adenosine inhibits platelet aggregation and platelet

adherence to endothelial cells via A2 receptor activation. 145.146

5 ) hcreased e n e s t o m : Adenosine may facilitate glucose uptake by cardiomyocytes and

stimulate glycolysis (indirectly, by increasing glucose-6-phosphate levels), thereby promoting the

production of highenergy phosphates. In addition, exogenous adenosine may replete energy stores

by acting as a nucleoside substrate for the creation of AMP, ADP m d ATP.'".'"

6 ) NeovaKldmzaa . -

: Adenosine is believed to increase the proliferation of endothelid ceïls and

to promote myocardid neovascularization undcr conditions of prolonged hyp~xia."~

7) P ~ ~ ~ Q . U ~ - n ~ n g .. .

: Adenosine, believed to be a mediator of the ischemic preconditioning

phenornenon, may reproduce the beneficial effects of ischemic preconditioning via an A, receptor

mediated proces~."~(~igures 4.5)

Notwithstanding the above hypotheses, the role of adenosine in myocardial preconditioning

has becorne most intriguing to b t h scientists and clinicians alike, owing to its profound protective

properties and possible implications in clinical practice. The following review will concentrate on

adenosine and its A, receptor effccts as they pertain to the mediation of ischemic preconditioning.

EXOGENOUS ADENOSINE STUDIES

To test the adenosine hypothesis, Olfsson and colleagues infused adenosine into the left

anterior descending artery of dogs following a 90 minute occlusion. After 24 hours, the size of the

infarct in the adenosine group was 10 percent of the myocardid region at risk cornparrd with 41

percent in the control group. In addition, regional and global left ventricular hmction was improved

in the adenosine group in cornparison with c ~ n t r o l s . ~ ~ Sirnilarly, in a canine model of 120 minute

left antenor descending artery ligation, Babbit and colleagues demonstrated decnased myocardiai

infarction and improved function with adenosine infusion. The protective effects of adenosine wen

lost, however, with 180 minute vesse1 occlusion."' Intravenous infusions of adenosine have also

been s h o w to be protective. Pitarys et ai found an 18 percent reduction in canine infarct size and

an improvement in regional myocardial function following a 90 minute occlusion of the left anterior

descending artery preceded by a one hour intravenous adenosine infusion at a rate of 150

~ g l k g l m i n . ' ~ As was the case with irhemic preconditioB&, adenosine has also been

demonstrated to preserve ventricular function independent of any effects on myocardial infarction.

Lasley et al. demonstrated the attenuation of in vivo myocardial stunning with direct intracoronary

adenosine administration in a porcine model of regional ischemia and reperfusion,lF) while Thourani

et al. revealed adenosine A, stimulation to prevent pst-ischemic cardiac dysfunction in isolated

perfused rat hearts.lY

Downey and colleagues employed an open chest rabbit mode1 of myocardial infarction to test

the adenosine hypothesis of ptecondit i~ning.~ In this model, chemically pnconditioned myocardial

regions demonstrated smaller infarct size compareci to non-preconditioned controls. To test the

adenosine hypothesis of preconditioning and to determine whether adenosine's effects were substrate

mediated or receptor mediated, the authors administered two broad acting adenosine receptor

blockers (8-psulphophenyl theophy lline and N-[Z-(dimethy lamino)eth y l]N-methy14(2,3,6,7-

tetrâh ydro-2,6-dioxo-l,34ipmpyl- 1 H-purh-8-y) pnor to prolonged ischemia. Adenosine receptor

blockade abolished the protective effects of ischemic preconditioning, resulting in infarct sizes

similar to those of non-preconditioned controls. To confinn their suspicions, the same group infused

exogenous adenosine directly into the coronary artenes in a blood perfused isolated rabbit heart

model. Intracoronary adenosine was found to protect the isolated heart to the same degree as

preconditioning.'" Moreover, the protective effects penisted well beyond the pmjected half life of

adenosine in blood (4 seconds), suggesting activation of a second messenger pathway via adenosine

receptor stimulation. To detemine which adenosine receptor was involved in the protective effects

afforded by adenosine, the authors employed both selective A, and A, receptor agonists. N6-

(phenyl-ZR-isopropy1)-adenosine (PIA), which is 100 times more selective for the A, than the A,

receptor, and 2chlor0-Nd-çyclopentylaQnosine (CCPA), which is 10,000 times more selective for

the A, receptor, were both able to repduce the beneficiai effects of ischemic preconditioning when

administered in isolated perfked rabbit hearts or intravenously in in-situ rabbit hearts preparations.

Conversely, 2-[4(2carboxyethyl)phenethylamino~'-NthyIcxdoa&nosine hydrochloride

(CGS 21680), a selective A, agonist, did not provide protection under similar ckurnstances.

Although A, selective agonists were found to k protective. the authors were unable to block the

protec tive effects of ischemic preconditioning with the highiy selective A, antagonist 8-cyclopen tyl-

1'3-dipropylxanthine (DFCPX) using a rabbit model. However, DPCPX has k e n show to block

the protective effacts of ischemic pnconditioning in the Such disc~pancies are not surprising

owing to the large degree of species variability in adenosine receptors and their effects. The resulu

of the aforementioned studies support the hypothesis that ischemic preconditioning is mediated by

the A, adenosine receptor and that the anti-infarct effects of ischemic preconditioning can be

reproduced with both exogenous adenosine and A, receptor agonists. In separate studies by Brown

et al and Stiles et al, adenosine A, receptors have been shown to be coupled to peaussis toxin

sensitive G prote in^.'^*'^ Similarly, Downey and colleagues demonstrated that pertussis toxin

blocked protection when administered inuavenously prior to preconditioning in an in-situ rabbit

heart model.'" Interestingly enough, partial protection has also been shown with stimulation of

muscarhic or cholinergie receptors coupled to the same G p r ~ t e i n . ' ~ ~ * ' ~ ~

Despite the data suggesting a receptor mediated effect of adenosine preconditioning, some

studies suggest that adenosine may also function via a substrate mediated effect. In isolated

retrograde perfused rabbit hearts, Bolling and coiieagues demonstrated myocardial fùnctional

recovery only in animals which received adenosine and not in animals which received adenosine

receptor ag~n i s t s . ' ~~

Although the vast majority of adenosine research has centred around its pnischernic and

ischernic effects, some studies have suggested a role for adenosine in the mediation of reperfusion

injury. Forman and coileagues evaluated the effects of adenosine in a closed chest canine

preparation subjected to 90 minutes of proximai left antenor descending coronary artenal occlusion

and 24 hours of r e p e m i s i ~ n . ' ~ ~ Intracoronary administration of adenosine 60 minutes following

reperfusion reduced infarct size by 75% compared to ischemic controls. These findings were later

conf i i ed by Homeister e t al in an open chest canine model of circumflex ~cc lus ion . '~~ Since the

canine heart is typically highly collateralized, Forman and colleagues set out to evaiuate the

reperfusion effects of adenosine in the more poorly collateralized rabbit heart. Various doses of

intravenous adenosine administered during the f m t 60 minutes of reperfusion were evaluated in a

rabbit model subjected to 30 minutes of circumflex coronary arterial occlusion.'" Both high and low

doses of adenosine significantly reduced histologically determined infarct size. Studies involving

selective Al receptor agonists revealed similar degrees of myocardial salvage following ischemia,

thus suggesting a receptor mediateci effect of adenosine on myocardial reperfusion injury.'"

Kitakaze and coileagues hypothesized that ischemic preconditioning increases adenosine

production during ischemia by augmenting 5'-nucleotidase activity. In ischemicaliy preconditioned

canine hearts, both ecto- and cytosolic-5'-nucleotidase activities were increased as was adenosine

release during prolonged ischemia and reperfusion." Protein kinase C (PKC) activity was also

found to increase during ischemia and repefiion.'"la The authors proposed this augmentation in

PKC activity to be a further stimulus for adenosine production.

ISCHEMIC PRECONDITIONING IN HUlMANS

Although various authors have documented some f o m of preconditioning in humans. the

degree of success is variable and resdts are somewhat controversial. Initial repts involved

observations of patients undergoing cardiac stress testing. In 1980 JaRe demonstrated a reduction

in ST segment depression in patients who undenvent two consecutive exercise tests separated by 30

minutes of walking and 20 minutes of rest.16' Similarly. Williams and coiieagues demonstrated an

increased exercise tolerance in patients undergoing the second of two consecutive periods of pacing-

induced angina separated by 5-15 minutes of reperfu~ion. '~~ Such findings led some clinicians to

propose a possible cardioprotective effect of stable angina prior to infarction. Muiler and colieagues

reviewed 775 patients who received pst-infarction reperfusion with either thrombolysis or

angioplasty and found that those patients with previous chronic angina demonstrated lower

reinfarction rates and lower in hospital moitality rates.''' Similarly, Kloner reported that patients

who experienced angina within 48 hours of infarction displayed smaller infarct sizes, fewer

complications of infarction, and lower in hospital mortaIity.ln

Subsequent studies would attempt to confirm the ability to precondition human myocardial

tissue in vitro. To demonstrate preconditioning, Walker and colleagues utilized a mode1 of isolated

right atrial trabeculae exposed to a 90 minute episode of rapid atrial pacing in conjunction with

h ypoxic pemision (contmls). Trabecular preparations exposed to a 3 minute episode of rapid pacing

and hypoxic pefision (followed by slow pacing and normoxic perfusion) pnor to the more

prolonged episode (preconditioned group) demonstrated a preservation of developed pressure in

cornparison to controls."' FinaUy, Ikonomidis et al reponed the ability to precondition culiured

human ventncular myocytes using a bnef 20 minute ischemic stimulus prior to a more prolonged

(90 minute) episode of irhemia." Reconditioned ceLls were less susceptible to ischemic injury and

demonstrated a marked high-energy phosphate preservative effect.

Despite such promising results, the clinical applicability of ischemic preconditioning

remained in question. Attempts to apply these findings to the clinical scenario included angioplasty

studies and studies of intermittent crossclamping in patients undergoing CABG. Kerensky et al.

examined Mne patients who experienced acute ST segment elevation during bailoon angioplasty with

complete resolution during the procedure. Seven of the nine had far less ST segment elevation with

the second balloon inflation, suggesting that preconditioning had occurred.'" Similarly, Deutsch and

colleagues demonstrated that in patients undergoing bailoon angioplasty, the second of two

consecutive 90 second balloon occlusions was associated with less anginal discornfort, less ST

segment depression, and a reduction in coronary sinus lactate pr~duction."~ Alkhulaifi and

colleagues randornized twenty patients undergoing elective CABG to receive either two 3 minute

crossclamp periods (each followed by 2 minutes of reperfusion) pnor to prolonged ischernia

(preconditioned group) or prolonged ischemia alone (control group). Intraoperative myocardial

biopsies revealed a preservation of tissue ATP levels in the preconditioned group and a significant

lowering of creatine phosphate 1evels.l"

Ho wever, subsequent studies of preconditioning in humans would yield contradictory resul ts.

In a review of 4,447 patients who suffered myocarrlial infarction, Barbash reported that previous

angina was associated with a higher in-hospital mortality-ln Sirnilarly. severai angioplasty studies

failed to show any advantage to intermittent bailwn occlusions for the purposes of

preconditioning. 178~'79 Moreover, initial favourable results were attn buted to the recmitment of

coronary artenal coiiaterals. Finally, Menasche et. ai. reported that patients preconditioned with 3

minutes of crossclarnping prior to institution of cardioplegia revealed increased levels of creatine

kinase MB and lactate release at the end of cardioplegic arrest.lm In addition, molecular biology data

previously shown to be related to the preconditioning proçess (i.e. expression of m-RNA for both

c-fos and heat shock protein 70) did not suggest a protective effcct of preconditioning.

Nonetheless, studies of ischemic preconditioning in humans, although inconclusive, have

been sornewhat promising. Perhaps variable results are attributable to the variable consequences

associated with the preconditioning stimulus, which in this case is ischemia. Ironically, the beneficial

effects of ischemic preconditioning may be maskeà by the detrimental effects of the initial brief

ischemic insult. Thus, researchers are stniggling to find a way to reproduce preconditioning without

the need for ischemia. A pharmacological mediator which could harness the beneficial effects of

preconditioning would be ideal in this regard.

Various agents have been shown to repmduce, to some extent, the beneficial effects of

ischemic preconditioning. Direct preconditioning via stimulation of opioid receptors has b e n

demonstrated in both animal1'' and clinical experirnent~!~~ Similarly, preconditioning-mimetic

effects have been shown with norepinephrine administration prior to prolonged ischemia and

reperfusion in isolated rat hem '" or superfuseci rat trabeculae." Nitrïc oxide (NO) and the NO

donor L-arginine have aiso been demonstrated to provide protection to isolated perfused rabbit hearts

exposed to ischemia and repemision.'" Inhalational anaesthetics such as isofiurane and sevoflurane

have been suggested to enhance the functional recovery of pst-ischemic reperfused myocardium,

possi bl y via activation of AT'-sensitive ion ~hanne1s.l~~" Other agents whic h have been proposed

to posess preconditioning effects include insulin," and monophosphoryl iipid A,''' in addition to

physical phenornenon such as thermal stunulationLgO and hyperdynamic circu~ation.'~'

However, none of the aforementioned have been found to be as consistent or as effective in

promoting myocardiai protection as adenosine administration or upmgulation. Adenosine, released

in significant amounts during myocardial ischemia, may represent an ideal mediator for the

protective effects of ischemic preconditioning.

Adenosine was füst administered to humans in the early 1900's and continues to be utilized

clinically as a first line antiarrhythmic agent. Recently. adenosine has reached the forefront of

clinical research due large1 y to its presumed cardioprotective properties.

EXOGENOUS ADENOSINE IN HUMANS

Physblogic Eflects

The physiologie efiects of adenosine are determined by the particula. type of receptor present

within the effector tissue.(Table 3) Adenosine A, receptors are present within the cardiomyocytes

which mediate the sinus slowing and AV-blocking actions of adenosine. Conversely, A, receptors

are found in both endotheiial and vascular smooth muscle cells and stimulate comnary vasodilatation

when activated? Although improving coronaiy blood flow, adenosine depresses myocardial

function acutely by reducing heart rate, slowing AV conduction, and antagonizing the inotropic

effects of catecholarnines. The resultant increase in oxygen supply and decrease in myocardial

oxygen demand has been suggested as a mechanism for some of the cardioprotective effects of

adenosine under normal physiologie conditions."

ElectrophyswCogïc Effects

Adenosine directiy shortens the atrial action potential duration, and suppresses the

automaticity of the SA node and other cardiac pacemakers, while slowing conduction through the

AV node and prolonging rehct~r iness . '~ In the ventricle, adenosine antagonizes the positive

chronotropy, dromotropy and inotropy induced by circulating cate~holamines.'~~

Regulation of Coronmy Bbod Flow

Adenosine is a potent coronary vasodilator. Various studies, both animal and human, have

shown that increased levels of adenosine are released during ischemia (likely due to degradation of

myocyte high energy phosphates), possibly in an adaptive role to stimulate corresponding

vasodilatation.'"'" Although initiai evidence was based largely upon measurements of adenosine

breakdown products (due to the extremely shoa half-life of endogenous adenosine), ment technical

advances have allowed for more direct methods of adenosine measurement. Fox and coileagues

showed that in 13 of 15 patients undergoing cardiac catheterization, pacing-induced angina

stimulated a ten-fold increase in coronary sinus adenosine leveis.'" The same group also studied

coronary sinus adenosine levels in patients undergoing cardiac surgery. Adenosine levels were found

to be five times those of control levels during cardioplegic ane~t.'~* Such reports were siMlar to

results from earlier animal studies employing models of intermittent coronary occl~sion.~'*'~~

Ironically, adenosine administration as an intravenous bolus has been reported to induce

angina-like chest ~ a i n . ' ~ ' ~ ~ Although this pain has been shown to be cardiac in origin, studies have

revealed no relation to coronary blood flow or myocardial ischernial"

Haemodynamic and Respwafory Effecls

Biaggioni and colleagues studied the physiologie effects of exogenous adenosine

administered to healthy volunteers. Adenosine infused intravenously at a rate of 140 ugkg/min

increased heart rate and systolic blood pressure, but decreased diastolic blood pressure resulting in

no change in the mean artenal pressure.198 Adenosine also induced a tachypnea which was not

related to bronchoconstrïction, hypoxia or hypotension. This respiratory stimuIation resulted in a

mild faIl in PaCO, and a corresponding rise in pH. Conversely, studies by Verani et al using similar

doses of adenosine in patients undergoing cardiac catheterization revealed a decrease in both systolic

and diastolic blood pressure.lW These findings were supported in studies by Srnits et al, where

adenosine was shown to induce a peripheral vas~dilatat ion.~ Subsequent studies by Watt and

colleagues revealed that both the cardiac and respiratory effects of adenosine were dose related, and

resolved immediately following termination of the infusion.201

ADENOSINE PRECONDITIONING IN HUlMANS

Despite an abundance of research into adenosine and its presumed cardioprotective

properties, much conhoversy exists with respect to its mechanisms of action and optimal mode of

application. Moreover, little evidence exists in human models with which to confirm or refute the

vast amount of animal data.

Adenosine pretreahnent @re-ischemic treatment)

Kerensky and colleagues examined nine patients who exptxienced acute ST segment

elevation during balloon angioplasty with complete resolution during the procedure. Seven of the

nine had far less ST segment elevation with the second balloon inflation, suggesting that

preconditioning had ~ccurred."~ In eleven other patients where adenosine was administered into the

coronary arteries prior to the first balloon inflation, the amount of ischemia noted during the first

inflation was reduced in oniy one patient during the second inflation. This finding suggested that

preconditioning had occurred prior to the first bailwn inflation, likely due to the effects of

adenosine. Lee and colleagues administered intravenous adenosine to elective CABG patients

immediately prior to the initiation of cardiopuhonary bypass. Adenosine pretreated patients had

improved cardiac indices and released l e s CPK during the firsî 24 postoperative hours in

cornparison to controls.*

Cardioplegic Adenosine freatment (ischemk treatment)

In an open label pilot study conducted by Mentzer and colleagues at the University of

Wisconsin, addition of exogenous adenosine to conventional cold hyperkalemic blood cardioplegia

in patients undergoing comnary bypass surgery resulted in a significant reduction in the requirement

for postoperative vasoactive dmgs (personai communication). Conversely, in a study by Fremes and

colleagues at the University of Toronto, CABG patients were administered varying doses of

adenosine in cardioplegiêm3 Although adenosine was show to be safe for administration during

cardiopulmonary bypass, no statistically significant effccts on outcome were demonstrated.

Adenosine post-îreatment (reperjùsion treahnent)

In a study by Houltz and colleagues, the effects of a pst-bypass adenosine infusion on

central hemodynamics, ST segment changes, and systolic and dias tolic function, were investigated

in 20 CABG patients. Adenosine caused a dose-dependent increase in hewt rate, c d a c output and

stroke volume with no changes in cardiac filling pressures. The mean ST segments wen slightly but

significantly depressed by adenosine. Analysis of lefi ventricular wall motion showed no ciifferences

in cornparison to controls? Sirnilarly, O wall and colleagues administered a non-hypotensive dose

of adenosine to 16 CABG patients for 4 hours following arriva1 to the intensive care unit. Although

adenosine increase heart rate and cardiac index, and decreased systemic vascular nsistance, no

differences were noted in ventricular function when compared to c o n t r ~ l s . ~ ~

Continuous udenosine tieatment

Acadesine (5-amino-1-bta-D-ribofuranosyl] imidazolexde) is a purine

nucleoside analogue belonging to a new class of agents termed adenosine regulating agents.

Acadesine has been shown to increase the availability of adenosine locally to ischemic tissues. In

a mu1 ticen tre prospective randomized aial, acadesine was administered to 633 patients undergoing

CABG by intravenous infusion starting 15 minutes before anaesthetic induction and continuing for

7 hours. as well as added to the cardioplegic solution. Although the incidence of myocardial

infarction by prespecified criteria was not different between groups, a pst-hoc subgroup analysis

using a more specified definition of myocardial infarction revealed a lower incidence of MI and a

lower incidence of adverse cardiovascular outcornes in patients who received the higher of two doses

(0.1 mg/kg/min). Moreover. in patients with Q-wave myocardial infarction, the high-dose acadesine

group had a lower peak median CKMB and area under the CKMB c ~ r v e . ~ " ~

Finally, in a multi-centre double blind, placebo controlled trial performed by Mentzer and

c ~ l l e a g u e s , ~ ~ ~ patients receiving high dose adenosine both as an inhavenous infusion pnor to and

following aortic crossclamp and as a cardioplegic infusion during crossclamp demonstrated a trend

towards decreased high dose dopamine requircments and decreased myocardial infarction. A

composite outcome analysis demonstrated that patients who received high-dose adenosine were less

likely to experience one of five adverse events including high dose dopamine use, epinephnne use,

insertion of intraaortïc ballwn purnp. myocardial infarction and death.

Thus, aithough there is some evidence to suggest that adenosine may be beneficial in humans

during coronary bypass surgery. available data is inconclusive and often controversial. Moreover.

the optimal timing of adenosine administration remains undetexmined. Unfortunately, the benefits

of adenosine are ciifficuit to determine in clinicai models due to adenosine's inherent systemic

(peripheral) hemodynamic effects. Our mode1 of human ventricular myocytes avoids such

limitations by permitting a clinically relevant evaluation of the cardioprotective effects of adenosine

in the absence of confounding hemodynamic aiterations.

SUMMARY OF STUDY RATIONALE, HYPOTHESES AND OBJECTIVES

Aortic crossclarnping during coronary bypass surgery results in global myocardial

ischernia." Although the detrimentai effects of ischemia are lessened with cardioplegia, adenine

nucleotides (ATP, ADP, and AMP) are degradeci while being used to maintain myocyte integrîty.

The resulting nucleosides (including adenosine) washout upon reperfusion, limiting nucleotide

res ynthesis resulting in poor postisc hemic rnyocardial func tion.

De novo purine synthesis is energetically costly, requiring 7 mol of either A T ' or GTP per

1 mol of AMP formed, and is very slow in organs such as the heart.6- Canine studies have

revealed that the depletion of cardiac ATP stores resulting from 15 min of ischemia requires

approximately 1 to 2 weeks for repletion and full restoration of cardiac f u n c t i ~ n . ~ ' ~ * ~ ~ ~ Thus

ventricular dysfunction secondary to myccardial stunning may reflect both ATP depletion as well

as the low capacity for de novo purine synthesis. Accordingly, any efficient method of c o n s e ~ n g

ATP stores, repleting ATP stores, or facilitating purine synthesis would be both energetically and

functionally advantageous. Adenosine kinase phosphorylation of adenosine is the most efficient

method of salvaging purines, requinng 1 mol of ATP to form 1 mol of AMP. Unfortunately, the

ability for direct purine salvage in hem muscle is rather ~ i m i t e d 6 ~ ~ ~ ~ ~ ~

Ischernic preconditioning is the most powefil endogenously mediated form of myocardial

protection. Adenosine. available as a phamacologic additive, may harness the beneficial effects of

ischemic preconditioning. Adenosine may normalize myocardial ATP stores by acting as a substrate

for and facilitating nucleotiàe resynthesis. In addition. adcnosine rnay act via a receptor mediated

mechanism to indirectly facilitate ATP production or prevent ATP degradation by facilitating a

second messenger p a t h ~ a y . ~ ' ~ - ~ ~ ~

Although animai &ta has been widely documented, little human data exists with which to

support or refute the adenosine hypothesis, and knowledge regarding the optimal time and method

of adenosine administration remains Lunitcd Elucidation of adenosine's mechanisms of effect dong

with those of ischemk preconditioning may enable the development of additional. more powerful

protective measures. Our model of isolated human ventricuiar myocytes provides an optimal method

of assessing the role of adenosine in human preconditioning and its mechanism of effect as

summarized in Figure 5.

We propose a series of cxperiments designed to assess the following hypotheses:

Endogenous preconditioning in hurnan ventricular myocytes is mediated via the release of

endogenous adenosine.

The protective effects of ischemic preconditioning in human ventricular myocytes can be

reproduced by the administration of exogenous adenosine.

Exogenous adenosine confers protection via a receptor mediated phenomenon.

The optimal time of ahnosine administration is prior to prolonged ischemia.

Exogenous adenosine confers protection to human ventncular myocytes via a protein kinase

C mediated pathway.

B y confimiing or refuting the above hypothesis, we hope to develop a model for the clinical

application of exogenous adenosine in patients undergoing coronary bypass surgery.

CHAPTER TWO: ENDOGENOUS PRECONDITIONING STUDIES

Prcconditionitcg is mediated vin denosine refeme in lturnnn ventriculnr nryocytes

SUMMARY

OBJECTIVE: T o determine the role of endogenous adenosine (ADO) in human preconditioning

(PC) . METHODS : Isolated cultures of human ventricular myoc ytes (n=8 platedgroup) were

stabilized in phosphate buffered saline for 30 minutes (S) followed by exposure to 90 minutes of

simulated ischemia O and 30 minutes of reperfusion (R)(ischemic Controls; IC). Certain plates

were exposed to a 20 minute pre-ischemic preconditioning stimulus using either anoxic (PO2*

mmHg; PCO) or hypoxic (Pop20 mmHg; PC20) pnxonditioning. In a separate group of

expenments, the supematant of anoxicaily preconditioned cells ( S m ) was applied to non

preconditioned cells for 20 minutes pnor to prolonged ischemia and reperfusion. Finally, non-

preconditioned cells were treated with the selective A l receptor inhibitor Sulfophenyl-theophylline

(SPT) prïor to and foiiowing anoxic preconditioning (PCO) as well as prior to and following the

administration of anoxically preconditioned supematant (SUPO) . Cellular viability was assessed

via Trypan Blue exclusion, and by measurement of cellular lactate release and intracellular ATP and

adenine nucleotide degradation products. Cellular supernatants were collected for the measurement

of adenosine concentrations with each intervention. RESULTS: PCO provided the greatest

endogenous protection as expressed by a decrease in Trypan Blue uptake (PCO: 20+/-5%; PC20:

3 1+/-4%; IC:39+/-6%; @.O0 1 ANOVADMR). The protective effects of anoxic precondi tioning

were abolished with SPT (3 8+/-5%). The supematant of anoxicall y pnxondi tioned cells (S WO) had

the highest concentrations of endogenous adenosine (PCO: 16.3 nmoVL; PC20: 6.7 n m o n ; K: 1.5

nmoVL, p 4 . 0 0 1 ANOVA; DMR) and provided partial protection to non-preconditioned cells whkh

was abolished with SPT (SURI: 32+/4%; SUW+Sm 41+-5%). Although lactate production was

not affected by PC, ATP levels fell following PC and following prolonged I and R.

CONCLUSIONS: Maximal ischemia is necessary for the maximal protective effects of PC. The

degree of ischemia is accuratel y reflected in supernatant AD0 concentrations. A D 0 mediates the

protective effects of PC. Despite its protective effects, the ischemic stimulus of PC creaies an initial

ATP fall, and may account for a lack of ATP preservation following 1 and R.

INTRODUCTION

This chapter describes experiments undertaken to establish the role of endogenous adenosine

in human preconditioning. We have developed a unique mode1 of simulated ischemia and

reperfusion in human ventricular cardiomyocytes. The quiescent nature of our myocytes exposed

to low volume ischemia simulates the low flow and noncontractile conditions encountered during

cardiopIegic arrest. Recently, we reportcd that ischemic preconditioning protected our human

cardiomyocytes h m a prolonged exposure to simulated is~hexnia.~'~ The following studies attempt

to detexmine: 1) the role of ischemia in human ischemic preconditioning; 2) the metabolic effects

of ischemic preconditioning; 3) the role of endogenous adenosine in human preconditioning.

MATERIALS and METHODS

lsosolation and Cuiîute of Hurnan Ventricub Cdwmyocytes

Cultures of human ventricular myocytes were established as described in Appendix 1.2'6218

Cells passaged 2 to 6 times, with a tirne h m primary culture of less than 60 days, were utilized for

this study. (Figure 6)

Experimenîd Design

A detailed description of our in-vitro technique of simulating "ischemia" and "reperfusion"

in human ventricular myocytes is availabte in Appendix 2?17(Figure 7) Briefly. following 30

minutes of stabilization in 15 ml of normoxic PBS (including MgCl, 0.49 mM, CaCl, 0.68 rnM, and

glucose 3.0 a; p02=150 mmHg), "ischemia'* was simulated by placing the cells into a sealed

plexiglass chamber flushed with 100% nitrogen to maintain anoxic conditions, while exposing the

cells to a low volume (1 -5 rnL) of deoxygenated PBS (p02=û mmHg) for a period of 90 minutes.

The volume of anoxic perfusate utilized was the minimum volume required to coat the cellular

monolayer for the prevention of cellular dehydration during the ischemic p e n d "Reperfusion" was

accomplished by exposure to 15 mL of nomoxic PBS for a perïod of 30 minutes. "Preconditioning7'

was simulated by exposing the cells to 20 minutes of "ischernia" and 20 minutes of "reperfusion"

pnor to prolonged (90 minute) "ischemia". To obtain two different graded preconditioning stimuli,

two PO, levels of preconditioning perfusion PBS were employed: pO, = O mmHg and 20 mmHg.

A small sample of deoxygenated PBS (2 m . ) was placed in a centre dish within the sealed

chamber to monitor temperature and to confirm anoxic conditions at the end of each "ischemic"

period The temperature was maintaineci at 37% throughout the expriment. A pH of 7.40 +/- 0.05

and an osmoldity of 290 +/- 20 mOsm/L was ensured with al1 solutions pnor to use.

Endogenous Preconditioning Studies

ExperUnentaC Protocols

Figure 9 s r n a r i z e s the experimental protocols employed to evaluate the effects of varying

endogenous preconditioning stimuli on cells undergoing prolonged "ischemia" and "repefision",

and the role of adenosine in this process.

Study 1 : Graàèd preconditioning

W e compared the protective effects of two grades of ischemic preconditioning on cellular

injury following prolonged "ïschemia7' and "reperfusion7'. The following groups were studied: 1)

incubation in PBS for 190 minutes (Non-ischernic Control; NIC); 2) stabilization, followed by

prolonged "ischemia" and "repefision" (Ischemic Control; IC); 3) s tabilization followed by

preconditioning with anoxic PBS @0,=0 mm.@ for 20 minutes, "reperfusion" for 20 minufes, and

prolonged "ischemia" and "reperfusion" (Anoxic Preconditioning; PCO); 4) stabilization followed

by preconditioning with hypoxic PBS (PO, =20 mmHg) for 20 minutes, "reperfusion" for 20

minutes, and prolonged "ischemia" and "reperfusion" (Hypoxic Preconditioning, PC2û). Metaboiïc

parameters were assessed in cells which underwent anoxic preconditioning (PCO).

Study 2: Supernatant precondinoning study

Supernatant was collected fiom cells which underwent stabilization for 30 minutes followed

by 20 minutes of preconditioning with anoxic ( S m ) PBS. This "preconditioned" supematant was

then applied to non-preconditioned cells for a p e n d of 20 minutes, after which the cells were

exposed to 20 minutes of "reperfusion" foiIowed by prolonged "ischemia" and "reperhision". As

non-preconditioned controls, we utilized cells which unâerwent incubation in the supernatant of

stabilized cells for 20 minutes. These cells were then "reperfi~sed" for 20 minutes followed by

exposure to prolonged "ischemia" and "repefision" (Ischemic Control; IC). Non-ischemic controls

were s tabilized in nonnoxic PBS for 190 minutes (Non-ischemic Control; MC).

Srudy 3: Meusurement of endogenous adenosine concentratr-011s

Adenosine levels were measured in the supernatants of cells which underwent either anoxic

(PCO) or hypoxic (PCZO) preconditioning. The supematants were collected immediately following

the 20 minute preconditioning stimulus and flash fnnen in Liquid Ritmgen. Following lyophilization,

the specirnens were reconstituted and assayed for adenosine content using step-gradient hi@-

performance liquid chromatography. The resultant values were quantified after evaluating a known

adenosine standard.

Stud j 4: Adenosine receptor antagonikt snidies

TO determine the d e of adenosine in human preconditioning. supematant from anoxically

preconditioned cells ( S m ) was applied to non-preconditioned cells dong with 100 pmoVL of the

adenosine receptor antagonist 8-(psulphophenyl) theophy lline ( S m ; Research Bioc hernicals

International; Natick, MA) prior to prolonged "ischemia" and "repefiion". Although non-

selective, SPT has been s h o w to possess six times the affinity for A, over A, receptors? SPT', in

addition to k i n g applied dong with the supernatant, was appiied to the non-preconditioned ceUs for

30 minutes prior to and 20 minutes foilowing exposure to the preconditioned supernatant. Ischernic

(IC) and non-ischemic (MC) control groups were identical to those outlined in study 3.

Assessrnent of CeUuCtrr Injury

Cellular injury was assessed using non-confluent plates of cardiomyocytes (approximately

337,000 cells per 9 cm diameter culture dish) cu1tured for 4 to 5 days after the latest passage.

Following the intervention of intetest, ce11 plates were incubated with 0.3% Trypan Blue dye

dissolved in nomal saline (Sigma Chernical Co.; St. Louis, MO) and assessed for injury under an

inverted light rnimscope (Nikon Canada Instnunent hc.; Mississauga, ON) at 200x magnification.

Injureci celis were unable to exclude the large molecular weight dye and stained blue.(Figure 8) The

number of bIue stained ceiis was counted from five standard locations on each plate and expressed

as a percentage of the total number of cells. Al1 counts were performed by a single observer who

was blinded to the intervention.

Biochemical Measurements

Selected experiments involved biochemical assays for extracellular lactate concentrations and

adenosine-triphosphate (ATP) content. Confluent cultures of cardiomyocytes (approximateiy

600,000 cells per culture dish) culturcd for 5 to 10 days h m the 1st passage were used for

bioç hernical anal ysis. Following removal from the culture dish. the extracellular fluid recovered

from each intervention was andyzed for lactate using an enzymatic method described in Appendix

3 (Stat-Pack rapid lactate test kit. Behring Diagnostics; La Jolla, CA). The remaining

cardiornyocytes were used to cietennine the concentrations of intracellular ATP following each

intervention of interest. (Appendix 3) The specimens were flash frozen in liquid nitrogen and then

freeze-dried. Specimens were anaiyzed by high performance Liquid chromatography with the

modifications desnibed by Weisel, et d l 9 of the step gradient technique developed by Hull-Ryde,

et al. and described in detail in Appendix 3 .=

The D N A in the ce11 extracts was ncovered in 5% perchloric acid and quantified using a

spectrop hotometnc, dipheny lamine colour reaction, with c d f thymus DNA as the standard

(Appendix 3).*' Extracellular lactate and intraceliular ATP values were then comcted for DNA

content from each plate.

Ischemic control cardiornyocytes, although untreated, were subjected to similar protocols

employing equivalent volumes of PBS for qua1 time periods with identical PO,. Baseline

biochemical measurements were made afkr removing the culture media and washing the cells with

normoxic PBS.

Statistical Analysis

The SAS S tatistical Package (SAS Institut+, Cary, NC) was employed for andysis of al1 data.

Data are expressed as the mean +/- standard deviation in the text and mean +/- standard emor in the

figures, with eight plates per group unless otherwise specified. Analysis of variance (ANOVA) was

used to simultaneously compare continuous variables at different time periods. When statistically

si gni ficant ciifferences were found, they were specified by Duncan's multiple range test. S tatistical

signi ficance was assumed for p4.05.

RESULTS

A. Endogenous Preconditioning Studies

Study 1: Graded preconditioning srudy

Figure 10 shows the results of Trypan Blue assessments for cellular injury. The most severe

injury was seen with cells which underwent prolonged ischemia and reperfusion only (IC). Hypoxic

preconditioning for 20 minutes with a PO, = 20 mmHg (PC20) significantly reduced the cellular

injury associated with prolonged ischemia and reperfusion. Anoxic preconditioning for 20 minutes

with a PO, = O mmHg 0) reduced cellular injury to a greater extent than did hypoxic

preconditioning (PC20) (NIC: 9+/-396, PCO: 20+/4%, PC20: 3 1+/-4%. IC: 39+/-6%; ANOVA:

p4.000 1 ; differences between each group pcû.05 by Duncan's multiple range test). ExtraceIlular

lactate concentrations in celis which underwent anoxic preconditioning (PCO) pnor to prolonged

"ischemia" and "reperfusion" were elevated irnmediately following preconditioning, although no

signifiant differences were found in comparison to ischemic controls during the same time periods

(Figure 11, upper panel). Intracellular ATP levels decreased significantly in the PCO group

immediateIy following preconditioning (PCO: 1.21+/-0.35, IC: 2.2+/-0.43 mmoVgDNA; p4.05).

Although during ischemia, the reduction in ATP was less profound in the preconditioned group, no

differences were found in ATP levels following prolonged "ischemia" or "reperfusion" in

comparison to ischemic controls (Figure 1 1, lower panel).

Study 2: Supernatant precondirioning study

Figure 12 (lower panel) demonstrates the results of Trypan Blue assessments from groups

pretreateà with the supernatants of cells which underwent anoxic (pO,=û d g ) preconditioning

(PCO). The most severe cellular injury was found in cells which undenvent prolonged ischemia and

reperfusion only (IC). Preincubation with the supernatant of cells preconditioned using anoxic PBS

(SUPO) significantiy reduced cellular injury however to an extent less than that seen with anoxic

preconditioning (PCO) (Stab: 12+/4%, SUPO: 28+/4%, IC: 41+1-5%; ANOVA pc0.0001;

differences between each group paû.05 by Duncan's multiple range test).

Study 3: Meusurement of endogenous adenosine concentrarions

Figure 12 (upper panel) demonstrates measured adenosine concentrations in supematants of

ce1 b whic h underwent either anoxic (PO, = O m d g ) or hypoxic (PO, = 20 mmHg) preconditioning.

HPLC analysis reveaied a p a t e r concentration of endogenous adenosine in the supematant of

anoxically preconditioned cells (SUPO) rather than hypoxically preconditioned cells (SUP20) (SUPO:

16.3, SUP20: 6.7, Non-ischemic Controls: 1.1 nrnoVL; p4.01, SUPû versus SUP20).

Study 4: Ahnosine receptor antagonist sîudies

As demonstrateci in Figure 12, cellular injury after prolonged "ischernia" and "reperfusion"

was significantiy reduced following pretreatment with the supernatant of anoxicaily preconditioned

cells (SUPO). The protective effects of the anoxicaüy precondi tioned supernatant (SUPO) were

abolished when the supematant and the non-preconditioned cells were first treated with the

adenosine receptor antagonist (Sm) (NIC: 1 W 4 , SUPO: 284-4, SUPO+SPT: 36+/-5, IC: 41+/-5

% Trypan Blue uptake; ANOVA: peû.0001; differences between groups p<0.05 by Duncan's

multiple range test).(Figure 13)

CONCLUSIONS

These studies demonstrate the protective effects of ischemic preconditioning on human

ventricula. rnyocytes undergoing ischemia and reperfusion, and the role of adenosine in this proce~s-

The following chapter will outline attempts to reproduce the protective effects of ischemic

preconditioning with the use of exogenous adenosine.

CHAPTER THREE: EXOGENOUS PRECONDITIONING STUDIES

Reprodiming the protcctive effccts of ischcmic preconnilioning using exogerzous denosine

SUMMARY

OBJECTIVES: To assess the protective effects of exogenous adenosine (ADO) in human

preconditioning (PC) and to determine the optimal dose and timing of exogenous A D 0

administration. METHODS: Isolated cultures of human ventricular myocytes (n=8 plates/group)

were stabilized in phosphate buffered saline for 30 minutes (S) followed by exposure to 90 minutes

of simulated ischemia (I) and 30 minutes of reperfusion (R)(Ischemic Controls; IC). A dose

response analysis was performed for exogenous AD0 and the optimal timing for AD0

administration was cietennineci by applying ADû prior to (Pretreatment), during (Ischemic

treatment), or following (Reperfusion treatment) i, or during al1 three phases (Continuous treatment).

To determine whether the effects of adenosine were secondary to a receptor or substrate mediated

effect, adenosine pretreatment was adrninistered with and without a pre-ischemic reperfusion p&od.

In addition, cells treated with exogenous AD0 were first treated with SPT. Cellular viability was

assessed via Trypan Blue exclusion, and by measurement of cellular lactate release and intracelldar

ATP and adenine nucleotide degradation products. RESULTS:Exogenous AD0 reproduced the

beneficid effects of PC. AD0 was most protective when administered prior to 1 at a dose of 50

umol, followed by a pre-ischemic reperfusion period. A D 0 administered pnor to 1 without a pre-

ischemic repefision period, or administered during 1, was partidly protective. No additional

protection was provided when ADO was applied continuously (NIC: IO+/-3, Pretreatment with pre-

ischemic reperfusion: 24+/4, Pretreatment without pre-ischemic reperfusion: 29+/-5 ; Ischemic

treatment: 33+/-3. Repemision treatment: 38+/-3, Continuous treatment: 25+/4, IC: 39+/4 %Trypan

Blue uptake; ANOVA p 4 . 0 0 1 ; differences between groups pe0.05 by Duncan's multiple range

test). AD0 prevented ATP degradation following 1 and R without the initial falt in ATP s e n with

PCO (NIC: 2.0+/-.3, Pretreaûnent: 1.9+/-0.3, Ischemic ueabnent: 1.2+/-0.4, Reperfusion treatment:

0.8+/-0.3, Continuous treatment: 1.7+/-0.3, IC: 0.7+/4.3 mmoVgDNA; ANOVA p<0.001;

differences between gmups @.O5 by Duncan's multiple range test). Although final lactate

concentrations following adenosine pretreatment did not di ffer from controls (MC: 0.34+/-0.2,

Pretreaûnent: 0.5+/-0.1, Ischemic treatment: 0.6+/4.2, Reperfusion treatment: 0.8+/-0.2, Continuous

treatmen t: 0.8+1-0.2. IC: 0.4+1-0. 1. moVgDNA; ANOVA p<0.00 1 ; differences between groups

p 4 . 0 5 by Duncan's multiple range test). AD0 signi ficantly increased lactate concentrations

immediately following its administration (ADENOSINE: Retreatment: 1.1+/-0.2, Ischernic

treatment: 1.5+/-0.3, Repefision treatrnent: 0.8+/-0.2. moVg DNA; Non-treatment CONTROLS:

Control S tabilizattion: 0.64+/4.28, Control Ischemia: 1.29+/-0.36, Control Reperfusion: 0.43+/4.11;

ANOVA pc0.000 1 ; p<0.05 ADENOSINE vs. Non-treatment CONTROLS). SPT abolished the

protective effects of ADO, and prevented ATP preservation and elevations in lactate.

CONCLUSIONS: AW effectively reproduced the protective effects of K. preserved ATP

concentrations, and increased steady state lactate production. perhaps by stimulating glycolysis.

AD0 is an effective substitute for PC in human cardiomyocytes.

INTRODUCTION

This chapter describes experiments designed to assess the ability of exogenous adenosine to

reproduce the protective effects of preconditioning in human ventricular myocytes exposed to

ischemia and reperfusion. AIthough the beneficial effects of exogenous adenosine have been widely

reported in animal models, Little human data exists. Moreover, the optimal method of adenosine

administration and its mechanism of effect have yet to be detemineci. The foilowing studies attempt

to determine: 1) the o p W dose and timing of exogemus adenosine in human preconditioning; 2)

the metabolic effects of exogenous adenosine in huma. preconditioning; and 3) whether the

protec tive effects of adenosine are receptor or substrate mediateci.

UATERULS and METHODS

Cultures of human ventricular myocytes (6 plates/group) were established as described in

Appendix 1.2'"18 Ceus passageci 2 to 6 cimes, with a tirne h m prirnary culture of less than 60 days,

were utilized for these studies. To simulate ischemia and reperfbion, cardiomyocytes were

stabilized in phosphate buffered saline for 30 minutes (S) aftcr removal h m the incubator, foilowed

by exposure to 90 minutes of simulateci ischemia (I) and 30 minutes of reperfusion (R)(Ischemic

Controls ; IC). Treatment groups were established accordingl y.

Experhentai Protocols (5,6,7)

Figure 14 demonstrates the protocols employed to determine the benefits and optimal

methods of exogenous preconditioning using adenosine, as well as the mechanisms underlying

adenosine mediateci cardioprotection.

Study 5: Optimal dose and timing of aàenosine

A dose-response analysis was undertaken using varying doses (0-200 pmoVL) of exogenous

adenosine (ADO; Sigma Chernical Co., St. Louis, MO) dissolved in normoxic o r anoxic PBS.

Adenosine was applied to the cells for 20 minutes following 30 minutes of stabilization, after which

the cells were exposed to 20 minutes of "reperfusion" followed by prolonged "ischernia" and

"reperfusion". Once the optimal dose of adenosine was detexmined (according to Trypan Blue

exclusion), the optimal timing was determined by incubating the cells with adenosine either prior

to "isc hemia" (Pretreatment). during "isc hemia" (Isc hemic treatmen t), during "reperfusion"

(Reperfusion treatment), or during al1 three phases (Continuous treatment). Non-ischemic controls

WC) undenvent stabilization in nomoxic PBS for 30 minutes, foliowed by exposure to adenosine

for 20 minutes. followed by 20 minutes of reperfusion, followed by exposure to adenosine for 120

minutes. Ischemic controls (IC) underwent stabilization for 70 minutes followed by prolonged

"isc hemia" and "reperfusion". In both ischernic and non-ischemic controls, PBS solutions were

periodically replaceci in accordance with treatment times to enable maximal generalizability beniveen

groups (Figure 14).

Study 6: Selective adenosine receptor antagonist studies

The folbwing series of experiments were intended to deïineate whether adenosine

preconditioning is dependent on a receptor mediated effec t or a substrate mediated effect. Firstly ,

the protective effects of adenosine preseatment were assessed with and without a pre-ischemic

reperfusion period. Secondly, the non-selective adenosine receptor antagonist SPI' was utilized.

Cells which were treated with adenosine either prior to (Pretreatment) or during (Ischemic treaûnent)

"ischemia" were exposed to SPT dissolved in PBS during both stabilization, adenosine pretreatment,

pre-isc hemic "reperhsion", a d o r "isc hemia". Non-ischemic controls were exposed to SPT for 30

minutes, followed by adenosine with SPT for 20 minutes, followed by SET for 20 minutes, followed

by AD0 with SPT for 90 minutes, followed by 30 minutes of reperfusion (MC). Ischemic controIs

were stabilized in normoxic PBS for '70 minutes followed by prolongeci "ischernia" and

"reperfusion" (IC) (Figure 14).

Assessrnent of Cellular Injury

Cellular injury was assessed using non-confîuent plates of cardiomyocytes (approximately

337,000 cells per 9 cm diameter culture dish) cultured for 4 to 5 days after the latest passage.

Foilowing the intervention of interest, ce11 plates were incubated with 0.4% Trypan Blue dye

dissolved in normal saline (Sigma Chernical Co.; St. Louis, MO) and assessed for injury under an

inverted light microscope (Nikon Canada Instrument hc.; Mississauga, ON) at 200x magnification.

Injured cells were unable to exclude the large molecular weight dye and stained blue. The number

of blue stained cells was counted fkom five standard locations on each plate and expressed as a

percentage of the total number of cells. Al1 counts were performed by a single observer who was

blinded to the intervention.

Bwchemical Measurements

For the assessrnent of extracellular lactate concentrations and adenosine-triphosphate (ATP)

content, confiuent cultuns of cardiomyocytes (approximately 600,000 ceils per culture dish) cultured

for 5 to 10 days from the 1st passage were utilized. Following removal fiom the culture dish, the

extracellular fluid recovered from each intervention was analyzed for lactate using an enzymatic

method describeci in Appendix 3 (Stat-Pack rapid lactate test kit, Behring Diagnostics; La Jolla, CA).

The remaining cardiomyoc ytes were used to detennine the concentrations of intracellular ATP

following each intervention of interest (Appendix 3). The specimens were flash frozen in liquid

ni trogen and then freeze-cirieci. Specimens were analyzed by high performance liquid

chmmatography with the modifications described by Weisel, et d219 of the step gradient technique

developed by Hull-Ryde, et al, and described in detail in Appendix 3?

The DNA in the ce11 extracts was recovered in 5% perchloric acid and quantified using a

spectrophotometric, diphenylamine colour reaction, with calf thymus DNA as the standard

(Appendix 3).*' Extracellular lactate and intracellular ATP values were then corrected for DNA

content from each plate.

Ischemic control cardiomyocytes, although untreated, were subjected to similar protocols

employing quivalent volumes of PBS for equal tirne periads with identical PO,. Baseline

biochemical measwements were made after removing the culture media and washing the ceils with

normoxic PBS.

Adenosine Assay

Ischemic supematants were flash frozen in liquid nitrogen, lyophilized and reconstituted

irnmediately prior to adenosine assay using stepgradient high performance Liquid chromatography

(HPLC) as detailed in Appendix 3. The resultant values were expressed as an adenosine

concentration against a h o w n adenosine standard.

Stafisticd Analysis

The SAS Statistical Package (SAS lnstitute, Cary, NC) was employed for anaiysis of al1 data.

Data are expressed as the mean +/- standard deviation in the text and mean +/- standard error in the

figures, with eight plates per group unless otherwise specified. Analysis of variance (NOVA) was

used to simultaneously compare continuous variables at different time penods. When statisticdly

significant clifferences were found, they were spccified by Duncan's multiple range test. Statistical

significance was assumed for pd.05.

RESULTS

Study 5: Optimal dose and timing of adenusine

Exogenous adenosine administration afforded significant protection against the injurious

e ffec ts of "isc hemia" and "reperfusion" (Figure 15, upper panel). Follow ing a dose-response

analysis based upon Trypan Blue assessments of injury, exogenous adenosine was found to be most

protective at a dose of 50 pnol. Adenosine lost its protective effects at doses equal to or above 100

pmol. At a dose of 50 pmol, the greatest degree of protection was afforded when adenosine was

applied pnor to "irhemia" (Pretreatment) followed by pre-ischemic repemision. Application of

adenosine during "ischemia" (Ischemic treatment) was protective to a lesser degree than was

adenosine pretreatment. The two protective effects were not found to be additive when adenosine

was administered continuously (Continuous treatment). Adenosine applied during "reperfusion"

(Reperfusion treatment) was not protective (Figure 1 5, upper panel). (NIC: 1 O+/-3, Pretreatment:

2W4, Ischemic treatment: 33+/-3, Reperfusion treatment: 3 8+/-3, Continuous treatment: 22+/-4,

IC: 39+/-6 %Trypan Blue uptake; ANOVA pcû.001; ciifferences between groups p4.05 by

Duncan's multiple range test).

Adenosine treatment resulted in a significant preservation of ATP following prolonged

"ischernia" and "repemision" (Figure 15, lower panel). Cornparison between groups revealed that

cells which were pretreated with adenosine or continuously treaîed with adenosine demonstrated the

greates t degree of ATP presewation follow ing prolonged "isc hemia" and "repefision" in

comparison to ischemic controls (IC). Unlike the case with ischernic preconditioning. ATP

concentrations imrnediately following adenosine pretreatment did not fa11 in comparison to conmls.

Application of adenosine during ischemia (Ischemic treatment) resulted in only partial preservation

of ATP. These preservative effects were non-additive when adenosine was applied continuously

(Continuous treatment). Adenosine applied during reperfusion (Reperfusion treatment) did not

prevent the degradation of ATP (Figure 15, lower panel) (NIC: 2.0+/--3, Pretreatment: 1.9+/-û.3,

Ischemic treatment: 1.2+/-0.4, Reperfusion treatment: 0.8+/-0.3, Continuous treatment: 1.7+/-0.3,

IC: 0.7+/-0.3 mmoVgDNA; ANOVA p4.001; differences between groups p 4 . 0 5 by Duncan's

multiple range test).

In cornparison to ischernic controls o, supernatant lactate concentrations following

"ischemia" and "repefusion" ('Final' lactate) were elevated in groups treated with adenosine either

continuously (Continuous treatment) or during reperfusion (Reperfusio~, treatment) (Figure 16)

(NIC: 0.34+/-û.2, Pretreatment: OS+/-0.1, Ischemic treatment: 0.6+/-0.2, Reperfusion treatment:

0.8+/-0.2, Continuous treatment: 0.8+/-0.2, IC: 0.4+/-0.1, moVgDNA; ANOVA pcû.001; differences

between groups p4.05 by Duncan's multiple range test). To determine the direct effects of

adenosine on lactate production, supernatant lactate concentrations were measured either prior to

ischemia, at the end of ischemia, or at the end of reperfusion, with and without adenosine treatment-

Under such circumstances, supernatant lactate levels were found to be elevated in al1 groups

immediately following adenosine treatment ('Post-Adenosine' lactate) in cornparison to non-

treatment controls (Figure 16) (ADENOSINE: Retreatment: 1.1+/-0.2, Ischemic treatment: 1 S+/-

0 -3. Reperfuion treatrnen t: 0.8+/-0.2, moVg DNA; Non-treatment CONTROLS: Control

Stabilization: 0.64+/-0.28, Control Ischemia: 1.29+/-0.36, Conhl Reperfusion: 0.43+/-0.11;

ANOVA p<0,0001; p4.05 ADENOSINE vs. Non-treatment CONTROLS).

Study 6: Non-selective adenosine receptor antagonist sîudies

To determine whether the protective effects of adenosine were receptor or substrate mediated,

cells were pretreated with adenosine (Pretreatment) with or without pre-ischernic reperfusion. As

demonstrated by Trpan Blue exclusion, pre-ischemic reperfusion was necessary for the protective

effects of adenosine pretreatment to be realized. (MC: IO+/-3, Pretreatment with pre-ischemic

reperfusion: 24+/4. Pretreatment without pre-ischemic reperfùsion: 29+/-5; IC: 39+/-6 %Trypan

Blue uptake; ANOVA p<0.001; differences between MC, Pretreat with pre-ischernic reperfusion,

Pretreat without pre-ischemic reperfusion and IC p4 .05 by Duncan's multiple range test) The

presence of protection despite such a 'wash-out* period further supported the receptor-mediated

hypothesis of preconditioning. Moreover, when cells treated with adenosine (either pnor to or

during ischemia) were simultaneously exposed to the non-selective adenosine receptor antagonist

Sm, the protective effects of adenosine were abolished as assessed by Trypan Blue exclusion and

measurements of intracellular ATP concentrations (Figure 15) (Trypan Blue: Pretreatment+SPT:

41+/-4, Ischemic tmatment+SPT: 38+/4, IC: 39+/-6, % Trypan Blue uptake; p=NS) (ATP:

Pretreatment+SPT.: 0.55+/-0.27, Ischemic treatrnent+SPT: 0.78+/-0.24, IC: O.'U+/-O. 29

mmoUgDNA; p=NS). Sirnilarly, to determine if the lactate elevating effects of adenosine were

receptor mediated, extracellular lactate levels were measured in cells simultaneously treated with

adenosine and SPT prior to "ischemia" (Pretreatment+SPT). Cells which were exposed to both

adenosine and SET revealed a significant reduction in pre-ischernic lactate concentrations in

cornparison to ceiis pretreated with adenosine alone (Pretreatment) (Figure 16) (Pretreatment: 1 A+/-

0.2, Pretreatmen t+SPT: 0.76+/-0.3 moUgDNA; p4.05).

CONCLUSIONS

Exogenous adenosine applied prior to ischemia with a pre-ischemic reperfusion p e n d

effectively reproduced the beneficial effects of preconditioning without the need for an ischemic

stimulus. Uniike ischemic prcconditioning, adenosine preserved intracellular ATP levels following

ischemia and reperfusion in cornparison to controls. In addition, adenosine stimulated lactate

production during its application, both during ischemia and during normoxia .

CHAPTER FOUR: PROTEIN KLNASE C STUDIES

A denosine precunditions huninn ventrkulnr myocytes via n P.'.-rncdinted p<ithivay

SUMMARY

OBJECTIVE: The aim of the following studies was to determine the role of pmtein kinase C in

the preconditioning sequence. METHODS: Isolated cultures of human ventricular myocytes (n=8

platedgroup) were stabilized in phosphate buffered saline for 30 minutes (S) followed by exposure

to 90 minutes of simulated ischemia O and 30 minutes of reperfusion (R)(Ischemic Controls; IC).

To determine the role of protein kinase-<= (PKC) in AD0 mediated protection, cells were treated

with the PKC agonist PMA. In addition, ceUs which underwent PC or AD0 pretreatment were

simultaneously incubated with the PKC antagonist Calphostin-C (Cal-C). Finally, isoform specific

PKC translocation and PKC activity were assesseci following ischemic preconditioning or adenosine

pretreatment. R d t s : PMA partiaiiy repmduced the protective effects of PUADO, and the effects

of al1 three treatments were blocked by Cal-C. AD0 stimulated a marked cytosolic to membrane

translocation of P X , and stimulated PKC activity. These effects were inhibited by SPT.

CONCLUSIONS: The protective effects of preconditioning are mediated via a second messenger

system which involves PKC stimulation and membranous translocation.

INTRODUCTION

Ischemic preconditioning and adenosine have been shown to afford significant

cardioprotection against the effects of ischemia and reperfusion via a receptor mediated process.

Extracellular receptors are often linked to intracellular effectors by a second messenger system.

Protein kinase C represents such a second messenger system.

Various studies have suggested that PKC activation is necessary for preconditioning to take

place. Ytrehus et al demonstrated b a t in isolated rabbit hearts, PKC stimulation using a phorbol

ester reduced infarct size foliowing 30 minutes of regional myocardial ischemia by 77%.

Conversely. treatment of preconditioned hearts with the PKC inhibitors polpixin B or stawosporine

abolished al1 protective effectsm Similar results were reprted by Armstrong et al in cul& rabbit

cardiomyocyte models of ischemia and r e p e r f u s i ~ n . ~ ~ Liu and coileagues reportecl that prevention

of PKC translocation using colchicine in rabbit myocardium prevented preconditioning? Similarly.

Speechly-Dick and colleagues demonstrated a reduction in infant s k when synthetic diacylglycerol

analogues were administered to rats pnor to prolonged myocardial ischemia. This protective effect

was blocked when rat hearts were treated with the PKC antagonist chelerythrhe immediately

following the preconditioning s t i m ~ l u s . ~ In human ventricular myocytes, Ikonomidis et al

reproduced the protective effects of preconditioning using the phorbol ester PMA. Conversely. the

protective effects of preconditioning were abolished when cells were treated with the PKC

antagonists c helerythrine or calph0stin-C following preconditioning. Immunofluorescent antibody

techniques in pnconditioned human ventncular myocytes demonstrated a redistribution of antibody

to the cytoplasmic and perinuclear membranes compatible with translocation of PKC?

Additional studies suggest a role for PKC in adenosine mediated preconditioning. Studies

involving various tissues including thyroid, cerebral cortex, myometnum and coilecting tubule have

demonstrated that activation of adenosine A, receptors stimulates phospholipase C? Kohl and

colleagues demonstrated that administration of A, receptor agonists to left atrial and papillary muscle

models of ischernia and reperfusion increased IP3 concentrations and decreased PIP2

concentrations." In human ventricular myocytes, ïkonomidis et al showed that adenosine

preconditioning was abolished when preconditioned cells were treated with the PKC antagonists

c helerythrine or caiphostin-C?

Nonetheless, other studies have questioned the role of PKC in preconditioning. Studies by

Ikeda and colleagues demonstratexi incnased cellular damage in cornparison to controls when PMA

was administered to hypoxic murine cardiac tells.= Similarly, in isolated pemiscd rat hearis, Yuan

and colIeagues showed that administration of PMA led to a dose dependent deterioration in

contractile function? Thus, the role of PKC stimulation in preconditioning requires further

elucidation. Moreover, confymatory data is necessary in a human model.

MATERIALS and METHODS

Cultures of human veneicuiar myocytes-were established as previously descnbed?*" Cells

passageci 2 to 6 times, with a time hom primary culture of less than 60 days, were utilized for this

study. Al1 cells were grown in 9.0 cm culture dishes and incubated under physiologic conditions

(Appendix 1). Cells used for microscopic assessments were grown to non-confluence

(approximately 223,000 cells per plate). Cells used for PKC activity and translocation studies were

grown to confluence (approximately 600,000 cells per culture dish) by culturing for 5 to 10 &YS

from the last passage.

Experimentd Protocols

Figure 17 demonstrates the protocois utilized to determine the role of a protein kinase C

(PKC) second-messenger pathway in ischernic and adenosine-mediated preconditioning.

S~udy 7: Protein Kinase-C (PKC) studies

To determine whether ischemic or adenosine-mediated preconditioning is dependent upon

protein kinase< (PKC) stimulation or translocation, 10 nmol of the PKC-stimulating phorbol ester

PMA (4gphorbol 12-myristate 13-acetate) (Sigma Chemîcai Co., St. Louis, MO) and 200 nmol of

the selective PKC antagonist Calphostin-C dissolved in normoxic or anoxic PBS (Cal-C; Sigma

Chernical Co., St, Louis, MO) were utilized. Non-preconditioned cells were exposed to PMA for

20 minutes followed by 20 minutes of pre-ischernic reprfusion prior to prolonged "ischemia" and

"reperfusion". Certain ceiis which underwent ischemic preconditioning or were treated with

adenosine or P M . prior to prolonged "ischemia" and "reperfusion" were exposed to Calphostin-C

during 30 minutes of stabilization, during preconditioning with ischemia, adenosine, or PM. , and

during ple-ischernic -ion (Figure 17). Non-ischemic controls were exposed to Calphostin C

for 30 minutes, followed by Calphostin C with adenosine or PMA for 20 minutes, followed by

Calphostin-C for 20 minutes, followed by 120 minutes of stabiiization. Ischemic controls were

stabilized in PBS for 70 minutes followed by prolonged "ischemia" and "reperfusion".

In a separate group of studies, cells which were stabilized for 30 minutes followed by

exposure to either 50 v o l of adenosine, 100 p o l of adenosine, or 50 pmol of adenosine with the

selective adenosine antagonist Sm, were assayed for PKC activity and isoform-specific

translocation. Cells exposed to 10 nmol of PMA were used as positive controls. Results were

compared to non-ischernic controls (NIC; negative controls) which underwent stabilization only.

62

P rotein Kinase C Analyses

Isoforrn specific translocation of protein kinase C (PKC) was demonstrated by performing

a %lot blot' analysis on cellular cytosolic and membrane fiactions using isoform-specific antibodies

for PKC-a and PKC-E. Western blot anaiysis using cherniluminescent detection demonstrated that

each antibody was specific for PKC with no evidence of non-specific background staining. Slot

blots were then scanned using a commerciaily available software program (Molecular Images;

Mississauga, ONT) and each band was assessed densitometrïcaliy, as outlined in detail in Appendix

3.

PKC activity was measured by in-situ phosphorylation of a PKC specific peptide substrate

using a modification of a method previously reporteci by Heasley and Johnson and detailed in

Appendix 3.-= Measured phosphorylation rates were standardized for ce11 protein measured using

the method of Lowry et almm The protein assay protocol is provided in Appendix 3.

Statisfical Analysis

The SAS Statistical Package (SAS Institutt?, Cary, NC) was employed for analysis of al1 data.

Data are expresseci as the mean 4- standard deviation in the text and mean +/- standard error in the

figures, with eight plates per group uniess otherwise specified. Analysis of variance (ANOVA) was

used to simultaneously compare continuous variables at different time periods. When statisticdly

significant differences were found, they were specified by Duncan's multiple range test. Statistical

significance was assumed for p<0.05.

RESULTS

Study 7: Protein Kinase-C (PKC) studies

To detemiine whether the receptor-mediated protective effects of ischemic preconditioning

or adenosine pretreatment are dependent upon protein kinase C stimulation and translocation. various

studies were employed Fi t ly , cells preconditioned with either anoxic PBS (PCO), PM& or

adenosine pretreatment (Pre~reatment), were simultaneously exposed to the selective PKC antagonist

Calphostin-C. Calphostin-C abolished the protective effects of anoxic preconditioning (PCO),

adenosine pretreatment, and PMA (Figure 18) (MC: Il+/-2. PCO: 2 M . PCOcCal-C: 34+14,

Pretreatment: 244-4. Pretreatrnent+Cal-C: 36+/-4, PUA: 28+/-4, PMA+Cal-C: 35+/-4, IC: 39+/-6,

% Trypan Blue uptake; p=NS, preconditioning+Cal-C versus IC; p4.05, PMA versus IC).

In a separate series of experiments, both PKC translocation and activity wexe assayed. Figure

19 displays a representative slot blot analysis which shows an isoform specific translocation of PKC

in cells exposed to 50 pmol of adenosine (Pretreatment), 100 pmol of adenosine. 50 pmol of

adenosine with SPT, or 10 nm PMA. Resulu were compareci to those of cells which underwent

stabilization in normoxic PBS only (NIC). Densitomeaic analyses revealed no changes in PKC-a

or PKC-E distributions with stabilization. Similarly, PKC-E distxibutions did not change with either

adenosine or the phorbol ester PMA. However, there was a marked cytosolic to membrane

translocation of PKC-a in ceIIs exposed to 50 pmol of adenosine (Pretreatment) or PMA. Cells

exposed to 100 pmol of adenosine prior to ischemia revealed a l a s marked translocation. Exposm

of the cells to 50 m o l of ahnosine with S m (non-selective adenosine receptor antagonist)

prevented differential translocation. Digitalized densitometry revealed the following

membrane:cytosoIic ratios for each group: PUA: 4.93; 50 pz01 aàénosine: 6.4; 100 pml ademsine:

1.85; 50 p o l adenusine + I 0 0 pnol SPT 2.27; NZC. 1.43-

In concomitant studies, we measured the effeçt of 50 pmol of adenosine, 100 pmol of

adenosine, 50 m o l of adenosine with Sm, 10 nrn PMA. or stabilization on total PKC activity.

Although b t h PMA and 50 p o l of adenosine stimulateci PKC activity, the effect of PMA was far

more potent (I?MA: 0.65+/4.07; Retreatment: 0.39+/-0.05; pe0.05, n=6/group).

CONCLUSIONS

The results of these studies suggest that both ischemic preconditioning and adenosine

preconditioning are mediateci by isoform-specific PKC translocation and activation.

CHAPTER FIVE: DISCUSSION

DISCUSSION

Operations of the heart represent the most commonly perfomied surgical procedures in North

America. Coronary bypass surgery (CABG) accounts for greater than 75% of such procedures.

Un fortunatel y, despi te recen t advances in myocardial protection, the prevalence of 10 w cardiac

output syndrome following comnary bypass surgery remains relatively high (approximately 9%).8

In the absence of intraoperative myocardial infarction, the development of low output syndrome

following CABG represents inadquate intraoperative myocardial protection.

Ischemic preconditioning is by far the most potent form of myocardial protection known.

The cardioprotective effects of ischemic preconditioning have k e n shown in various species.

including h ~ r n a n s . ~ ~ ~ ~ However, more recently, the protective effects of ischemic preconditioning

in humans have been c d e d into question. Menasche et. ai. reported that patients preconditioned

with 3 minutes of crossclamping prior to institution of cardioplegia revealed increased levels of

creatine kinase MB and lactate release at the end of cardioplegic a m ~ t . ' ~ In addition, molecular

biology data previously shown to be related to the preconditioning process (Le. expression of m-

RNA for bath c-fos and heat shock protein 70) did not suggest a protective effkct of preconditioning.

Studies such as this dong with the risks of repeated crossclamping (including intraoperative

infarction and cerebral embolic disease) emphasize the need for identification of pharmacologie

mediators that could safely and effectively hamess the kneficiai effects of ischemic preconditioning.

Such mediators could be applied in the form of a simple additive to be administered in conjunction

w i th cardioplegia during cardiac surgery.

Adenosine may represent such an additive. Evidence in support of such a possibility was first

introduced by Przyklenk and colleagues who reported that protection was afforded to non-ischernic

myocardial regions adjacent to those which undenrent ischemic preconditioning? The authors

suspected that preconditioning induced adenosine release which in turn initiated a sequence of

cellular signalling events, resulting in protection from a subsequent prolonged ischemic episode.

Using a microdialysis technique, van Wylen and colleagues found increases in adenosine and other

solubIe purines in canine myocardial interstitial fluid during the ischemic and reperfusion phases of

preconditioning." Since the role of adenosine in human preconditioning remains unknown, we

endeavoured to study both endogenously released adenosine and exogenously administered

adenosine in our human cellular mode4 of simulated cardioplegic arrest. Such a model permits an

evaluation of adenosine treatment in human cardiomyocytes in the absence of altemate ce11 types (i.e-

endothelial cells), and independent of the hemodynamic effects associated w i th adenosine infusion.

Humun cardiomyocyte cell culture model

The cardiomyocytes employed in these studies have been extensively evaluated in previous

reports.99a8 Our cells were passaged 2-6 times and were cultured for up to 60 days h m the time

of pnmary cul tue. These cardiomyoc ytes ntain many characteristics of freshly isolated cells. but

have distinct ciifferences. Following enzymatic digestion and passaging, the cells change their shape,

lose their striations. and become quiescent. Despite an abundant supply of mitochondria and

contractile proteins. the saxomeres become disnipted during division and do not reestablish their

characteristic functiond format The cardiomyocytes in culture are easily differentiated from other

ce11 types. Endothelid cells are oval-shaped (15 X 20 pm) and fibroblasts are spindie-shaped (4 X

80 pm), compared to the rcctangular and much larger cardiomyocytes (40 X 80 p). In addition,

endothelial cells grow poorly in the medium employed for cardiomyocytes, whereas fibroblasts have

a much faster doubling time in culture and are easily identified as a spindle-shaped contaminant.

The quiescent nature of our cardiomyocytes is likely the result of isolation techniques which

cause a breakdown of myofibrillar organization. These cells may simulate the cardioplegically

arrested heart encountered during cardiac surgery. The cellular concentrations of troponin 1, troponin

T and the MB isofonn of creatine kinase are similar to that seen ~ i - v n t o ? ~ The rnetabolic response

of these cells to ischemia aiso closely resembies our intraoperative findings during cardiac

s ~ r g e r y . " ~ ~ ~ ~ Therefore, despite their quiescent state. we believe that these cells are

phenotypicaily cardiomyocytes and provide a unique opportunity to evaluate the cellular response

to ischemia and reperfusion as well as the effects of pharmacological additives such as adenosine,

Our model of "ischemia" and "reperfusion" is similar to the effects of global ischemia on the

myocardium. Although the volume overlying our cells during "ischemia" exceeds that found in the

globally ischemic heart, reduction of the volume of ischemic PBS from 10 mL to 1.5 mL resuited

in a marked increase in the products of ischemic metabolism, a decrease in the extracellular pH, and

an increase in ceii injury. Thus, our model may actualiy represent a form of low-flow ischemia

analogous to iimited cardioplegic perfusion during cardiac surgery.

Endogenous Preconditioning

The ability to endogenously precondition our celis against the detrimental effects of

prolonged "ischemia*' and "reperfusion" is similar to the effect seen Ut vivo.273'6*21z Ikonomidis et

al. previously demonstrated that the beneficial effects of preconditioning were dependent on the

duraiion of the ischemic stimulus, such that the greatest degree of protection was conferred with a

20 minute ischemic stimulus applied pnor to a more prolonged episode of ischemia and

repemision? In our studies, the degree of ischemia was similarly found to be crucial in regulating

the protective effects of ischemic precondi tioning, suc h that anoxic preconditioning (pO, =O d g )

conferred greater protection than did hypoxic preconditioning (p0, =20 mmHg) as assessed via

Trypan Blue exclusion. Thus we have determined that the ischemic stimulus of preconditioning

cannot be minimized (in an effort to limit the detrimental effects of ischemia) without reducing the

degree of protection afforded- Although lactate levels were elevated immediatel y after the i SC hemic

preconditioning stimulus, the levels were similar in both preconditioned and ischemic control groups

following both "ischemia" and "repefision".

Not surptîsingly, intracellular ATP levels were found to decrease significantly immediately

following the ischemic stimulus of preconditioning. We refer to this phenornenon as an ATP 'debt'.

Despite this debt, however, the rate and degree of ATP degradation in preconditioned cells during

prolonged ischemia was significantly reduced in cornparison to ischemic controls. Thus, both groups

demonstrated similar degrees of ATP degradation following "ischemia" and "repefision", implying

some recovery of ATP levels in the preconditioned group, and emphasizing the possible benefits of

a pharmacological substitute which couid presumably precondition without creating an initial ATP

'debt'. This hypotthesis was substantiated when exogenous adenosine administration was found to

preserve ATP levels compared to ischemic controls.

In similarity to previous reports,2s we demonstrated that the protective effects of ischemic

preconditioning could be transferred to non-preconditioned cells via the supernatant of

preconditioned cells. Moreover, to support Our hypothesis that the crucial protective mediator was

indeed adenosine, we demonstrated, for the first time, the existence of significant and differing

adenosine concentrations in the supematants of varïably preconditioned cells. Once again, the

supematants of anoxically (PO, =O mmHg) preconditioned cells yielded greater concentrations of

adenosine than did ' the supernatants of h ypoxically precondi tioned cells. Conversel y, the

supematants of non-preconditioned cells yielded the lowest arnounts of adenosine and conferred no

protection. The concentrations of adenosine recovered from the hypoxicaily preconditioned cells

(6.7 nmol) and from the non-preconditioned cells ( i .1 nrnol) were below the published dissociation

constant (Kd) for the adult myocardial adenosine A, receptor (1.5 to 3.0 nrn01)."~""* In contrast,

adenosine concentrations in the supernatant of anoxically preconditioned cells (16.3 nmol) greatly

exceeded the reported Kd for the A, receptor. These findings demonstrate once again that maximal

ischemia is necessary for the greatest protection, and that the degree of ischemia and the degree of

protection are both appropriately reflected by the amount of adenosine generated and released with

precondi tioning.

To detennine whether endogenous (ischemic) preconditioning functions via an adenosine-

mediated receptor pathway, cells undergoing supernatant preconditioning were simultaneously

incubated with the non-selective adenosine receptor blocker SPT. In the presence of SPT, the

protective effects of ischemic preconditioning were abolished, implying an adenosine-mediated

receptor phenomenon. Unfortunately, due to the non-specific nature of SPT, we were unable to

establish the specific receptor subtype (ie. A, vs. Ad involved in the preconditioning cascade. 213315

Exogenous Preconditioning

The application of exogenous adenosine was an attempt to facilitate the clinical applicability

of ischemic preconditioning. As previously reported by Ikonomidis and colleagues," adenosine

effectively reproduced the beneficial effects of ischemic preconditioning. However, since the

optimal timing of adenosine treatment was previously undetennined, we treated our cells with

varying doses of adenosine either prior to (Pretreatment), during (Ischemic treatment), or following

(Reperfusion treatment) ischemia, or during al1 three phases (Continuous treatment). We determineci

that adenosine was most protective at a dose of 50 pmol applied pnor to ischemia (pre-aeatment)

and followed by prc-ischemic reperfusion. This was in contrast to the nanomolar quantities of

adenosine required for endogenous preconditioning. The need for higher exogenous doses in order

to penetrate the cellular monolayer in our isolated cardiomyocyte mode1 rnay account for this

discrepancy. Moreover, the absence of several ce11 layers rnay leave exogenous adenosine exposed

to degrading factors and may preclude any accumulation with pre-isc hemic 'washout ' . Finally ,

additional unknown mediators may be released during ischemic preconditioning which may

potentiate the reçeptor mediatedeffects of endogenous adenosine, possibly by enhancing adenosine's

availability at its membranous receptors.

No protection was afforded with adenosine doses equal to or greater than 100 pmol, possibly

due to the phenomenon of receptor down-regdation which has been documented with other receptor

types including adrenergic r e c e p t o r ~ . ~ ~ ~ ~ Moreover, the higher dose of 100 pmol inhibited PKC

activation and did not stimulate isoform-specific PKC translocation. in contrast to the 50 pmol dose.

A report by Amistrong and colleagues documented similar findings when adenosine was applied at

increasing doses in a mode1 of rabbit cardiomyocytes. The authors suggested that the decreased

effects of adenosine at increasing concentrations was due to the activation of inhibitory receptor

subtypes .24

Administration of adenosine during ischernia (ischemic treatment) had a slight protective

effect which was not as great as that seen with adenosine prrîreaîment. This discrepancy was likely

due to the absence of a nomoxic reperfusion period (pnor to ischernia) in the ischemic treatment

group, a condition which seems to be necessary for the maximal effect of adenosine and the second

messenger systems of preconditioning.

For the fmt time, we demonstrateci bat adenosine pretreatment paxtïally lost its effectiveness

when not followed by pre-ischernic (nonnoxic) reperfusion. This finding illustrated two important

points. Firstly, the receptor mediated hypothesis of adenosine preconditioning was iurther supporteci

by the fact that protection was afforded despite the absence of adenosine imrnediately pnor to

prolonged ischemia. Secondly, the presence of adenosine for pmlonged periods in super-

physiological quantities may facili tate receptor downregulation as did the higher 100 m o l dose of

adenosine pretreatment This is not a factor in ischemic preconditioning, presumably due to the fact

that adenosine is present in much smaller quanti ties (i.e. nanomolar rather than micromolar).

Nonetheless, for preconditioning to be achieved, adenosine must be present in sufficient quantities

for a sufficient p e n d of time. The absence of this feature may have accounted for the lack of

protection realized in previous studies of in-vivo preconditioning since constant myocardial

perfusion may have prevented the accumulation of adenosine for a smcient length of the. The

administration of exogenous adenosine allows for control of tissue exposure for the achievement of

optimal preconditioning.

Unlike previous reports in the literature 1@*16530<3M, adcnosine applied during reperhision

(reperfusion treatment) had no measurable effect. We suspect that adenosine pretreatment provided

the maximum attainable pmtective effect since continuous treatment did not provide any additional

benefits.

The effects of adenosine pretreatment were receptor mediated since protection was afforded

despite a p e n d of pre-ischemic nperfision (at which time no adenosine was present) and since the

protective effects were abolished by simultaneous incubation with receptor antagonists. Using the

same principle. we confimicd that the mild pmtective effects conferred with ischemic adenosine

treatment were also likely secondary to receptor activation, rather than a direct substrate-mediated

effect as has been previously hypothesized (Le. adenosine was not con ferring protection by acting

as a substrate for the production of high energy phosphates). Nonetheless, although a direct subsaatt

mediated effect was not detected in our experiments, we cannot completely exclude the possibility

that such an effect was present-

Unlike the case with ischemic pteconditioning, adenosine pretreatment resulted in a

significant preservation of intracellular ATP levels following prolonged "ischemia" and

"reperfùsion". This finding may be due to the fact that no ATP 'debt' was incurred during the

exogenous adenosine pnconditioning process. Although adenosine pretreatment did not affeçt final

lactate concentrations (foilowing prolonged "ischemia" and "reperfusion") compared to controls,

adenosine did increase extracellular lactate concentrations immediately following its application.

This phenornenon is k l y due to a previously ~ported stimulatory effect of adenosine on glycolysis

@y directly increasing glucose 6-phosphate levels), dong with an increase in glucose uptake and

~tilization."'~" Such an effect may m e r facilitate ATP production. The giycolytic effect was

found to be receptor mediated since the elevation in lactate was abolished upon SPT treatmen t.

Protein kinase-C has k e n implicated as an important second rnessenger in animal studies

of the ischemic preconditioning p h e n o r n e n ~ n ? ~ ~ * ~ ~ ~ Thus. we evaluated the hypothesis that human

ischemic and adenosine preconditioning are meditated via this pathway. In concert with previous

reports,= we found that the protective effects of ischemic preconditioning and adenosine

pretreatment were &pendent upon PKC stimulation, as protection was abolished in the presence of

the PKC antagonist Calphostin-C. Monover, the protective effects of preconditioning were partially

reproduced by PKC stimulation using the selective agonist PMA. Our dot-blot analyses confinn that

adenosine exposure results in the translocation of PKC-a corn the cytosolic to the membrane

fraction. The weaker effect seen with 100 pmol may have been secondary to receptor d o m -

regulation. Once again, the receptor mediated effixts of adenosine were demonstrated when the

addition of SPT decreased membrane translocation. Although the extent of translocation was

similar between PMA and adenosine pretreatment, total PKC activity as measured by an in vitro

phosphorylation assay was significantly higher following exposure to PMA. Nonetheless, the

protection afforded by ischemic and adenosine preconditioning was greater than that seen with PMA.

This finding may suggest that preconditioning ac ts via more than one second messenger pathway .

Although various mechanisms may contribute to the protective properties of adenosine in

vivo, our model of isolated ventricular myocytes confirms a preconditioning effect of adenosine

which is independent of altemate protective mechanisms and altemate ce11 types. Thus, adenosine

was protective despite the absence of any effect upon coronary vasodilatation, adrenergic inhibition,

and endotheliai protection. Moreover, the isolated ce11 model and the short time course precludes

any neovascularization&pen&nt effect, Nonetheless, we cannot definitively exclude the presence

of other effector mechanisms of ischemic preconditioning which may or rnay not act independently

of adenosine receptor stimulation and PKC activation? Such possible alternate mechanisrns may

include the synthesis of cardioprotective pmteins in response to thennal1'= or ischemic41N stimuli

(i-e. heat shock proteins), modulation of free radical production,3*4' alteration in the production of

prostanoids and other inflaxmnatoxy mediators.'"' promotion of intennediary m e t a b ~ l i s r n , ~ ~ ~ andlor

modulation of specific ionic flux (Le. potassium, calcium).w1u7

In an effort to M e r explore the possible pmtective effects of adenosine, and to determine

the optimal mode of administration of exogenous adenosine. we undertook a phase II prospective

75

evaluation in patients undergoing elective coronary bypass surgery (CABG)." Since adenosine's

protective effects were hypothesized to be both receptor and substrate mediated, and since iate

benefits could be related to a free radical-scavenging pathway, the effects of exogenous adenosine

were evaluated both prior to and during the ischemic crossclamp period, as well as during

reperfusion. Thirty-three patients undergoing elective CABG using tepid (29' C) 4:l blood

cardioplegia were wigned to receive adenosine, while 40 patients received no adenosine (control

group). Among the patients given adenotine, 21 received a 10 minute precrossclamp intravenous

infusion at 100 pollkglmin via the venous resemoir of the cardiopulmonary bypass circuit,

followed by a 500 pmol infusion via the first 500 mL of high potassium cardioplegia (Low Dose).

The remaining 12 patients received a 200 pmol/kg/min p~crossclamp and reperfusion adenosine

infusion, in addition to a 2 mM cardioplegic infiision throughout the crossclamp perïod (High Dose)).

M a l and coronary sinus blood samples dong with lefi ventricular biopsies were obtained pnor

to (pre-crossclamp), during (crossclamp), and following (post-crossclamp) crossclamp to enable

evaluation of adenosine levels, high-energy phosphate levels and metabolic parameters.

Postoperative hemodynamic parameters (pulse ratdrhythm. systolicldiastolic blood pressure, mean

artenal pressure, pulmonary artend pressure, cardiac output, cardiac index, systernic vascular

resistance) were monitored to evaiuate the clinical benefit, if any, of adenosine administration.

The pre-crossclamp intravenous adenosine infusion induced controllable hypotension in the

high but not the low dose patients, although elevated senim adenosine levels were not measurable

in either group. During the cardioplegic adenosine infusions, senim adenosine levels increased

dramatically in both groups (High Dose: pre-crossclamp=1.49+/-0.14 nmoYg serum,

crosscIamp==1182.59+/-9.6 nmoVg senun; Low Dose: pre-crossclamp=l .S+/-û.36 nmoYg serum,

76

crosscIamp=-466.03+/-64.7 nmollg se-; pc0.01). Similarly. markedly elevated tissue levels of

adenosine were found in myocardial biopsy samples during the cardioplegic infusion only (Low

Dose: pre-crossclamp=û. 19+/-0.11 pmoVg; crossclamp--l.38+/-û.24 pmollg; p 4 . 0 1) . Arterial-

coronary sinus differences suggested myocardial metabolism of adenosine dwing the cardioplegic

infusion. In comparison to controls where tissue ATP levels decreased by 15% during crossclamp.

tissue ATP levels were preserved in both the low dose and high dose adenosine groups with

crossclamping (Law Dose: pre-crossclamp = 2 1.7+/-3.5 )cmol/g, post-crossclamp = 20.6+/-5.l

pmoVg; High Dose: pre-crossclmnp = 26.8+/4.2 junoUg, post-crossclamp = 29.5+/4.7; Contn,Is:

pre-crossclamp = 17.9+/-3.2 pmoYg, post-crossclamp = 14.7+/-2.5 pmoVg; p<0.05). Patients

receiving adenosine tended to produce more lactate during the pre- and early XCL periods in

comparison to controls (Pre-crossclamp: Low Dose = -0.09+/-0.08 mmoYL, High Dose = -0.244-

0.06 mmol/L, Control = 0.16+/-0.1 mmoVL; Crossclamp: Low Dose = -0.3+/-0.06 mmoVL, High

Dose = -0.7+/-0.12 mmolL, Control= 0.15+/-0.1 mrnoYL, @.OS). No significant ciifference in

coronary flow augmentation was noted with adenosine administration. Moreover, no metabolic or

hemodynarnic differences were noted between groups following XCL removal, and no clinical

benefit was attnbutable to adenosine administration.

Although previous clinical studies of adenosine administration during cardiac surgery are far

from conclusive, emtic results may be attributable to inadequate methodologies and/or technical

shortcomings. Based upon our clinical experiences, we have found that due to the extremely short

half life of adenosine, an observable pharmacologie effet likely depends upon the presence of large

doses delivered in sufficient quantities and at appropriate rates directiy into the coronary artenes.

Moreover, although cardioplegic adenosine delivery may npresent the most convenient method of

77

chical administration, our studies suggest that a pre-ischemic @re-crossc1amp) infusion is necessary

for the protective effects of adenosine to be realized. This feature may be due to the requirement for

second messager (G protein, PKC, etc) activation, a phenornenon which is more than likely oxygen

dependent. Finally, the absence of a treatment benefit with clinicai adenosine administration in some

studies may be secondary to the patient population chosen for inclusion in some studies. Since the

results of contemporary coronary bypass surgery in low risk patients are excellent, such patients

Likely have Little to gain from additionai intraoperative protective measures. If any conclusive benefit

of adenosine supplementation is to be found, it will likely be seen in high risk patients requiring

urgent CABG, or in patients with poor ventricular function, for whom current protective measures

are less than optimal.

Al ternate Clinid Applications

Donor Heart Preservation

Contemprary methods of donor hart paxvation allow for maximal ischemic storage times

of four to six hours. Storage times exceeding 6 hours are associateci with an increase in tissue edema

and vascular endothelid injury. Donor shortages. however, have necessitated the search for organs

in distant locals, such that rctneval times rnay far exceed 6 hours. Thus, improved mthods of organ

preservation are necessary in order to maximize storage times. Two groups have reported that

addition of adenosine to a continuous hypothermie infusion system for donor canine hearts has

allowed for successfbl preservation and transplantation for up to 24 hours following organ

r e t r i e ~ a l . ~ ' ~ Our data in human ventricular myocytes suggests that such an application of

adenosine may be clinically favourable for cardiac storage prior to transplantation.

Reducfion of pustbypass t r . i n requiremenr

Coagulopathy following cardiopulmonary bypass is a well described phenornenon. The rnost

Iikely aetiology is platelet dysfunction. Passage of platelets through the membrane oxygenator

secondary to platelet activation and aggregation initiated by the membrane oxygenator of the bypass

circuit. During their study of adenosine's cardioprotective effects, Mentzer and coileagues found

that patients receiving cardioplegic adenosine experienced significantly less blood Ioss and had a

lower incidence of blood product transfusions (personai communication). This effact may have been

secondary to the inhibitory effects of adenosine on platelet aggregation and activation

Off Pump Coronary Bypars Surgery

Contemporary clinical practice advocates the increasing use of off-pump coronary bypass

operations in the hope of minimizing costs, and more importantly, minimizing the complications

associated with cardiopulmonary bypass. Due to the inability to make use of cardioplegic arrest

during such procedures, bypass gr& are constr~.~cteâ on beating h e m with continuous coronary

flow. However, in order to facilitate optimal visualization for construction of crucial distal

anastomoses, perfusion of isolated coronary arteries is intempted using either silastic ligatures or

vascular clamps. Regardless of the mechanism utilized, such interventions render the heart ischemic

along the particular coronary distribution. To iimit resultant rnyocardid ischemia or infarction,

various surgeons have advocated the use of intexmittent bnef coronary occlusion, or ischemic

precondi timing, pnor to prolonged coronary occlusion. Al though recen t studies have no t

demonstrated a beneficial effect of such clinical preconditioning, perhaps the use of a direct

intracoronary adenosine infusion pnor to final coronary occlusion may facilitate improved hinctional

outcornes following off pump surgery.

Second Window of Protection

The 'second window of protection' describes a phenomenon whereby the protection afforded

by preconditioning occurs up-to 24 hours following the initial s t i rn~lus.~ ' This effect is believed

to involve the upregulation of protein synthesis, and in particular the production of cardioprotective

'heat shock proteins' which have been shown to be produced in response to thermalLR andior

ischemicm stimuli. Although we did not investigate the possibility of such an effect in our mode1

of human ventricular myocytes, the existence of such a phenomenon may posess significant merit

and clinical applicability. In fact, in patients undergoing elective coronary bypass surgery,

administration of adenosine 24 h o m pre-operatively may afford a degree of myocardial protection

which is additive to that afforded by intraoperative adenosine administration, for an overall enhanceci

effect.

SUMMARY OF INVE!STIGATIONS AND ORIGINAL CONTRIBUTIONS

The aforernentioned series of experiments have attempted to define the mechanisms and

benefits of myocardid preconditioning in a human mode1 of simulated "ischemia" and "mperiùsion".

In doing so, we have emphasized the importance of exogenous adenosine as a possible

pharmacologie substitute for ischemic preconditioning. We have shown:

1. Ischemic preconditioning protects human cardiomyocytes from prolonged ischemia and

reperfusion through an adenosine-rwieptor, protein kinase-<= mediated pathway.

2. A maximal ischemic stimulus is necessary for the maximal protective effects of ischemic

preconditioning to be realizeà, resulting in the degradation of ATP prior to prolonged ischemia and

reperfusion (ATP debt).

3. Exogenous adenosine applied pnor to ischemia effectively mimics the protective effects of

ischemic preconditioning through a receptor mediated pathway involving protein kinase C activation.

4. Exogenous adtnosine pte~erves intracellular ATP levels duxing prolonged ischemia without

first i n c d n g an ATP debt.

5. Exogenous adenosine facilitates lactate production likely by stimulating glycolysis which,

in h m , may conûibute m e r ATP.

CONCLUSIONS

Adenosine pretreatmen t effectively protects human ventncular myoc ytes €rom the injurious

effects of ischemia and repefision. However, clinical trials are necessary to further define the

beneficial effects of adenosine in humans. Ongoing prospective randomized trials of adenosine

administration in patients with poor ventncular function may help to identify the clinically relevant

benefits of adenosine in cardiac surgery. Finally. M e r studies are necessary to determine the

mechanismfs whereby stimulation of protein kinase C by ischemia or adenosine affords protection

against ischemia and reperfkion injury, as well as the final eff-orls involved in this phenornenon.

APPENDIX ONE

Isolcrtion and cukre of humun venhlculer cardiomyocytes

Technique of isoluîion and c u b e of human ventriculm cadwmyocytes

Bnefly, 20 mg biopsies were obtained from the nght ventricular outflow tract of patients

undergoing corrective surgery for tetralogy of Fallot. After washing the specimen in phosphate

buffered saline (PBS; NaCl: 136.9 mmoYL, KCI: 2.7 rnmoVL, Na$W04: 8.1 mmoYL, KHm, 1.5

rnrnoVL; pH: 7.4) al1 connective tissue elements were removed and the remaining myocardial cells

were separated by enzymatic digestion using a mixture of 0.2 % -sin Wfco Laboratories; Denoit,

MI) and 0.1 % collagenase (Worthington Biochemical Corp.; Frcehold, NJ). The separated cells

were seeded ont0 cell culture dishes and cultured at 37C and 5% CO2 in Iscove's modified

Dulbecco's medium (GIBCO laboratones; Grand Island NY) containing 10% fetal bovine serum, 100

U/ml penicillin, 100 m g l d streptomycin. and 0.1 rnM &mercaptoethanol). Purification was

achieved using a dilution cloning technique. Enzymatically isolated cells were seeded at a low

density (50-100 cells per 9 cm diameter culture dish) to enable morphological identification of

individual cardiomyocytes by their rectangular shape and large size (4ûx80 pm), and separation

from alternate ce11 types such as fibroblasts and endothelid cells. Using a Pasteur pipette, single

cardiomyocyte colonies were then transfemd to a separate culture dish. Celi cultures were inspected

daily, and any contaminateci dishes were discarded. Culture purity of p a t e r than 95% was

demonstrated for each ce11 passage with fluo~scent monoclonal antibody s t a i ~ n g for actin (ENZO

Biochemical Inc.; New York, NY) and human ventricular myosin heavy chah (Rougier Bio-Tech

Ltd.; Montreal, QUE). Ceils passaged 2 to 6 times, with a tirne h m primary culture of less than 60

days, were utilized for this study.

APPENDIX 'Iwo

Ischemih and Regemswn Model

Technique for ceR cuhre ischemia and reperfirswn

The technique used for simulating ce11 culture ischemia and reperfusion was defined

previously by Tumiati, et d2" Following stabiiization in perfusion PBS (phosphate-buffered saline

as defined in Appendix One with the addition of MgQ, 0.49 mM, CaCl, 0.68 mM and glucose 3.0

rnM) at 37°C for 30 minutes. ischemia was simulated by placing the cells into an air-tight plexiglass

chamber (Figure 7) flushed with 100% nimgen and exposing them to a iow volume (1.5 mL) of

anoxic (PO fl mmHg) or hypoxic (Pq =20 mrnHg) pemision PBS at 37'~ for 90 minutes.

Deoxygenated PBS was pnpared in 100 mL quantities by degassing normoxic PBS with 5% CO,

and 95% N2 until the measured reachcd O or 20 mmHg, and the measured pCQ reached 10

mmHg (Blood Gas Analyzer Mode1 IL1312, Instrumentation Laboratory, Milan, My). During this

process, pemision PBS was passed through two oxygen traps including a 1% w/v solution of NaS03

in deionized water flrap #1, Figure 7) followed by a bicarbonate buffer (Na,C03 20 mM, NaHCO,

20 rnM, Trap #2, Figure 7). The solution pH was adjusteci to 7.40+/-0.05 and the osmolality

corrected to 290+/-10 mOsm/L using 1.0 M NaOH and NaCl, respectively. In order to verify the

desired conditions within the nitrogen chamber, 2 mL of anoxic perfusion PBS was also placed in

an open dish within the chamber and tested to ensure the absence of oxygen 5 minutes from the end

of each ischemia experiment. Reperfusion was accomplished by exposure to 15 mL of normoxic

37'C perfusion PBS for 30 minutes.

Bwchem&al Measurements

(Lac fate, ATP, denine nuckotide degradation produch, and PKC assays)

Protein KUuzse C Anaiyses

Isoform specific translocation of protein kinase C (PKC) was demonstrated by performing

a 'slot blot' analysis on cellular cytosolic and membrane fraçtions. Following the intervention of

interest, cells were washed, scraped, and resuspended in 50 pL of 50 pmol/L TRIS-buffered saline

(150 mmoVL NaCl in 50% glycm>l, pH=7.2). Cells were then sonicated and centrifuged at 14,000

rpm for 5 minutes. Following removal of the cytosolic soluble supernatant haction, the pellet was

resuspended in 50 p L of TRIS-buffered saline to yield the membrane enriched Fraction. Both

fractions were then divided into qua1 aliquots of 25 each. One aliquot was employed for

determination of protein concentrations after which the equivalent of 20 pg of protein for each

sample was placed in the slot blot apparatus. Foilowing protein transfer to nitrocellulose, each blot

was exposed to an isoform-specific antibody for PKC-a and PKC-e. Western blot analysis using

chemiluminescent deteetion demonstrated that each antibody was specific for PKC with no evidence

of non-specific background staining. Slot blots were then scanned using a cornmercialiy available

software program (Molecular Images; Mississauga, ONT) and each band was assessed

densitometrically .

Rotein kinase C activity was mcasured using a modification of a previously reported as sa^.^

Confluent cultures of cardiomyocytes were exposed to the treatment of interest for 20 minutes. The

cells were then f in&, scraped and muspendeci in M pL of 50 p m o L TRIS-buffered saline.

Following sonication, 10 pL of each ce11 extract was added to 15 of reaction buffer for 60

minutes. The reaction buffer consisted of equal concentrations of a lissamine rhodamine B-labelled

peptide containing a PKC-specific phosphorylation site (epidermal growth factor receptor,

RKRTLRIU.), an activating solution (Phosphatidyl-L-serine, lm-), and a buffer containing 10

rnrnoUL ATP, 50 mmoVL MgCl,, 0.5 mrnoVL CaCb , 0.01% Triton X-100 and 100 rnrnoüL

TRIS(hydroxymethy1)-amino methane, pH=7A (PIERCE Biotechnoiogy; Rockford, IL).

Following incubation, the reaction mixture was fractioned through a DEAE-sepharose

column quilibrated with 20 mmoUL HEPES (pH=7.9 at 4OC), 20% glycerol and 1 IMIOVL EDTA.

&ter binding to the positively charged column, phosphorylated peptide was eluted with a 2 mmoYL

NaCl-HEPES buffered saline solution. The absorbence of the eluted fluid was then measured using

a spech'ophotometer (Beckmann Ltd.; Fulierton. CA) at 570 m. Reaction buffer that had not been

exposed to any cell extracts was also placed on the column, and the eluate used as a negative control.

Ce11 extracts exposed to 10 nmoVL PMA (4fbphorbol 12-myristate 13-acetate) (Sigma Chemical

Co., St. Louis, MO), a PKC stimulating phorbol ester, were employed as positive controls.

Absorbence was subsequently comcted for protein content and expressed in relative units for

absorbencdmg protein.

1. Naylor CD, Slaughter PM: Cardiovascular health and services in Ontario. An ICES atlas. 1999; 16- 198.(Abstract)

2. Lear S, Stachenko S: 1997 Canadian Cardiovascular Society Consensus Conference. 1997;(Abstract)

3. Vamauskas Ea: Twelve year foilow up of survivai in the randomized European Coronary Surgery Study. New England Journal of Medicine l988;3 19:332-337.

4. Califf RM, Haml FEJ, LEC KL, et ai: The evolution of medical and surgical therapy for corcmary artery disease: A 15 year perspective. IAMA 1989;261:2077-2086.

5. Myen WO, Gersh BJ, Fisher LD, et al: Medical versus early surgical therapy in patients with triple vesse1 disease and mild angina pectoris. Annais of Thoracic Surgery l987;44:47 1486.

6. Brown AC, Hutter AM, DeSanctis RW: Prospective study of medical and urgent surgical therapy in randomizable patients with unstable angina pectoris: Results of in-hospital and chronic mortality and morbidity. American Heart Journal 198 1; lO2:959-964.

7. Parisi AF, Khuri S, Deupree RH, Sharma GVRK, Scott SM, Luchi RJ: Medical compared with surgical management of unstable angina: 5 year moRaljîy and morbidity in the Veteran's Administration Study. Circulation 1989;80: 1 176-1 189.

8. Chnstakis GT, Birnbaum PL, Weisel RD, et al: The changing pattern of coronary bypass surgery (CABG). Circulation 1989;80: 15 1-16 1.

9. Maharajh GS, Mastem RG, Keon WJ: Cardiac operations in the elderly: who is at risk? Ann Thorac Surg 1998;66: 1670-1673.

10. Tsai-Tsung-Po., Nessim SS, Kass RM, et al: Morbidity and mortality after coronary artexy bypass in octogenerians. Annals of Thoracic Surgery 1991;51:983-986.

11. Mohan R, Amsel BJ, Waiter PJ: Coronary artery bypass graftmg in the elderly: a review of studies in patients older than 64,69, or 74 years. Cardiology 1992;80:215-225.

12. Fremes SE, Christakis GT, Weisel RD, et al. A clinical trial of blood and crystalloid cardioplegia. Jomal of Thoracic and Cardiovascular Surgery 1984;88:726-74 1.

13. Teoh KH, Chnstakis GT, Weisel RD, et ai. Accelerated myocardial metabolic recovery with temiinal w a m blood cardioplegia (hot shot). Journal of Thoracic and Cardiovascular Surgery l997;g 1:888-895.

14. Hayashida N, Ilconornidis JS, Weisel RD, et al. The optimal cardiopiegic temperature. Annals of Thoracic Surgery l994;58:% 1-97 1.

15. Hayashida NT lkonomidis JS, Weisel RD. et ai. Adequate distribution of warm cardioplegi solution. J Thorac Cardiovasc Surg 1995; 1 10:800-8 12.

16. Rao V, Cohen G, Weisel RD, et al. Optimal flow rates for integrated cardioplegia. J Thorac Cardiovasc Surg 1998; 115226-235.

17. Chirac P: De Motu Cordis. Adversaria Anaiytica 1698; 121

18. Hemck JB: Clinid fatutes of sudden obstruction of the coronary arteries. Journal of the Amencan Medicd Association 19 l2;59:2O 15

19. Blumgart HL, Giiligan DR, Schlesinger MJ: Experirnental studies on the effects of temporary occlusion of the coronary arteries: The production of myicardial infarction. Arnéncan Heart Journal 1941;21:374

20. Jennings RB, Schaper J, Hill ML, Steenbergen U, Reimer KA: Effect of reperfusion late in the phase of ischemic injury: changes in ce11 volume, electrolytes, metabolites and ultrastructure. Circulation Research 1985;56:262-278.

21. Reimer KA, HiII ML, lennings RB: Rolonged depletion of ATP and of the adenine nucleotide pool due to delayed resynthesis of adenine nucleotides following reversible ischemic myocardial injury in dogs. Journai of MoIecular and Cellular Cardio1ogy 198 1; l3:229-239.

22. Swain Sabina RL, McHale PA, Greenfield JCJ, Holmes EW: ProIonged myocardial adenine nucleotide depletion after brief ischemia in the open chest dog. American Journal of Physiology 1982;242:H8 18-H826.

23. Braunwald E, Kloner RA: The stunned myocardium: prolonged postischemic ventricula. dysfunction. Circulation 1982;66: 1 146- 1 149.

24. Mahan E, Korestune Y, Correti MT Chako VP, Kusuoka H: Calcium and its roIe in myocardial ce11 injury during ischemia and reperfusion. Circulation 1989;80: 17-22.

25. Bolli 1, Jeroudi MO, Patel BS, et al: Marked reduction of free radical generation and contractile dyshinction by antioxidant therapy begun at the t h e of repemision: evidence that myocardial stunning is a manifestation of repefision injury. Circulation Research 1989;65:607-622.

26. Reimer KA, Murry CE, Yamasawa 1, Hill ML, Jennings RB: Four brief periods of myocardial ischemia cause no cumulative ATP loss or necrosis. Amencan Journal of Physiology 1986;251:H1306-H1315.

27. Murry CE, Jennings RB, Reimer KA: Rcconditioning with ischemia: a delay of lethal ce11 injury in ischemic myocardium. Circulation l986;74: 1 124-1 136.

28. Ikonomidis JS: Preconditioning Wunian Ventricular Cardiomyocytes. 1995;30(Abstract)

29. Mitchell MB, Winter CB, Locke-Winter CR, Bane rjee A, Harken AH: Cardiac preconditioning does not q u i r e rnyocardial stunning. Ann Thorac Surg 1993;55:395-400.

30. Cohen MV, Liu GS, Downey JM: Preconditioning causes improved wall motion as well as smaiier infarcts after transient coronary occlusion in rabbits. Circulation 1991;84:341-349.

31. Flack JE, Kimura K, Engelmm RM, et al: Preconditioning the heart by repeatted snuining improves myocardiai salvage. Circulation 199 1;84:IIi-369-III-374.

32. Shinikuda Y, Iwamoto T, Mallet RT, Downey HF: Hypoxic preconditioning attenuates stunning caused b y repeated coronary occlusions in dog heart. Cardiovasc Res l993;27: 565-570.

33. Landymore RW, Bayes AJ, Murphy JT, Fiis JH: Preconditioning prevents myocardial stunning after cardiac transplantation. Ann Thorac Surg 1998;66: 1953- 1957.

34. Przykienk K, Bauer B, Ovize M. Kloner RA, Whittaker P: Regionai ischemic "preconditioning" protects remote virgin myocardium h m subsequent sustained coronary occlusion. Circulation 1993;87:893-899.

35. Thannhauser SJ, Bommes A: Experimentelle Studies Ober den Nucleinstoffwechsel. II. Mitteilmg. Stoffwechselversuche mit Adenosin und Guanosine. Joumal of Physiological Chemistry 19 14;9:336-343.

36. Drury AN, Szent-Gy0rgyi.A. The physiological activity of adenine compounds with wpecial reference to their action upon the mamrnalian heart. Journal of Physiology of London 1929;68:213-237.

37. Lindner F, Rigler R: Uber die Beeinflussung der Weita der Herzteranzgefasse durch Produkte des Zellkem stoffwechsels. Pfieugers Archives 193 1;226:697-708.

38. Bennet DW, Dxury AN: Further observations relating to the physiological activity of adenine compounds. Journal of Physiology of London 193 1;72:288-320.

39. Fleisch A, Weger P: Die gefasserweitemde W i h g der phosphoryiierten S to ffwechselprodukte. Pfleugers Archives 1938;239:362-369.

40. Honey RM, Ritchie WT, Thompson WAR: Action of adenosine upon the human heart. Q J Med 1930;23:485-489.

41. Jezer A, Oppenheimer BS. Schwarîz SP: The effect of adenosine on cardiac irregulantis in man. American Heart Journal 1934;9:252-258.

42. Rothman H: Der Einfluss der Adenosin phosphosaure auf die Herztatigteit. Archive Experimenta Pathologika Pharamakologica 193 1;155: 129-138

43. Wayne EJ, Goodwin IF, Stoner HB: The effect of adenosine triphosphate on the electrocardiogram of man and animals. British Heart Journal 1949; 1 15567.

44. Wedd AM: The action of adenosine and certain related compounds on the coronary flow of the perf'used heart of the rabbit Journal of Pharmacologic Experimental Therapy 193 1 ;4 1 :355-366.

45. Wedd AM, Fenn WO: The action on cardiac musculature and the vagomimetic behaviour of adenosine. Journal of Pharmacologic Experimental Therapy 1933;47:365-375.

46. Berne RM: Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood flow. Amencan Journal of Phy siology 1963;204:3 17-322.

47. Gerlach E, Becker F: AnonymousTopics and perspectives in adenosine research. Berlin: Springer-Verlag , 1987:

48. Olsson RA: Changes in content of purine nucleoside in canine myocardium during coronary occlusion. Circulation Research 1970;26:301-306.

49. Bardenheuer H, Schrader J: Supply to demand ratio for oxygen determines formation of adenosine by the heart. Amencan Journal of Physiology 1986;250:H173-H180.

50. Berne RM: The role of adenosine in the regulaîion of coronary blood flow. Circulation Research 1980;47:807-8 13.

51. Olsson RA. Patterson RE: Adenosine as a physiological rrgulator of coronary b l d flow. R o p s s in Molecular Subcellular Biology 1976;4:227-248.

52. Hershfield MS. Kredich NM: S-adenosylhomocysteine hydrolase is an adenosine-binding protein: a target for adenosine toxicity. Science Washington DC 1978;202:757-760.

53. Olsson RA, Saito D. Steinhart CR: Compartmentalization of the adenosine pool of dog and rat hearts. Circulation Research 1982;50:6 17426.

54. Schrader J. Schutz W. Bardenheuer H: Role of S-adenosylhomocysteine hydrolase in adenosine metabolism in mammalian heart. Biochemistry Journal 198 1; l96:65-70.

55. Sanin A. Rall TW: The effect of adenosine and adenine nucleotides on the cyclic adenosine 3',5'-phosphate content of guinea pig cerebral cortex slices. Molecular Pharrnacology 1970;6: 13-23.

56. Londos C, Wolff k Two distinct adenosine-sensitive sites on adenylate cyclase. Roceedings of the National Academy of Science USA 1977;745482-5486.

57. Belardinelli L, Isenberg G: Isolated atrial myocytes: adenosine and acetylcholine increase potassium conductance. Amencan Journal of Physiology 1983;244:H734-H737.

58. Kurachi Y, NakajimaT, Sugimoto T: On the mechanism of activation of muscarhic K+ channels by adenosine in isolated aüial cells: involvement of GTP-binding proteins. Pfleugers Archives 1986;407:264-274.

59. Schrader J: Metaboüsm of adenosine and sites of production in the heart. In: Berne RM, Rail TW, Rubio R, eds. Regulatory hction of adenosine. The Hague: Martinus/Nijhoff, 1983:133-156.

60. Bertolet BD, Hill JA: Adenosine: diagnostic and therapeutic uses in cardiovascular medicine. Chest 1993;lM: 1860-1871.

61. Bardenheuer H, Schrader J: Supply to demand mtio for oxygen detemines formation of adenosine by the heart. American Journal of Physiology 1980;H389-H398.

62. Olsson RA, Pearson JD: Cardiovamilar pwinoceptors. Physiological Reviews l99O;70:76 1-845.

63. Holian A, Owen CS, Wilson D F Control of respiration in isolated mitochondria: quantitative evaluation of the dependence of respiratory rates on [ATP], [ADP) and [Pi]. Archives of Biochemistry and Biophysics 1977; 18 1 : 164-17 1.

64. Gamble WJ, La Farge CG, Fyler DC, Weisul J, Monroe RG: Regional coronary venous oxygen saturation and myocardial oxygen tension following abrupt changes in venaicular pressure in the isolated dog heart. Circulation Research 1974;34:672-68 1.

65. Holtz J, Grunewald WJ, Manz R, Restorff WV, Bassenge E: Intracapillary hemoglobin oxygen saturation and oxygen consrmiption in different layers of the left ventricular myocardium. Pfleugers Archives 1977;370:253-258.

66. Sparks HV, Bardenheuer H: Regdation of adenosine formation in the heart. Circulation Research l986;58: 193-201.

67. Lloyd HGE, Schrader I: The importance of the transmethylation pathway for adenosine metabolism in the hearts. In: Gerlach E, Becker BF, eds. Topics and perspectives in adenosine research. Berlin: Springer-Verlag, 1987: 199-207.

68. Achterberg PW, De Tombe PP, Hamisen E, De Jong JW: Myocardiai S-adenosylhomocysteine hydrolase is important for adenosine production during normoxia. Biochimica Biophysica Acta 1985;840:393-400.

69. Olsson RA: Ligand binding to the adenine analogue binding protein of the rabbit erythrocyte. Biochemistry 1978; 17:367-375.

70. Kwong FYP, Davies A, Tse CM, Young JD, Henderson PJF, Baldwin SA: Purification of the human exythmcyte nucleoside transporter by immunoaffînity chromatography. Biochemistry Journal 1988;255:243-249.

71. Paterson ARP, Kolassa N, Cass CE: Transport of nucleoside dnrgs in animal cells. Phannacological Therapy 198 1 ; 12:s 15-536.

72. Plagemann PGW, Wohlheuter RM: Permeation of nudeosides, nucleic acid bases, and nucleo tides in animal cells. C m n t Topics in Membrane Transport 1980; l4:225-230.

73. Plagemann PGW, Wohlheuter RM: Nucleoside transport in mammalian cells and interaction with intracellular metabolism. In: Berne RM, Rail TW, Rubio R, eds. Regulatory function of adenosine. The Hague: Nijhoff, 1983: 179-201 .

74. Olsson RA, Gentq MK, Snow JA: Steric requirements for binding of adenosine to a membrane carrier in canine heart. Biochimica Biophysica Acta 1973;3 11:242-250.

75. Paterson ARP, Oliver JM: Nucleoside transport: Inhibition by pnia~benzylthioguanosine and related compounds. Canadian Journal of Biochemistry 197 1 ;49:27 1-274.

76. Heller LJ , Sherin JC, Mohrrnan DE, Dole W. Adenosine receptor blockade potentiates cardiac interstitial adenosine during metaboiic stimulation in isolated rat rearts. Circulation 1987;76:IW327(Abstract)

77. Mckenzie JE, Steffen RP, Haddy FJ: Effect of thwphyiline on adenosine production in the canine myocardium. American Journal of Physiology 1987;252:H204-H2 10.

78. Paterson ARP, Jakobs ES, Ng CYC, Odgard RG, Adjei AA: Nucleoside transport inhibition in-vitro and in-vivo. In: Gerlach E, Becker BF, eds. Topics and Perspectives in Adenosine Research. Berlin: Springer-Verlag, 1987:89-101.

79. Hopkins SV, Goldie RG: A species difference in the uptake of adenosine by heart. Biochemical Phmacology 197 1 ;20:3359-3365.

80. Parkinson FE, Clanachan AS: Subtypes of nucleoside transport inhibitory sites in heart: a quatitative autoradiographic analysis. European Journal of Pharmacology 1989; 163:69-75.

8 1. Achterberg PW, Stmve RJ, De Iong JW: Myocardial adenosine cycling rates during normoxia and under conditions of stimulated purine release. Biochern J 1986;235: 13- 17.

82. Bontemps F, Van Den Berghe G, Hers HG: Evidence for a substrat+ cycle between AMP and adenosine in isolated hepatocytes. Proc Natl Acad Sci USA 1983;80:2829-2833.

83. Maguire MH, Lukas MC, Rettie 3F: Adenine nucleotide salvage synthesis in the rat heart. Pathways of adenosine salvage. Biochim Biophys Acta l972;262: 108-1 15.

84. Deussen A, Moser G, Schrader J: Contribution of coronary endothelial cells to cardiac adenosine production. Pfleugers Arch 1986;406:608-614.

85. Gorman MW, Bassingthwaighte JB, Olsson RA, Sparks HV: Endothelid ce11 uptake of adenosine in skeletal muscle. Am J Pfiysiol 1986;250:H482-H489.

86. Kubler W, Spieckerman PG, Brctschneider HJ: Muence of dipyridamole on myocardial adenosine metabolism. J Mol Ce11 Cardiol 1970; 1 :23-28.

87. Catravas JD: Removai of adenosine from the rabbit pulmonary circulation, in vivo and in vitro. Circ Res 1984;54:603-611.

88. Liu MS, Feinberg H: Incorporation of adenosine-8-14C and inosine-8-14C into rabbit heart adenine nucleotides. Am J Physiol 197 1 ;220: 1242-1248.

89. Becker BF, Gerlach E: Unc acid, the major catabolite of cardiac adenine nucleotides and adenosine, originates in the coronary endothelium. In: Gerlach E, Becker BF, eds. Topics and Perspectives in Adenosine Research. Berlin: Spnnger-Verlag, 1987:209-221.

90. Ronca-Testoni S, Borghini F: Degradation of perfused ahnine compounds up to uric acid in isolated rat heart. J Mol Celi Cardiol 1982; 14: 177-180.

91. Arnold ST, Cysyk RL: Adenosine export fiom the liver: oxygen dependency. Am J Physiol 1986;25 1 :G34-G39.

92. Manfredi P. Holmes EW: Purine salvage pathways in myocardium. Annu Rev Physiol 1985;47:691-705.

93. Chechik BE, Baumal R, Sengupta S: IAX:alization and identity of adenosine deaminast positive cells in tissues of the young rat and calf. Histochern J 1983;15:373-387.

94. Geisbuhler T, Altschuld RA, Trewyn RW, Ansel AZ, Lamka K. Brierly GP: Adenine nucleotide metabolism and compartrnentalization in islated adult rat heart celis. Circ Res 1984;54:536-546.

95. Bardenheuer H, Whelton B. Sparks HV: Adenosine reluise by the isolated guinea pig heart in response to isoproterenol, acetylcholine, and acidosis: the minimal role of vascular endothelium. C k Res l987;6 l:59~-600.

96. Bunistock G, Buckley NJ: The classification of receptors for adenosine and adenine nucleotides. In: Paton DM, ed. Methods in Pharmacology. Methods Used in Adenosine Research. New York: Plenum, 198% 193-2 12.

97. Van Calker D, Muller M, Harnprecht B: Adenosine regulates via two different receptors, the accumulation of cyclic AMP in culnired brain cells. J Neurochem 1979;33:999-1005.

98. Fain JN, Malbon CC: Regulation of adenylate cyclase by adenosine. Mol Ce11 Biochem 1979;25:143-151.

99. Cam CS, Hill RJ, Masamune H, et al. Evidence for a role for both the adenosine Al and A3 receptors in protection of isolated human atrial muscle against simulated ischernia Cardiovasc Res 1997;36:52-59.

100. Liang BT, Jacobson KA: A physiological role of the adenosine A3 receptor: sustained cardioprotection. Proc Nati Acad Sci USA 1998;95:6995-6999.

101. Tracey WR, Magee W, Masamune H, et al: Selective adenosine A3 receptor stimulation reduces ischemic myocardial injury in the rabbit heart. Cardiovasc Res 1997;33:410-415.

102. Hechter O: Concerning possible mechanisms of hormone action. Vit Hom 1955; 13:293-346.

1 03. Graziano MP, Gilman AG: Guanine nucleotide-binding regulatory pro teins: mediators of transmembrane signaling. Trends Pharmacol Sci 1987;8:478-48 1.

104. Neer ET, Clapham DE: Roles of G protein subunits in transmembrane signalling. Nature Lond l988;333: 129-134.

105. Rodbell M: Programmable messengers: a new theory of hormone action. Trends Biochem Sci 1985; lO:461-464.

106. Kitakaze M, Hon M, Kamada T: Role of adenosine and its interaction with alpha adrenoreceptor activity in ischemic and reperfusion injury of the myocardium. Cardiovasc Res l993;27: 18-27.

107. Thornton JD. Liu GS, Downey Jnk Pretreatment with pertussis toxin blocks the protective effects of preconditioning: evidence for a G-protein mechanism. J Mol Ce11 Cardiol l993;25:3 11-320.

108. Lasley RD, Mentzer RM: Pertussis toxin blocks adenosine Al mediated protection of the ischemic rat heart. J Mol Ce11 Cardiol l993;25:8 15-821.

109. Liu Y, Downey mi: Reconditioning against infarction in the rat heart does not involve a pertussis toxin sensitive G protein. Cardiovasc Res l993;27:608-6 1 1.

110. SziIvassy 2, Ferdinandy P, Bor P, Jakab 1, Lonovics J, Koltai M: Ventricular overdrive pacing induced anti-ischemic effect: a conscious rabbit mode1 of preconditioning. Am J Physiol 1994;266:N2033-HXM 1.

1 11. Iwase T, Murakami T, Tornita T, Miki S, Nagai K, Sasayama S: Ischemic preconditioning is associateci with a delay in ischemia induced reduction of beta-adrenergic signai transduction in rabbit hearts. Circulation 1993;88:2827-2837.

112. Fu LX, Kirkeboen KA, Liang QM, Sjogren KG, Hjalmarson A, ïiebekk A: Free radical scavenging enzymes and G protein rnediated receptor signalling systems in ischemically precondi tioned porcine myocardium. Cardiovasc Res 1993 ;SE6 12-6 16.

1 13. Jochem G, Nawrath H: Adenosine activates a potassium conductance in guinea-pig atrial heart muscle. Experientia Basel 1983;39: 1347- 1349.

114. Peralta EG, Ashicenazi A. Winslow JW, Ramachandran J, Capon DJ: Differential regulaîion of PI hydrolysis and adenylyl cyclase by muscarhic nceptor subtypes. Nature Lond 1988;334:434-437.

115. Isenberg G, Serbai E, KIockner U: Ionic channels and adenosine in isolated heart cells. In: Gerlach E, Becker BF, eds. Topics and Perspectives in Adenosine Research. Berlin: Spnnger-Verlag, 1987~323-335.

116. Kurtz A: Adenosine stimulates guanylate cyclase activity in vascular smooth muscle cells. J Bi01 Chem 1987;262:6296-6300.

117. Dolphin AC, Forda SR, Scott RH: Calcium dependent currents in cultured rat dorsal mot ganglion neurones arr inhibited by an adenosine analogue. J Physiol Lond 1986;373:47-61.

118. Dolphin AC, Prestwich A: Pertussis toxin reverses adenosine inhibition of neuronal glutamate release. Nature Lond 1985;3 16: 148-150.

119. Londos C: On multiple targets for fat ce11 receptors. In: Gerlach E, Becker BF, eds. Topics and Perspectives in Adenosine Research. Berlin: Springer-Verlag, 1987:239-248.

120. Barret ET, Schwartz RG, Francis CK, Zaret BL: Regdation by insulin of myocardial giucose and fatty acid metabolism in the conscious dog. J Clin Invest 1984;74: 1073-1079.

121. Eckel J, Pandalis O, Reinaur H: ùwl in action on the glucose transport system in isolated cardiocytes from adult rat. Biochem J 1983;212:385-392.

122. Long CJ, Stone TW: Adenosine reduces agonist induced production of inositol phosphates in rat aorta. J Pham Pharmacol 1988;39: 1010-1014.

123. Streb H, Irvine RF. Bemdge MJ, Schulz 1: Release of calcium from a non-mitochondrial intracellular store in pancreatic acinar cells by inositol 1.4,S-triphosphate. Nature 1983;306:67-69.

124. Takai Y, Kishimoto A, Kikkawa U, Mori T, Nishizuka Y: Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activaid phospholipid dependent proiein kinases system. Biochem Biophys Res Commun 1979;91: 1218-1224.

125. Newton AC: Protein kinase C: Structure, function and regulation. J Bi01 Chem 1995;270:28495-28498.

126. Coussens L, Parker PJ, Rhee L, Yang-Feng TL: Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 1986;233:859-866.

127. House C, Kemp BE: Rotein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 1987;238: 1726-1728.

128. Newton AC: Rotein kinase C. Seeing two domains. Cuxr Bi01 1995;5:973-976.

129. Taylor SS, Radzio-Andzelm E: Three protein kinase structures define a common motif. Structure 1994;2:345-355.

130. Newton AC: Interaction of proteins with lipid headgroups: lessons from protein kinase C. Annu Rev Biophys Biomol Sûuct lW3;22: 1-25.

13 1. houe M, Kishimoto A, Takai Y, Nishinika Y: Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mamrnalian tissues. IL Proenzyme and its activation by calcium-dependent protease b m rat braui. J Bi01 Chem l977;252:76 10-76 16.

132. Goto M, Cohen MV, Downey IM: The role of protein kinase C in ischemic preconditioning. Ann NY Acad Sci 1996;793: 177-190.

133. Kennely PJ, Krebs EG: Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Bi01 Chem 1991;266: 15555-15558.

134. Mochly-Rosen D, Koshland DE: Domain structure and phosphorylation of protein kinase C. J Bi01 Chem 1987;262:2291-2297.

135. 0'Bria.n C, Ward NE: Stimulation of the ATPase activity of rat brain protein kinase C by phospho acceptor substrates of the enzyme. Biochemistry 1991;30:2549-2554.

136. OIT JW, Newton AC: Intrapeptide regulation of protein kinase C. J Bi01 Chem 1994;269:8383-8387.

137. Limas CJ, Limas C: Involvement of microtubules in the isopmterentol induced down regulation of myocardial B-adrenergic raceptors. Biochun Biophys Acta 1983;735:18 1-184.

138. Kraft AS, Anderson WB, Cooper HL, Sando JJ: Nature 1983;301:621-623.

139. Berne RM, Rubio R: Coronary Circulation. In: Beme RM, Sperlakis N, Gieger SR, eds. Handbook of Physiology, Washington DC: Arnerican Physiology Society, 1979:873-952.

140. Hon M, Kitakaze M: Adenosine, the hem, and coromary circulation. Hypertension 199 1 ; l8:565-574.

14 1. Belardinelli L, Isenberg G: Actions of adenosine and isoproterenol on isolated mammaiian ventricula. mywytes. Circulation Resemh 1983;53:287-297.

142. Sato H, Hon M. Kitakaze M: Endogenous acfenosine blunts beta-adrenoreceptor rnediated inotropic response in the hypoperfused canine myocardium. Circulation 1992;85: 1 594- 1603.

143. Cronstein BN. Lmin RI, Belanoff J, Weissmann G, Hirschhom R: Adenosine: an endogenous inhibitor of neutrophil-mediated injury to endothelial ceus. J Clin Invest 1986;78:760-770.

144. Cronstein BN, Kramer SB, Weissmann G, Hirschhom R: Adenosine: a physiological modulator of superoxide anion generation by human neutrophils. J Exp Med 1983; 158: 1 160- 1 177.

145. Newman WH, Becker BF, Heier M, Nees S, Gerlach E: Endothelium-mediated coronary dilitation by adenosine does not depend on endothelial adenylate cyclase activation: studies in isolated guinea pig hearts. Pfleugers Arch 1988;413: 1-7.

146. Kitakaze M, Hon M, Sato H: Endogenous adenosine inhibits platelet aggregation during myocardial ischemia in dogs. Circ Res 199 l;69: l4ûZ- 1408.

147. Mainwaring R, Lasley R, Rubio R, Wyatt DA, Mentzer RM: Adenosine stimulates glucose uptake in the isolated rat heart. Surgery 1988;13:445-449.

148. Wyatt DA, Edmunds MC, Rubio R, Berne RM, Lasley RD, Mentzer RM: Adenosine stimulates glycolytic flux in isolated perfûsed rat h e m by Al-adenosine receptors. Am J Physiol 1989;257:H1952-H1957.

149. Beme RM: Adenosine: a cardioprotective and therapeutic agent. Cardiovasc Res i993;27:2

150. Olfsson B, Forman MB, Puett DW: Reduction of reperfusion injury in the canine preparation by intracoronary adenosine: importance of the endotheliwn and the no-reflow phenornenon. Circulation l987;76: 1135-1 145,

15 1. Babbitt DG, Virmani R, Forman MB: Intracoronary adneosine administered after reprefusion limits vascular injury after prolonged ischemia in the canine model. Circulation 1989;80: 1388-1399.

152. Pitarys CJ, Vimani R, Vildibill HD, Jackson EK, Forman MB: Reduction of myocardial reperfusion inj ury by intravenous adenosine adrninistered during the earl y reperfusion period. Circulation 199 1;83:237-247.

153. Lasley RD, Zhou 2, Hegge JO, Bunger R, Mentzer RM: Adenosine attenuates in vivo myocardial stunning with minimal effects on cardiac energetics. Basic Res Cardiol l998;93:303-3 12.

154. Thourani VH, Ronson RS, Jordan JE, Guyton RA, Vinten-Johansen J: Adenosine A3 pretreatrnent before cardioplegic arrest attenuates postischemic cardiac dysfunction. Ann Thorac Surg l999;67: 1732-1737.

155. Downey JM, Guang SL, Thomton JD: Adenosine and the anti-infarct effects of preconditioning. Cardiovasc Res 1993;27:3-8.

156. Schwartz ER, Mohn M, Sack S, Anas M: The role of adenosine and its Al-receptor in ischernic preconditioning- Circulation 199 l;84:II-L9 L (Abstract)

157. Brown AM: Regdation of heartbeat of G-protein coupled ion channels. Am J Physiol 1990;259:H1621-H1628.

158. Stiles GL: Adenosine receptors: physiologie regulation and biochemical mechanisms. News Physiol Sci 1991;6: 161-165.

159. Thornton JD, Downey JM: Pretreatment with pertussis toxin blocks the protective effects of preconditio~ng: evidence for a Gi-protein mechanism. J Mol Ce11 Cardiol 1992;

160. Lasley RD, Wylen VL, Mentzer RM: Evidence for the role of Gi-proteins in attenuating myocardiaf isc hemia damage. Circulation 1990;82:m-759(Abs tract)

161. Bolling SF, Childs KF, Ning XH: Adenosine. J Surg Res 1994;57:591-595.

162. OIafsson B, Forman MB, Puett DW: Reduction of reperfusion injury in the canine preparation by intracoronary aâenosine: importance of the endothelium and the "no-reflow" phenornenon. Circulation l987;76: 1 135-1 145.

163. Homeister JW, Hoff PT, metcher DD, Fantone JC, Lucchesi BR: Combined adenosine and lidocaine administration limits myocardial repemision injury. Circulation 1990;82:595-608.

164. Norton ED, Jackson EK, Vinnani R, Forman MB: Effect of intravenous adenosine on myocardial reperfusion injury in a mode1 with low myocardial blood flow. Am Hem J 1991;122:1283-1291.

165. Norton ED, Jackson EK, Turner MB, Vinnani R, Forman MB: THe effects of intravenous infusions of selective A-1 nxeptor and A-2 receptor agonists on myocardial reperfusion injury. Am Heart J 1992;123:332-338.

166. Belardinelli L, West A, Crampton R, Berne RM: Chronotropic and dromotropic effects of adenosine. In: Berne RM, Ra11 TW, Rubio R, eds. Regulatory function of adenosine. Boston: Martinus Nijhoff, 1983:377-396.

167. Kitakaze M, Kitabatake A: Increased 5'-nuclwtidase activity caused by protein kinase C enhances adenosine production in hypoxic caniiomyocytes of rats. Circulation 199 1 ;84:II-620(Absmct)

168. Strasser RH, Walendzik H, Marquetant R: Prolongeci myocardial ischemia provokes an induction of protein kinase C activity . Circulation 199 1 ;84:II-620(Abstract)

169. Jaffe MD, Quinn NK: Warm up phenomenon in angina pectoris. Lancet L980;2:934-936.

170. Williams DO, Bass TA, Gerwitz H, Most AS: Adaptation to the stress of tachycda in patients with coronary artery disease: insight into the mechanism of the warm up phenomenon. Circulation 1985;7 1:687-692.

17 1. Muller DWM, Topo1 EJ, Califf M. Relationship between antecedent angina pectoris and short term prognosis after thrombolytic therapy for acute myocardial infarction. Am Heart J 1990; 1 19:224-23 1.

172. Kloner RA, Shook T, Rzyklenk K: Previous angina alters in-hospital outcome in TIMI 4. Circulation 1993;88:149

173. Walker DM, Walker JM, Pattison C, Pugslet W, Yellon DM: Preconditioning protects isolated human muscle. Circulation lW3;88:I- 138

174. Kerensky RA, Kutcher MA, Braden GA: The effects of intracoronary adenosine on preconditio~ng during coronary angioplasty . Clin Cardiol 1995; 1 8:g 1-96.

175. Deutsch E, Berger M, Kussmaul WG, Hirshfeld JW. Hemnann HC, Laskey WK: Adaptation to ischemia during percutaneous transluminal coronary angioplasty. Clinical, hemodynami-c and metabolic features. Circulation 1990;82:205 1

176. Aikhulaifi AM, Yeiion DM, Pugsley WB: Reconditioning the human heart during aorto-coronary bypass surgery. Eu J Cardiothor Surg 1994;8:270-276.

177. Barbash GI, White HD, Modan M. Van de Werf F: Antecedent angina pectoris predicts worse outcome after myocardial infarction in patients receiving thrombolytic therapy: Expenence gleaned from the International Tissue Plasminogen Activator / Sheptokinase Mortaiity Trial. J Am Col1 Cardiol l992;2O:364l,

178. Denys BG, k J, Reddy PS, Urctsky BF: 1s the= adaptation to ischemia during PTCA? Eur Heart J 1992; l3:246

179. DeBniyne B, Vantrimpont PI, Goetua SM, Stockbrockx J, Paulus WJ: Can coronary angioplasty serve as a mode1 of myocardial preconditioning? Circulation 1992;86:I-378

180. Perrault LP, Menasche P, Bel A, et al; khemic preconditioning in cardiac surgery: a word of caution. J Thorac Cardiovasc Surg 19%; 1 12: 1378-1 386.

181. Liang BT, Gross GJ: D k t preconditioning of cardiac myocytes via opioid receptors and K-ATP channeh Circ Res 1999;84:1396-1400.

182. Tomai F, Crea F, Gasparâone A, et al: Effects of naloxone on myocardial ischemic preconditioning in humans. J Am Col1 Cardiol l999;33: 1863-1869.

183. Parikh V, Singh M. Possible role of cardiac mast cells in norepinephrine-induced myocardial preconditioning. Methods Find Exp Clin Pharmacol 1999;21:269-274.

184. Musters RJ, van der Meulen ET, Zuidwijk M, et ai: PKC-dependent preconditioning with norepinephrine protects sarcoplasmic reticulwi function in rat trabeculae following metabolic inhibition. J Mol Ceil Cardiol l999;3 1: 1083-1094.

185. Horimoto K, Saltman AE, Gaudette GR, Knikenkarnp IB: Nitric oxide generating beta adrenergic blocker nipradilol preserves pst-ischemic cardiac function. Ann Thorac Surg 1999;68:844-849.

186. Toller WG, Montgomery MW, Pagel PS, Hettrick DA, Warltier DC, Kersten JR: Isofluraneenhanced recovezy of canine stunned myocardium: role for protein kinase C? Anaesthesiology l999;g 1 :713-722.

187. Novalija E, Fujita S. Kampine JP, Stowe DF: Sevoflurane mimics ischemic preconditioning effects on coronary flow and nitric oxide release in isolated hearts. Anaesthesiology 1999;91:701-712.

188. Baines CP, Wang L, Cohen MV, Downey IM: Myocardial protection by insulin is dependent on phosphatidyl-inositol3-kinasen but not protein kinase C or K-ATP channels in the isolated rabbit heart. Basic Res Cardiol 1999;94: 188- 198.

189. Zhao L, Elliot GT: Pharmacologie enhancement of tolerance to ischemic cardiac stress using monophosphoryi lipid A. Ann N Y Acad Sci 1999;874:222-235.

190. Ito H, Shimojo T, Fujisaki H, et al: Thermal preconditioning protects rat cardiac muscle cells fiom doxombicin induced apoptosis. Life Sci 1999;64:755-76 1.

191. Mahgoub JMA, Guo JH, Gao SP, et al: Hyperdynamic circulation of artenovenous fistula preconditions the heart and iimits infarct size. Ann Thorac Surg 1999;68:22-28.

192. Leman BB, Belardinelli L: Cardiac electrophysiology of adenosine: basic and clinical concepts. Circulation 199 l;83: 1499-1509.

193. Song Y, Thedfoni S, kmi;ir7 BB, Belardinelli L: Adenosine sensitive afterdepolarizations and tnggered activity in guinea pig ventricular myoçytes. Circ Res 1992;70:743-753.

194. Fox AC, Reed GE, Glassmand E, Kalünan AJ, Silk BB: Release of adenosine from human hearts during angina induced by rapid atrial pacing. J Clin Invest 1974;53: 14-47-1457.

195. Fox AC, Reed GE, Meilman H, Silk BB: Release of nucleosides from canine and human hearts as an index of pnor ischernia. Am J Cardiol 1979;43:52-58.

196. S ylven C, Jonzon B, Edlund A: Angina pectoris-like pain provoked by i.v. bolus of adenosine: relationship to coronary sinus blood fiow, heart rate and blood pressure in healthy volunteers. Eur Heart J 1989; 10:48-54-

197. Sylven C, Beermann B, Jonzon B, Brandt R: Angina pectoris-like pain provoked by intravenous adenosine in healthy volunteers. BMJ 1986;293:227-230.

198. Baggioni 1, Olafsson B, Robertson RM, Hollister AS, Robertson D: Cardiovascular and respiratory effécts of adenosine in conscious man: evidence for chemoreceptor activation. Circ Res 1987;61:779-786.

199. Verani MS, Mahmarian JJ, Hixson JB, Boyce TM, Staudacher U Diagnosis of coronary artery disease by controlled coronary vasodilitation with adenosine and thallium-201 scintigraphy in patients unable to exercise. Circulation 1990;82:80-87.

200. Smits P, knders JWM, Thien T: C a e i n e and theophyüine attenuate adenosine induced vasodilitation in humans. Clin Phannacol Ther l990;48:4 10-4 18.

201. Watt AH, Bayer A, Routledge PA, Swift CG: Adenosine induced respiratory and h a r t rate changes in young and elderly adults. Br J Clin Pharmacol l989;27: 165- 17 1.

202. Lx HT, Lafaro RJ, R e d GE: Pretreaiment of human myocardium with adenosine during open heart surgery. J Card Surg 1995; 10:665-676.

203. Cohen G, Feder-Elituv R. Iazetta J, Fremes SE: Phase 2 studies in adenosine cardioplegia. Circulation 1998;98:11225-11233,

204. Houltz E, Ricksten SE, Milocco I, Gustavsson Tt Caidahl K: Effects of adenosine infusion on systolic and diastoiic left ventncular function after coronary artery bypass surgery: Evaluation by cornputer assisted quantitative 2-D and doppler echocardiogmphy. Anaesth Analg 1995;80:47-53.

205. Owall A, Ehrenberg J, Brodin LA, Juhlin-Dannfelt A, Sollevi A: Effects of low dose adenosine on myocardial performance after coronary artery bypass surgery. Acta Anaesthesiol Scandin 1993;37: 14-0-148.

206. Anonymous. Effects of acadesine on the incidence of myocardial infarction and adverse cardiac outcornes after coronary artery bypass graft surgery. Multicentre Study of Perioperative Ischemia Research Group. Anaesthesiology l995;83:658-673.

207. Mentzer RM, Byiniuk V, Khuri S. et al: Adenosine myocardial protection: pxelimïnary results of a phase II clinical trial. Ann Surg 1999;229:643-649.

208. Hi11 JA. Utterback DB. Keim SG, Dugger D, Mayfield WR, Shryock JC: Validation of a new rnethod for sarnpling human plasma adenosine in vivo. Cor Art Dis l992;3:963-971.

209. Mauser M, Hoheister HM, Neinaber C, Schaper W: Influence of ribose. adenosine and "AICAR" on the rate of myocardiai adenosine trîphosphate synthesis during reperfusion after coronary artery occlusion in the dog. CUc Res 1985;56:220-230.

210. Harmsen E, Detombe PP, Dejong JW. Actiterberg PW: Enhanced ATP and GTP synthesis h m hypoxanthine or inosine after myocardial ischemia. Am J Physiol 1984;246:H37-H43.

21 1. Kloner RA, De Boer L W , Braunwald E: Recovery from prolonged abnomiaüties of canine myocardium saivaged from ischemic necrosis by coronary reperfusion. Roc Nat1 Acad Sci USA 198 l;78:7lSS-7156.

212. Boliing SF, Childs KF, Ning XH: Adenosine's effect on myocardial functional recovery: substrate or signal? J Surg Res 1994;57:591-595.

2 13. Armstrong S. Ganote CE: Adenosine receptor specificity in preconditioning of isolated rabbi t cardiomyocytes: evidence of A3 receptor involvement Cardiovasc Res 1994;28: 1049- 1056.

214. Liu GS, Thomton JD, Van Winkle DM, Stanley AWH. Olsson RA, Downey JM: Protection against infarction afforded by preconditioning is mediated by Al adenosine receptors in rabbit kart. Circulation 199 1;84:350-356.

215. Liu GS, Richards CS, Olsson RA, Muliane K, Walsh RS, Downey JM: Evidence that the adenosine A3 receptor may mediate the protection afforded by preconditioning in the isolated rabbit heart. Cardiovasc Res 1994;îS: 1057- 106 1.

216. Ikonomidis JS, Tumiati LC, Weisel RD, Mickle DAG, Li RK: Preconditioning in human ventricular myocytes with bnef episodes of simulated ischemia. Cardiovasc Res 1994;28: 1285-129 1.

217. Tumiati LC, MicWe DAG, Weisel RD, Williams WG, Li RK: An in-vitro mode1 to siudy myocardial ischemic injury. J Tissue Cult Methods 1994; 16: 1-9.

2 18. Li RK, Shaikh N, Weisel RD, Williams WG, Mickle DAG: Oxyradical-induced antioxidant and lipid changes in cultured human cardi~rn~ocytes. Am J Physiol 1994;266:H2204-H2211.

219. Weisel RD, Mickle DAG, Finkle CD, Tumiati LC, Madonik MM. Ivanov J: Delayed my ocardial metabolic recovery after blood cardioplegia Ann Thorac Surg l989;48:503-507.

220. Hull-Ryde EA, Lewis WR, Veronee CD, Lowe JE: Simple step gradient elution of the major high energy compounds and their metabolites in cardiac muscle using high performance liquid chromatography. J Chromatogr 1986;377: 165- 174.

221. Burton K: A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:3 15-323.

222. Ytrehus K, Liu Y, Downey JM: Preconditioning protects isc hemic rabbit heart by protein kinase C activation. Am J Physioll994;266:Hl145-H1152.

223. Armstrong S. Downey IM, Ganote CE: Reconditioning of isolated rabbit cardiomyocytes: induction by metabolic stress and blockade by the adenosine antagonist SPT and Calphostin C, a protein kinase C inhibitor. Cardiovasc Res 1994;28:72-77.

224. Armstrong S, Ganote CE: Preconditioning of isolated rabbit cardiomyocytes: effects of glycolytic blockade, phorbol esters and ischemia. Cardiovasc Res 1994;28: 1700-1706.

225. Liu Y, Ytrehus K, Downey JM: Evidence that translocation of protein kinase C is a key event during ischemic preconditioning of rabbit myocardium. J Mol Ce11 Cardiol 1994;26:661-668.

226. Speechly-Dick ME, Mocanu MM, Yellon DM: Protein kinase C. Its role in preconditioning in the rat. Circ Res 1994;75:586-590.

227. Kohl C, Linck B, Schmitz W, Scholz H, Scholz J, Toth M: Effects of carbachol and (-)-N6-phenylisopcopyladenosine on myocardial inositol phosphate content and force of contraction. Br J Pharmacol 1990; 10 1 : 829-834.

228. Ikeda U, Arisaka H, Takayasu T, Takeda K, Natsume T, Hosoda S: Protein kinase C activation aggravates hypoxic myocardial injury by stimulating Na+/H+ exchange. J Mol Ce11 Cardiol 1988 ;20:493-5Oo.

229. Yuan S, Sunahara FA, Sen AK: Tumour promoting phorbol esters inhibit cardiac function and inhibit redistribution of protein kinase C in perîused beating rat heart. Circ Res 1987;61:372-378.

230. Heasley LE, Johnson GL: Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells. J Bi01 Chem 1989;264:8646-8652.

231. Heasley LE, Johnson GL: Detection of nerve growth factor and epidermal growth factor-regulated protein kinases in PCl2 cells with synthetic peptide substrates Mol Pharmacol l989;35:33 1-338.

232. Ikonomidis JS, Shirai T, WeiseI RD, Ivanov J: Endogenously released adenosine mediates human venaicular cardiomyocyte precondi tioning through protein kinase C. Am J Physiol 1996;

233. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurements with the folin-phenol reagent. J Bi01 Chem 1951;193:265-275.

234. Kloner RA, Yellon DM: Does ischemic precondi tioning occur in patients? J Am Col1 Cardiol lW4;24: 1 133-1 142.

235. Lawson CS: Does ischemic preconditioning occur in the human heart? Cardiovasc Res l994;28: 146 1-1466.

236. Yellon DM, ALkhulaifi AM, Pugsley WB: Reconditioning the human myocardiurn. Lancet 1993;342:276-277.

237. Van Wylen DGL: Effect of ischemic preconditioning on interstitial purine metabolite and lac tate accumulation during myocardial ischemia. Circulation 1994;89:2283-2289.

238. Walker DM, Walker JM, Pugsley WB, Pattison CW, Yellon DM: Preconditioning in isolated superfuseci human muscle. I Mol Ce11 Cardiol 1995;27:1349-1357.

239. Li RK, Weisel RD, Williams WG, Mickle DAG: Methods of culninng cardiomyocytes h m human pediatric ventricular myocardium. J Tiss Cult Meth 1992;14:93-100.

240. Li RK, Mickle DAG, Weisel RD, et al. Human pediatric and adult ventricular cardiomyocytes in culture; assessrnent of phenotypic changes with passaging. Cardiovasc Res 1996;32:362-373.

24 1. Arend U, Sonnenberg WK, Smith WL, Spielman WS : A 1 and A2 adenosine receptocs in rabbit cortical collecting tubule ceils: modulation of hormone stimulated c-AMP. J Clin Invest l987;79:7 10-7 14.

242, Musser B, Morgan ME, Leid M, Murray TF, Linden J, Vesta1 RE: Species cornparison of adenosine and beta adrenoreceptors in mammalian atrial and ventricular myocardium. Eur J Pharmacol l993;246: 105-1 1 1.

243. Lee HT, Thompson CI, Hemandez A, Lewy JL, Belloni FC: Caràiac desensitization to adenosine analogues after prolonged R-Ph infusion in vivo. Am J Physiol 1993;27:565-570.

244. Matsuyama T, Sato H, Kitabatake A, et al: Blunted cardiac response to exercise induced sympathetic stimulation in non-failing aortic regurgitation; insight into role of cardiac dilation in h yporesponse of failing hearts. Jap Circ J 1992;56: 1 17- 127.

245. Delehanty JM, Himura Y, Elam H, Hood WB, Liang CS: Beta adrenoreceptor downregulation in pacing-induced heart failwe associated with increased interstitial NE content. Am J Physiol 1994;266:H930-H935.

246. Takaro T, Peduzzi P, Deûe KM, et al: Sumival in subpups of patients with left main coronary disease: Veteran's Administration Cooperative Study of surgery for patients with coronary artecial occlusive disease. Circulation l982;66: 14-22.

247. Gross GJ, Auchampach JA: Blockade of ATP sensitive potassium channels prevents myocardial preconditioning in dogs. Cin: Res 1992;70:223-233.

248. Cohen G, Rao V, Weisel RD: J NY Acad Sci 1999;

249. Cohen G, Shirai T, Weisel RD. et al: Optimal myocardial preconditioning in a human mode1 of ischernia and repefision. Circulation 1998;98: 137-143.

250. Whalley DW, Wendt DJ. Grant AO: Basic concepts in cellular cardiac electrophysiology. Pace 1995; 18: 1556-1574,

251. Marber MS, Latchman DS, Walker JIM, Yeilon DM: Cardiac stress protein elevation 24 hours after bnef ischemia or heat s tms is associated with resistance to myocardial infarction. Circulation 1993;88:1264-1272.

252. Cmie RW. Kamiazyn M. Moc M, Mailer K: Heat shock mponse is associated with enhanced postischemic ventrïcular recovery. Circ Res 1988;63:543-549.

253. Knowlton A, Brecher P. Apstein CS: Rapid expression of heat shock protein in the rabbit after brief cardiac ischemia, J Clin Invest lWl;87: 139-147.

Table 1 . Charncteristics of P, and P, purinomceptors

11 Linked to prostaglandin synthesis 1 No 1 Yes

Recognize adenosine

Recognize ATP

Antagonized by met hylxant hines

Potentiated by inhibition of adenosine transport (ie. dipyrid)

Linked to adenosine cyclase

Yes

No

Yes

Yes

Yes

No

Yes

No

No

N o

Table 2. Anributes of A , and A, adcnosine receptors and of the P site

Action on adenylate cyclase 1 Inhibit 1 Stimulate 1 Inhi bit

Location in ceII I Surface I Surface I Interior

GTP dependence 1 Yes 1 Yes 1 No

Molecular mas of ligand- binding peptide, kDa

Alkylxanthine inhibition

Transduction protein 1 Gi 1 Gs 1 None

Toxin for NAD' ribosylation 1 PTX CTX 1 None

- 35-38

Yes

of G protein I I I

Gi, inhibitory G protein; Gs, stimulatory G protein; PTX, toxin of Bordetella pertzissis, CTX, toxin o f Vibrio cholerae.

45

Yes

22

No

Table 3. Cnrdinc effects of denosine

A, Receptor Effects Direct

Decrease SA node automaticity Decrease AV node conduction Decrease atnal contractility Decrease atnal action potentiai duration Suppress norepinephrine release

Indirect Attenuate chronotropic, dromotropic, and inotropic effects of catecholamines Suppress catecholamine-induced triggered ventricular afterpotentials

A, Receptor Effects Vasodilation Decrease blood pressure

?A, or A2 Receptor Effects Increase ventilation Cause chest paiddiscomfort

1

Ribose Moiety

- Purine Base

OH' P - O - p - O - P - O

I I I I I I

Figure 1. A: Schematic structure of Adenosine combining a purine base and a ribose moiety. B: Schematic structure of Adenosine Triphosphate combining adenosi n e and three phosphate groups.

Adenine Nucl A ~ X A ~ ' T > AMp a

SAH 1-9 Homocysteine + A D 0 '"" a AD0

eotides

XPYRIDAMOLE

Inosine

Calcium channel

I(+channel G'IP G i

Figure 2. Adeiiosine Metabolism. The cardiac adenosine system is compnsed of three components; (1) formation; (2) receptor complex effects; and (3) degradation. I - Adenosine (ADO) can be formed intracellularIy via the adenosine triphosphate (ATP) or S-adenosylhomocysteine (SAH) pathway, or extracellulady via breakdown of adenine nucleotides. 2 - The adenosine receptor (ADO- R) is coupled to ion channels via the guanine binding regdatory proteins (Gi). Theophylline (THEO) derivatives act as competitive antagonists for the adenosine receptors. 3 - AD0 can be transported into the cell and then degraded via deamination to inosine or phosphorylated to adenosine monophosphate (AMP). Dipyridamole can block the cellular uptake of ADO, thus prolonging its effect. ADP=adenosine diphosphate; cAMP=cyclic AMP; GTP=guanosine triphosphate.

exogenous adenine

ATP

a

ADP 9

C

1

de novo

=)AMP <=> IMP f

g n d . o \ *do :=> na.=> ,=> UA

Figure 3. Purine Metabolism. Ado=adenosine; Hx=hypoxanthine; Ino=inosine; UAwric acid. a=ATP conwming reactions; h x i d a t i v e phosphory lation; c=myokinase; d=S-nudeotidase; e=Ah@ deaminase; f=adenylosuccinate synthase and lyase; g=adenosine kinase; h=adenosine deaminase; +purine nucleoside phosphorylase; j=xanthine dehydrogenase; kguanine phosphoribosyl transferase; kadenine phosphonbosyl transferase.

Figure 4. Summary ofthe adenosine-protein kinase C mechanism ofischemic preconditioning. Brief ischernia results in thedegradation o f adenosine triphosphate (ATP) through adenosine diphosphate (ADP) and adenosine monophosphate (AMP) to adenosine. Adenosine freely di fises across the cell membrane to interact wi th surface adenosi ne recepton.(A 1 ). Adenosine receptors are believed to be coupled to inhibitory guanosine triphosphate binding proteins (Gi proteins) consisting of a, p. and y subunits. The activated a subunit sti mu1 ates membrane bound phosphol i pase C (PLC) to conven membrane phosphatidylinositol biphosphate (PiP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 induces intemal mobilization of calcium stores from sites such as the sarcoplasmic reticulum (SR). As the intracellular calcium concentration rises. inactive cytosolic protein kinase C (PKCinact) translocates to ce11 membranes and is activated by DAG (PKCact). Activated PKC may now mediate the cardioprotective response through modulation of final eRector/s such as ion channels. intermediary metabolic pathways, and gene expression.

Figure 5. Simplified siimmary of the adenosine-protein kinase C mechanism of ischemic preconditioning. Brief iscliemia results in the degradation of adenosine triphosphate (ATP) to form adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine. Adenosine diffises across the ce11 membrane to interact with extracellular adenosine receptors (AI ). Through a series of intermediary steps including G protein activation and hydrolysis of membrane pliospliolipids, protein kinase C (PKC) is activated. Activated PKC goes on to pliospliorylate intra- or extracellular final effectors tlierebye conferring protection.

Figure 6. Representative photomicrographs of primary cultures of human pediatric (A) and adult (B) ventricular cardiomyocytes. (tOOx magnification; reprinted from Li, et al?)

W p #1 'Lkp #2 Humidifier 37'C 4' C 37'C

Sdf-rciling port 1 Cbimber (37*C)

Temperature probe

Figure 7. Schematic diagram of sirnulated "ischemia" and "reperfùsion" model. Culture dishes of hurnan ventricular cardiornyocytes are placed in an air-tight plexiglass chamber. To ensure anoxic conditions. 100% nitrogen (Na gas bubbled through two oxygen traps is utilized to continuously flush the sealed chamber thereby displacing any ambient oxygen. Four culture dishes are placed in the chamber which is equipped with a central sampling dûh to enable venfication of anoxic conditions and to allow temperature monitoring with each ischemialrepemision experiment. (Reprinted from Tumiati, et

Figure 8. Liçht micrograph of cardiomyocytes stained with Trypan Blue. Left PanneL- cardiomyocytes stabilized in phosphate-buffered saline for 30 minutes show little evidence of ceIluIar injury. Middle Pc~mel: cardiomyocytes preconditioned with 20 minutes of "ischemia" followed by 20 minutes of "repertùsion" reveal relatively few injured cells (denoted by arrows) following prolonged "ischemia" and "repertùsion". Righr Patmel: non-preconditioned cardiornyocytes reveal large numbers of injured cells (denoted by arrows) followuig prolonged "ischemia" and "reper£Ùsion". (200x magnification; scale bar=20ltm; Reprinted from Ikonomidisg4)

Normoxic PBS Simulated Ischemia pot = O mmHg I-] Simulated Ischemia: p02 = 16 m m H Normoxic PBS + SPTADA

NIC [ 1

SUPO 1-- Indicntes application of precontiitiomd

SUP Precond [ 1 1 1 w

Adenosine Antagonist mllllllllllllllll(l(llllllllllllllllllll-• 1

Figure 9: Endogenous preconditioning s~udies: In study 1) cells underwent either anoxic (PCO) or hypoxic (PC 16) preconditioning for a period of 20 minutes pnor to prolonged ischemia and reperfùsion. In study 2) non-preconditioned cells were preconditioned for a period of 20 min. using the supematant of cells which underwent either anoxic (SUPO) or hypoxic (SUP16) preconditioning. In study 4) supernatant from anoxically preconditioned cells was treated with either SPT or adenosine deaminasé (ADA) and applied to non-preconditioned cells which were pre-treated with SPT or adenosine deaminase. Al1 groups were compared to non-ischemic controls (NIC) which underwent 190 min. of stabilization, and ischemic controls (IC) which undenvent 70 min. of stabilization followed by prolonged "ischemia" (90 min.) and "repemision" (30 min.).

% TRYPAIV BLUE UPTAKE -L ru W P O O O O

3r p 4 . 0 5 vs. Stabiiization vs. IC @ 50 min, vs. PCO and IC (ZJ Rep

'ri

7 *

rtr p4.05 vs. PCO @ 50 min, 1, R vs. IC @ 1, R + pd.05 vs. PCO @ 1

IC@I,R

d l d o

C ' - O 0

P Isch emia Reper fusior.

Figure 11 : Upperpanel: Extracellular lactate levels were sigùficantly elevated at 50 minutes in the anoxic preconditioning group (PCO), however not significantly. Extracellular lactate concentrations following both "ischemia" and "reperfusion" did not differ between groups. Lower panel: Intracellular ATP levels decreased siguficantly in the anoxic preconditioning g o u p (PCO) in cornparison to ischemic controls (IC; pc0.05) ('ATP debt'). However intracellular ATP levels following both "ischemia" and "reperfusion" did not differ between groups.

NIC SUPO SUPI6 IC

Figure 12 : Lower Panel: Preconditioning with the supematant of anoxically preconditioned cells (SUPO) reduced cellular injury to a greater extent than did preconditioning with the supematant of hypoxically preconditioned cells (SUP 16)(p<0.05). Both forms of supematant preconditioning sibwificantly reduced cellular injury compared to ischernic controls (IC) (pc0.05) (NIC: Non-ischemicControls). W p e r Panel: HPLC anaiysis revealed a greater concentration of endogenous adenosine in the supematant of anoxically preconditioned cells (SUPO)(p=O.O 18, SUPO vs. SUP 16). The supematant of cells which underwent stabilization only revealed the lowest endogenous adenosine concentrations.

Sc 11~0.05 va SPT, ADA, IC m + p<0.05 vs. SUPO, SPT, ADA, IC I I

SUPO SPT ADA

Figure 13: The protective effects of anoxically preconditioned supernatant (SUPO) were abolished when the non-preconditioned cells and the supernatant were first incubated with either SPT or adenosine deaminase (ADA) (NIC: d on-ischemic controls; IC: Ischemic controk) (*p<0.05 vs. SPT, ADA, and IC; +pcO.05 vs. SUPO, SPT, ADA, IC).

1 O Normorie PBS . Simulateci Irchenia p02= O minHg 1 NLC [ 1 1 1

Figure 14: Exogenous preconditioning studies: In sîudy 5 ) exogenous adenosine was applied to cells either prior to (Pretreat), during (Ischemic treat), or following (Reperfusion treat) prolonged "ischemia" and "reperfusion", or during alf three phases (Continuous treat). Cornparisons were made with cells which undenvent stabilization in normoxic PBS for a total of 190 min. (Non-ischemic controls; NIC) and with cells which underwent stabilization for 70 min. followed by prolonged "ischemia" and "reperfusion" (Ischemic controls; IC). In study 6) celis treated with adenosine either prior to or during ischemia were simultaneously treated with SPT (Pretreat + SPT and Ischemic Treat + SPT, respectively). Cornparison was made with cells which undenvent stabilization in SPT and adenosine only (NIC + SPT). (A: Adenosine)

O

Figure 15: L

3c p4.05 vs, REP, IC + p4.05 vs. ISCH # p4.05 vs. +SPT

*

+p4.05 vs. PRE, ISCH, REP, CONTIN, IC .k * 114.05 vs. ISCH, REP, IC

# ~ 4 . 0 5 vs. REP, IC & p4.05 VS. -SPT Jt

#

&

PRE ISCH REP COIVTIN IC

7per Panel: Exogenous adenosine was most protective when administered at a 4 dose of 50 uinol pior to ischeinia (PRE). Application of adeiosine dunng ischemia (ISCH) was protective to a significantly lesser degree. The two protective effects were not found to be additive when adenosine was administered continuously (CONTIN). Adenosine administered dunng reperfiision (REP) was not protective. Al1 groups were compared to both ischemic controls (IC) and non-ischemic controls (NIC). Al1 protective effects were abolished when SPT was applied to adenosine treated cells, regardless of timing. Adenosine and SPT had no effect on non-ischemic controls (NIC). Lower Panel: Both PRE and CONTiN groups revealed a preservation of ATP following "ischeinia" and "reperfusion" in cornparison to ischemic controls (IC). The ISCH group revealed preservation of ATP to a lesser degree. Simultaneous administration of SPT abolished the ATP-preservative effects of adenosine. Adenosine applied during reperfusion did not afford ATP-preservative properties.

+ p9.05 v r coriitrponding COhT # @.OS v r eorrcsponding POST-ADENOSINE

NIC PRE ISCH R E ' COIVTIN XC

Figure 16: Extracellular lactate concentrations following "ischemia" and "reperfusion" (FINAL) were elevated in cells which received adenosine either continuously (CONTIN) or during reperfusion (REP)(*p<O.OS). In evaluating the direct effects of adenosine (POST-ADENOSINE), lactate levels were elevated immediately following adenosine administration in al1 groups compared to untreated controls (CONTROL) (+p<0.05 vs. corresponding CONTROL). SPT blocked the lactate elevating effects of adenosine (ADENOSINE+SPT) (pc0 .O5 vs. corresponding POST-ADENOSINE). (NIC: Non-ischemic controls; PRE: Pretreatment; [SC: Ischemic treatrnent; IC: Ischernic controls)

Normoxic PBS . Simulated Ischemia PO,= O mmHg

-C

Figure 17: Protein kinase C studies: Non-preconditioned cells were exposed to PMA for 20 minutes followed by 20 minutes of pre-ischemic reperfusion prior to prolonged "ischemia" and "reperfusion". Certain cells which undenvent ischemic preconditioning (PCO) or were treated with adenosine (A) or PMA pior to prolonged "ischemia" and "reperfusion" were also exposed to Calphostin-C (Cal-C) dunng 30 minutes of stabilization, dunng preconditioning with ischemia, adenosine (PRE), or PMA, and during pre-ischemic reperfusion. Non-ischemic controls (NIC) were exposed to Calphostin-C for 30 minutes, followed by Calphostin-C with adenosine or PMA for 20 minutes, followed by Calphostin-C for 20 minutes, followed by 120 minutes of stabilization.

CAGC CAGC

NIC

-c

CAL-C

, CAGC AfPMA+

CALC .

A/PMA+ CALC

* pdl.05 vs. NIC, IC + p4.05 vs- PMA

NIC PCO PRE PlMA IC

Figure 18: The protective efYects of preconditioning with either ischemia (PCO), adenosine (PRE), or PMA (PMA) were abolished with the addition o f Cal-C (+Cal-C) (*p<O-05 vs. NIC, IC). (Cal-C: Calphostin-C; A: Adenosine; NIC: Non-ischemic controls)