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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
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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.
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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
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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.
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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.
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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)