Dioision 0f Cardiova!X&r s~l?@~'y The Toronto Hospital

The Centre for Cardiov8sc\1k Research

A thesis submitted in conformity wîth the reqiiirements for the degee of Doctor of Pbilosophy.

Graduate Department of the hsühte of Medicai Scieneg University of Toronto

Copyright by Vivek Rao, MD (199%)

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UnivaSay of Tomnt0

ABSTRACT

BACKGROUND: ~ o v a S c u l a r disease remains an imporiant cause of morbidity aiad

mortaiity in North America. Surgical tnatment of cardiovasculaf disorciers has emeqed

over the past 40 yavs and is now a pnwm, enrctve therapy. Cardiop1egic arrest of the

heart enables surgeons to operate on a motionless field, but is associated with a degree of

perîopaative injury. Sensitive measures of myocardial mefabolism and hction meal a

deiayed recovery following surgery which may lead to postoperative morbidity and rnozfality.

These Senes of investigations were designeci to evaïuate the recovery of aembic

myocardial metabohm following cardioplegic arrest. W e believe that improuing the

transition from aMerobic to aerobic membolism may resuit in enhancd functional recovery

and impved patient outcornes following cardiac surgery. W e hypothesized that insulin

would stimuîaîe myocardial pynivate dehydrogenase activity and lead to improved metabolic

recovexy foiiowing s i m W ischemia and repemision.

MEEIODS: W e ernployed isoiated, cultured human v e n û i ~ cardiomyocytes to examine

the effects of ischemia and repenusion on myocardial metabolism. W e measured the degree

of cellular injury, intraceiluiar iactaîe accumulation, extraceliular lactate and pyruvate

release, intracellular high energy phosphate levek and the activity of rnitochondrial ppvate

dehydrogenase (PDH). We investigated the effect of preischemic exposure to insulin on the

recoveq of myocardial metabolism following ischemia and repemision.

RKSïETS: Prekhemic exposun to iasulin (10 RTn) was found to confer pmtection

q a h t ischemia, Iasulin was found to stimuiate riiitocbiondrial PDH dwhg stabjlizatïm lad

pnserved the activity of PDH folîowing iscbemia and npmision. Insuiin aposure reduœd

extmdular iaçtite rd- and impfoved the m o n of high en= phoqhîes. The

mechanism of insulin' effd on myocarraal PDH was fwnd to be mediated by Protein

Kmase CU stimulation of the PDH phoaphatlse &unit.

CONCLUSIONS: Preischemic exposure ta iasulia improved human cardiomyocyte tolerzil~lce

to ischemia and repemison. These inv- 0 prwide the nrst duect evidenœ that

insuiin can stimulate human myocardial pynrvate dehydrogenase and lcad to improved

metabolic recovery foîiowing ischemia. Novel -es of mya?udial protection can be

developed b exploit the beneficial metabolic e f f e of pynniate dehydrogenase stimulation.

This work would not be posi%Ie without the assbtauce of my many fnends and

coiteagues within the DiVison of CardiOvaScular Surgexy at the University of T010nto.

1 wouîd Mce to thanlr my mentor aab supaviaor, Dr. Richard D. WeiseI, for his

tirelessguldance,supportande~xwnagemcnt* IomforeMgraoefultohimfariatroducing

metothepl;ictiœofacademic~urg~andhopetojustifyhiscommittmmttaa~eintbe

years to corne.

I wouid iike to thank my Program Diredor in the Division of CardiOvaSculaf Surgery,

Dr. Christopher M. Feindel, f a his support and vaiuabie advice throughout my clinid and

research training. 1 am indebted to the members of my thesis cornmittee for their valuable

input and suggestions: Dr. D d d A.G. Mickie (Chical Biochemistry) and Dr. Brian H.

Robinson (Medical Genetics).

1 greatly appreaate the love and cornfort of my fàmily and especiaiiy my wife Zena

whose nurnerous sacrifices throughout my braining have aliowed me to pursue my goals.

Lastiy, I wouid like to thank the Heart and Shroke Foundation of Canada for

providing me with Research Fellowships in suppoa of this work.

Table of Contents

List of Abbreviations

ChuparOiu: KNOWLEDGE Tû DATE

1.1 Myocardial Protection for - surgery

1.1.1 In-ve Myocardiai Physiology 1.1 -2 Persistent Anaerobic Mefabolism and the

Recovery of Left Ventricuiar Functicm 1.1 -3 Aerobic versus Anaeaobic Glucose Melabolkm 1.1 -4 Stimitlating Glycolysis During Ischemia 1. i .5 ûptïmai Delivery of Cardioplegia 1.1.6 Stllnulating Myocairlial MetaboliSm with

Cardioplegic Additives

1.2 The Pyruvate Dehydrogenase (PDH) Cornplex

1.2.1 Structure and Function of the PDH Complex 1.2.2 Effect of khemia on PDH Activity 1.2.3 Irwlin and the Pynrvate Dehydrogenase Complex 1.2.4 nie Role of a Second Messenger

1 -3.1 Signal Transduction Pathways 1.3.2 The Discovery of Protein Kinase C 1.3.3 Isoform Specinc Pmperties of Protein Kiaase C 1 -3.4 Pharmaco10gic Moâiilation of Protein Kirme C

1.3.5 Protein giiiase C and Ischemic Reamditioning ... 35

chapter Ibo: INSULIN STXnIULATES MYOCARDIAL PYRUVATE IDEEYDROGENASE AND PROTECrS ISOLATED HUlMAN VENTRI- CARDIOMYOCYTES FROM SlMWATED ISCEKEMIA . .A6

2.1 Introduction . ..47

2.2 Methods . . -48

2.2.1 Human Ventriculaf Cardiomyocyte Culture . . -48 2.2.2 Simulated Ischemia and Repafusion . ..49 2.2.3 Expenmental ProtocoIs .. -50 2.2.4 Assesment of Cellular Injury . ..50 2.2.5 Biochemical Measuremerits ... 51 2.2.6 Statisticai Andysis . ..53

2.3.1 Assessrnerit of Cellular Injury 2.3.2 Insulin on PDH Activity 2.3.3 Insulin Effect on Intermediate Metabolites 2.3.4 In& Wect on Adenine Nucleotides

2.4 Conclusions . . .56

KINASE C-a DEPENDENT

Introduction

Methods

3.2.1 Insuiin Effect on Protein Kinase C 3.2.2 PDH Activity FoiIowing PKC Modulation 3.2.3 Statistical Andysis

Results

3.3.1 The Efféct of Insulùi on Protein Kinase C 3.3.2 PDH Activity Following PKC Modulation

Conclusions

chpar Fow: ADDITIONAL INVESTIGATIONS

4.2.1 Endotherial CeU Study 4.2.2 Porcine Model of Orthotopic Transplantation 4.2.3 Donor Operation 4.2.4 m e n t Opaation 4.2.5 Biochemical M-ts 4.2.6 Statisiical Analysis

4.3.1 Endothelial Cell Study 4.3.2 Porcine Study

QIoprer me: DISCUSSION

htroductim

Cell culture Model

5.2.1 Human Ventricuiar Cardiornyocytes 5.2.2 Sirnuiaieci Ischemia and Reprfbsion

Insulin Mediated Cardioprotection

5 -3.1 Reduction of Cellular Injury 5.3.2 Metabolic E f f ~ of Insuiin Exposure 5.3.3 Hemodynamic EffecfS of Insulin Exposure 5.3.4 Mechanism of the Insulin méct

Additional MectS of InsuIin Exposure

5.4.1 Insulin and Fatty Acid Metabolism 5.4.2 Insulin and the N a + Exchanger

Cell-specific Effeds of Insulin

Summary and ûrighd Contributions

Future Amis of Research

APPENDIX ONE APPENlDIX TWO

AdenOSiae AdaaoBinedipbphk Analysis of vPiPnce Adenosine triphosphate Adenosine tnphoaphatase Bovine Semm Aibumin -nazyarterybyppspgraftjng Calcium Ion Calcium Chlaride Calphosfin C Chdaythrine confaence Intervaf Creatine kinase MBfrsiictionofcreathekinase carbcm dioxide Creatine phosphate Counts per million Degms CeISius Change in absorbane 1.2-Diafylgiycerol Dichloroaawe DirnethyIsulphoxide DeoxyribonucIeic acid 1.2-DiOcfanoylglyœrOl "and others" Flavin adenine dinucleotide F I a . adenine dinucleotide (reduced) Fluoroscein isothi~cyanate Gram Guanosine triphosphate binding protein Hydrogen ion Hydrochioric acid N-[2-hydroxyethyI]piperaZine-N'-[2-etbanesulfonic] acid High @ofmance liquid chrornatography Sulphuric acid Hypoxanthine Immunoglobulin G hosine Inosïtol 1,4,5-triphosphate Insuiin receptor substare-1 Inteniaticmai Units Potassium ion Potassium chloride Potassium phosphate (monobasic) Potassium phosphate (dibasic)

kDa LAD m- M MARCgS Mo" lw% min mol mOsm mRNA

P Pa PKC PiMA FTCA RACKS rpm RNA SAS SEM SOD SPT

modalton L& anterior descending conniary arOay Müli- ( 1 0 Moles pr Litre Myristolstpri, ahninerich, C-kinase abstraie MagiiesiumTon Magnesium Chloride Minutes Mole (6.023 x 1 p particies) Mïlïïosmoles Messenger RNA Nano- (lm Nitrogen Sodium Ion Sodium bicarbonate Sodium bisulphite Sodium carbonate Sodium chloride Sodium hydroxide Disodium phosphate Sodium phosphate Dihydronicotinamide adenine dinucleotide (oxicii.lpn) Dihydrrmiwtinamide adenine dinucleotide (reduced) 2'-Gmeth yladenosine ûdds Ratio OXygen Percent (parts of a hundred) Phosphate b u f f ' saline Perchloric acid @Ci03 R(-)N6-@hmy1-2R-iSopf0~y1)-adenosine Phosphatidyl4,5-bisphosphate pi- (109 Negative logarïthm of hydrogen ion concentration Protein kinase C Phorbol 12-myristate 13-acetate Percutaneuus û'ansiuMnal coronary angiuplasty Receptor for activafed C-kinase subtype Revolutions per minute RibonucMc acid StatisticaI Analysis Systems Standard error of the mean Superoxide dismutase 8-psulphophenyl theaphylline M i c m (106)

Figure 2: The relationship betwrea myOcaLdial lirgte release, oxygeri extraction and acid release during rrpahisioa ancl the developmeat of postoperative low output syndrome @AIS) in 614 paticats uadagoing isolated ammary aaay bypass surgery. Patients who dtveloped LûS (11-36) had sigaincantly higher ladate release imrnedhtdy d c c~ossciamp removal (XCL OFF') and at fÏve miautes of repahision (5'). However, net myocanfial lactate Aease at ten minutes of repenusion (10') was not ciiffixent between groups. There were no diffefences in myocardial oxygen extracton or acid release at any t h e point. (Adopredfr<m 2kommici;is et ol.9

... Pape 41

Figure 3: Myocardial giucose me!aboiism. ... Page 42

Figure 4: The mitochondrial eiectron nansport (nspiratary) cchain. NADH is oxidized by complex 1 and coenzymeQ, a lipid soIuble quinone. Comp1cx II consis& of succiaatc C reduc&se and cœnzyme-Q nductase and transfers reduchg equilavents h m FADH, to comp1ex III (cytochrome c reductase). Cornplex III then transfers electrcms to complex IV. cytochrome c oxidase (COX). COX thai reacts directly with rno1eda.r oxygen to fomi hydroxyl ions (OH-) which react with the protans pmduced by the complex 1 reaction to f m water (',O). For each NADEI molede, three molecules of ADP are phosphorylatprl to form ATP, whüe two m01ecules of ATP are produoed for each molecuie of FADHZ.

... Page 43

Figure 5: The pyruvate dthydmgenase cumplex. The El mbunit acts to decarbaxyiate pyruvate to form CO, and thiamine pyrophosphate (TPP-EI).?~~ E2 subunit transfers the acetyl- group from TPP to a lipoy1 =factor and then subsequently to cofactor A, fomiing acetyl-CoA. The E3 subunit is a fiavin q u i M g enyzme which oniciizes the Lipoyl cofactor h m E2 by transfming elecirons to NAD+. The cornplex is regulated by two additionai enzymes: PDH kinase and PDH phosphaîase.

. . .Page 44

Figure 6: The insulin naptor compln. nie iaPulin rccepor cornplex is a fetrameric traasmembrane glycopotein corisistuig of two 120 kDa O and two 90 kDa B subdts. The two ar-sububits are linLtd by diSulphi& bonds, are entinly extradulat and amtain tbe insuün bmding sites. Each Bsubunit is ïinkd to an a-subunit by a disulphide bond, crosses the p h n a membrane and contains a turr,sine kinase domah in its intraceUuhr poition. The asockfion of insului protein with i reqtm lotimulates the tyrosine kinase activity of the Bsubimit and d t s in autopbobphorylation of the second BsubUIut, Autophosphorylaticm of the &subunit s t i m m the kinase advity of the feceptortowardsothasubstratepmteins, includingiiisulinreceptorsubsaatel w-1). ... Page 45

Figure 7: Representative photomiaograpbs of primary cultures of human pediatnc (A) and adult (B) venhricuîar cardiomyocytes. (2OX magnification, reprinted ficm Li et a P )

. . .Page 58

Figure 8: Schematic diagram of the quiprnerit required to simulate "ischemia" and reperfusion. 100% nitmgen gas (NJ is bubbled through two oxygen trapB prïur to flushing a seaïed plexigiass chamber. Four plates of cultured ceils can be placed in each chamber which is equipped with a cent& sampling dish to ensure the absence of oxygem and to monitor temperature. (Reprint& from Turniati et aP)

... Page 59

Figure 9: Representaîive photomimgraphs of hurnan ventriah cardiomyocytes foliowuig assesrnent for cellular viability using 0.4% tiypan blue dye. In control non-ischemic ceils (far left panel) no d u h r injury is visible. In contrast, the œlls in the far nght panel have undergone 90 minutes of ischemia and 30 minutes of repemision resulting in approximately 50% cellular injury.

. . .Page 60

Figure 10: Assessment of cellular injury foilowing ischemia and repenuSon. At an ambimt glucose conceritration of 5 mmoYL, thae was no sipnincant effkzt of insulin treatment. Howeveq exposure to 10 IU/L of insulia at an arnbient gïuoose concentration of 100 mmoYL reduced cellular injury with no fiirther protection demonstrated as the insulin concentration increased to 100 W.

. . .Page 61

FigureAl: Assessmentofdalari~uryfo~girdiemiaandreppfusi~~~. Eighercell injury was o b d as the gipcoae cmamtratiion incfeaSed @y tweway ANOVA, glirçcnre @cf F=6.48, p <0.0001). Tbe addition of 10 IUL of insuiin reduced d u i a r injury (anclin @kt F=U.26, p < 0.0001). Duncan's muitiple range test spccifird di&naaa betweai irisulin ami oon-însdh tnated p u p s at glruxwc ooncentratiioas of 50 and 1 0 mmollL.

...Page 62

Figure 12: Pymmte dehydrogemse (PDH) activity folîouhng thirty minutes of scposure to glucose and insulia (STABïUUTION); irimty minutes of ISCaEMIA and thirty minutes of REPERFUSION. h s u h expanne increasd PDH aictlvity at both levels of giucose @or to ischeda lad preventeû PDH Wvaticm diaing repemuicm. (Results wmpved to mm-isGhemic amtrolvaiues obtirineri at bgseline or h m d s nposed to equrvalent volumes of nornioxic PBS for equivdent the @mis.)

... Page 63

Figure 13: Pynivate dehydrogenase (PDH) activity following thirty minutes of srposure to mannitol (5 or 100 mmoVL) and insulin (O or 10 WL). Lnsulin resdted in simiiar PDH stllnuiation at both canœntrations of mannitol, indicating that the stimulatory e f fe of insuiin is indepaiderit of giucose.

... Page 64

Figure 14: Intraœlluiar lactate accumulation fouowing thirty minutes of stabibtion, ninety minutes of ischemia and thïrty minutes of repemiSion. Lactate extraction during stabiIïzaticm and repahision was highest in ceils exposed to 100 rnM and insulin. Howeva, insulin reduced iaciaîe accumiilation foUowing ischemia in cells exposai to a glucose concentration of 100 mM. Intracelluiar lactate increased in ail groups foiiowing ninety minutes of ischemia. (Results compared to non-ischemic wntrol va- obtained at baseline or h m c& erposed to equivalent volumes of normoxic PBS for quivalent tirne peaiods.) . . .Page 65

Figure 15: ExtraœUuiar ladate reiease into the supaaatuit wer each plate. Lactate rel*ise increased significantly with ischemia and remained elevakd durhg repafusion, suggestiag peEweflt aaaaobic metabolism. Insulin treabneat r e d d lactate dease at boîh glucose concentrations. (Results compered to non-ischemic control values obtained at beseline or h m cells exposed to eqiiivaent vohnes of normoxic PBS for eqiiivamt tirne periods.)

... Page 66

Figure 16: Iatracelfular adenosine tnphospbate (ATP) levels following -011, after niriety minutes of isdiemia and afkr thirty minutes of qerfkicm. ATP fell sïgnincantly in aU groups, but wpo ktta pserved in cells expoBed to 100

glwse and 10 Nn insulia. R e d t s apressed as a pacentage of colltrol values obtained at baseline or f b m d s exposai to equivaient volumes of normoxic PBS for equivalent the periods.

... Page 67

Figure 1 7 Intradluiar totai adenhenucleotide (TAN) Ieveis foilowing stabïhtion, aRg ninety minutes of ischemia and a f k thitty minutes of repafusion. TAN f a sigdcantly in ali groups, but was better pmserved in œUs exposed to high glucose and insulin. Mts expressed as a percentage of control values obtained at baseiïne or h m ceb exposed to equivalent volumes of nmoxic PBS for quivalent tirne periods.

... Page 68

Figure 18: In-situ fluorescent immunohistochemistry demonstrating the distn%ution of protein Ianase C using a 1:40 dilution of rabbit anti-human anti-pmtein kinase C IgG antibody. Panel A demonstrates gemdhed cytopbnic staining in œlis exposed to 5 mM gimse alone. Ceils exposed to 10 IW/L of insulin with 100 a glucose (panel B) dispiay a distribution of PKC staining to the sarcoIemnal membranes.

... Page 113

Figure 19: In-situ fluorescent imrnunohista:hemisûy demonstrating the distn'bution of protein kinase C using a 1 :40 dilution of rabbit anti-human anti-protein kinase C,, IgG antibody. Panel A demonstrates generaüzed cytaplasmic staullng in ceiis exposeü to 5 a glucose done. Cells exposed to the phorboI ester PMA (1 PM; panel B) or adaosine (50 FM, panel C) display a ndistribution of PKC to the perinuclear and sarcolemnd membrane. Ischemic preconditioning @anel D) d t s in a similar redistribution of PKC stainiag. (Reprinted h m Ikonomidis et aiP)

... Pape 114

Figure 20: Slot blot analysis d e m e g insuiin's ened on protein kinase C WC!) tmslocafion. Each lam is bloüed with U) pg of protein. The left paad shows no e&d of iasulin, PMA or adePosine on PKC* distributiou. The r&ht panel demonshates that insuiin, PMA and adenosiue cause a translocaticm of PKC-cu to the membrane fracfim. The adenOSU1e mediateci PgC-cr taasîocaîion was iihiaited by the suicinosine nceptor antagonist SPT.

...Pape 115

Figure 21: Measuriernent of total piobin kinase C (PKC) activity usllig an in vin0 phosphorylation assay. nie phosphorylation of a PKC sp&c peptide (epidermal growth fador mcepor, R K R . ) is mm*tsured ~01OrimetRcally as spectrophotornetric absorbanœ at 570 nm and comcted for protM cuntent. The p h h l estez PMA resuited in a sigmficant stimuiation of PKC activity compared to 5 glucose alone. A signincant interactive effect between insulin and giucose and total PKC activity was fomd (gluCose*illSUlin, F=S.l4, p-0.035 by nnANOVA). Insulin shulated PKC activity with a greata e f b t observed at an ambient giucose concentration of 100 mM.

...Page 116

Figure 22: The activity of mitochondrial ppvate dehydrogenase (PDH) activity following protein kinase C (PKC) madukition. The phorbol ester PMA stimulated PDH activity to a si* extent as high glucose and insulin. CeU exposed to high glucose and insului in the pfe~ence of the PKC antagonists calphosth C (CALC) and chelyrauine (CHEL) demonstrated PDH activities simiiar to control values (5 a glucose).

...Pape 117

Figure 23: Twenty-four hour survival foilowing ninety minutes of ischemia and repemision. Endotheliai d s (EC) displayed incnased sensitivity to ischemia compareci to cardiomyocytes (CM). Insulin treatment conferred protection to cardiomyocytes, but not to endothelial tek.

... Pape 166

Figure 24: Endothelial celi pynivate dehydrogenase (PDH) activity and extracellular lactate release foiiowing exposure to insulin. Insuiin stimuiated PDH activity and reduced extracellular lactate release.

... Page 167

Figure 25: Effect of continuous low flow donor blood pgfusion during three hours of hypothermie stmage. Measurements were obtained prbr to organ procurement (PRE); afta the initiai cafdjopllegic infusion ( P B ) ; after the completion of the lefi atrial (LA), ngbt aîrial (RA) and pulmonary arterial (PA) anastomoses; a m removai of the aortic crossclamp QCL) and every 15 minutes during reperfusion. Hearts in the BLûûD pemised group displayed persistent anaerobic metabolism with greater iactate and acid release during cardiuplegk arrest. There were no differences during the repemision perid.

... Page 162 XV

Figure 26: Effect of continuous lm flow donor blood pafusion during three hom of hypothermie storage. Perfiisicm of daior bLood (BLOOD) led to an impnmd recovery of left ventricular devdopeû prrssure compared to ncm-pesrwtd mm1 heam (CONT).

...Page 163

Figrire 27: Effect of conlinuous low flow dona blood pemision enhanced with insulin. Insulin treatment (lNS: 10 MIL) d t e d Bi an e a r k rec~very of aembic metabolism and improved the remvery of lefi vmtricular deweloped pmsure compared to blood alone (BLOOD) or non-pemised control hearts (CONT).

. . .Pape 164

Figure 28: U p p r PuneZ: Myocardial ladate flux during and afkr cardioplegic arrest. Patients who d v e d iasuün erihanced CafdiopIegia displayed lactate exîmction immediately a f k r crosscm removal compared to persistent lactaie release in the placebo grwp. Lowiier P d Left vmtricular fuaction was betm preserved in the insulin cardiople& group afta two hours of Rperfusim.

...Pape 165

CârdiovaScuIaf disease is the most prevaient chronic disease in North Amexka and the

leaduig cause of morbidity and rnOrfality.l3 Athemclen,tic coronary artay disease accounts

forwer6096 o f a l l d e a î h s d u e t o c a r d i a c ~ . '

Medical treatment remains the prefened theiapy for the initial management of angha

pectmkY UnfOTa1Rately, many patients continue to have symptoms despite maximai medical

therapy and are forced to severeiy restrict their aCtiYities of d d y living.

Surgeons once held the N e f that the heart would never be able to withstand a

surgicai insuit. The famous surgeon, Thdore BiIfroth, was once quoted " Those who

attempt to operate on the heart are doomed to fail and to lose the esteem of their

colieagues" .' Nevertheles, surgical repair of congeniîai and acquired heart disease contintml

to interest swgeons for many years. Ludwign Rehn is cndited with perfkmhg the first

cardiac operation in 1897 by repairing a stab wound to the heart with direct suture c ~ o ~ u f e . ~

Improving the blood supply to the heart proved to k fàr more dificuit as ail

techniques employed usually resulted in vascular fibrosis and loss of patency. Claude Beck,

at the Cleveland Qinic, developed methods to indirectly revascularize the heart by suturing

adjacent structures such as pericardium, pericardiai fat and omentum in an aüempt to develop

mUaW blood flow.' Arthur Vieberg, a Canadian surgeon at The Royal Victoria Hospital

in Montteal, first describeci in 1946 a kchnique of implanthg the left internai rnammary

(thora&) artexy (LITA) into a myocardial tunnel hopig to establish coiiateral circulaîion with

the left anterior descendkg coronary artery (LAD).8 The success of this operation was

quexied by many but was subsequently validated in 1960 by Mason Sones at the Cleveland

ciinic who performed se1ecfive coronary arteriography and àemonstrated patent collateral

2

communications betwem the LJTA and the LAD in two patients who were operated on 5 and

6 yars pior to aiigi~graphy.~

The fifit dued cofoaary artaial aaastomosis was actiially desaibed in an animal

modd by Alexis Carrel in 1910.n Using a cryopresewed camtid artery graft, Caml

pesfmed an anastomosis between the descendhg t h d c aorta and the left coronary artery.

Caml commenteci that it took him 5 minutes to perform the anastomosis, but that intractable

ventricuiar fibrillation developed after only thne minutes following interruption of the blood

flow to the coronary artery. He M e r commented that the continuous beating motion of

the heart made dissection difficult and that such an operation would have to take less than

three minutes in order to be sucasfd.

in 1955, Melrose descnbed the fïrst elective amst of the heart using a potassium rich

"cardioplegicn sol~tion.~' This solution provided a motionles, bloodless field for surgical

intervention. Unf~rtunately , the h . potassium concentration (240 mmol) resuïted in satere

cardiac injury.12 Reintroduction of a crystailoid solution with a lower potassium

concentration in the late 1970's enabled surgeons to electively arrest the heart with much less

ischemic injury than with intermittent aor&ic crossclamping (occlusion). "

The benefits of hypothennia were fbsî espoused in 1950 by Dr. Wilfred G. Bigelow

at the University of Toronto.'' In a series of classical experiments, Bigelow and wlleagues

were able to show that moderate hypothennia (25-28C) mis able to protect the heart h m

ischernic injury. This technique, with or without intermittent aortic crossclamping was

widely used d h g the early years of cardiac surgery.

In 1967, Favalm described the use of autogenous saphenous vehs to bypass stenotic

lesions of the coronary arteriesOu Coronary artery bypass grafong (CABG) is now the most

3

commody ~ m e d surgical procedure in North Ameria2 Several, large randomhd

cliinicaI trials have demonstrated the supaiority of CABG over medical treatment for

p1011ging Me and reducing symptoms in subsets of patients with athemsclerotic ainnary

severe triple v d a>raiary artery disease,' unstable an- and left ventricular

dysfiinction.' Since its original description by Favaioro, s e v d advances have been made to

reduœ the morbidity and mortality associated with CABG.Ia Most importa;lltly, advances in

myocardial ptectionWl9 have dowed surgeons to opadte on an increagIlgly high risk

patient population without a signifiant increase in paioperative complications.

Unfortunatey, several subgroups continue to be at increased risk for perkpedve

morbidity and mortality. These include fernales,= patients with poor Preoperative left

ventncular func t i~n~~ and those who prrsent for s u r g q with either unstable angîna or a

reoent myacardial infarcti~n.~~- Improved methods of @operative rnyocardial protection

are required to reduce the risks of surgery for these preCanow high risk patient populations.

Modifications to the delivery and composition of cardioplegic formulatjcms may lead

to improved myocardial. protection. Despite the success of contemporary coroaary bypass

surgery, sensitive indices of myocardial metabolhm and fwiction rweal delayed recovery

following cardioplegic The investigations descrii in this thesis w a e designeci

to investigate the me&bolic sequeiae of cardioplegic amst and to stimulate the recovery of

normal aaobic metabolism foliowing a pexiod of ischemia and npemision. Stimulating the

recovery of aerobic metabolism rnay lead to an eariier recmery of left ventricular functicm

and lead to improved surgical outcornes following coronary artery bypass surgefy.

the meîabolic respoases to simitlatPA cardiaplegic amat (iihemia) and qxdkireperfusion. 'Ibe use

of an isolatCui d culture model removes the possliIe Confouding &ects of d e r d types

and organ systems. In addition, the extracellular environment can be carefiilly co~ltroiied to

an extensive metabolic evafuation of isotatpri human cardiomyocytes subjec&d ta simutated

low fiow cardioplegic arrest and repemLpion. However, the in via0 redts of these

investigations require confinnation in a whole organ or in vivo model.

6

1.1 MY0CARDIA.L PROTECTION M)B CORONARY BYPASS SURGERY

The first heart opaations were PafOrmed cm the beabing heart. The results of these

procedure~waenotdydependerrtonthetechnicalsucce~sofUieoperati~~~, butalsoonthe

ability of the surgeon to reduœ intnoperatve myocardial injury. The advent of

of surgery and repair both cornpiex amgenital and aquired kart discase.

Sinœ Melrose first described the use of a 'cardioplegica solution to electively amst

the heart," th= have beea many advances in both the composition and delivery of

cardioplegia. The ultimate goal of any peziopesative myoprotective stmtegy is to provide the

surgeon with a motiodess, bloodless field in which to @mm a technidLy pafect conniary

aoastomosis while at the same tirne, preventing any injury to the heart which rnay result in

metaboiic or fwicticmai abnonnalities in the postoperative @od. In order to meet the

myocardid nutrient tequirements during cardiac surgery, it is cnicial to understand the

physiology of the arrested h m .

Z~cu)per4tr*~e Myoc(Udial PIrySiology

The heart npresents less than 0.5% of body weight, yet it accounts for over 7% of

the body's resting oxygen consumption." Myocardial oxygen consumption (MVOa can be

readily caicuiated using the Fick equation if coronary blwd flow (CBF), arterial (C.03 and

coronary sinus (COQ) oxygen contents are knom (MVQ = CBF x CC&-C-OJ). Cardiac

muscle extracts much more oxygen in the normal state than other organs, and thus increased

myocardial oxygen consumption is primarily achieved by increases in coronary blood flow.

The left ventncle ansumes approximaîeIy 8 mL of 4 per 100 g of myocardium per minute

in a normal human subject at rest. Durkg potassium i n d d arrest, oxygen conswnption

of MVQ are: heart nite, stroke work (the ana within the pressure-volume loop, which

incorporates afterld) and inotropic state. The relationship between M V a and heart rate,

stroke work and inotrapic state is almost iinear?" MVO, is greater when a low stn,ke

volume is ejeded against high pnssures than when large m k e volumes are ejected agaïmt

low aortic pressures.

During cardiac surgery, myocardial oxygen consumption varies widely. The lowest

MVG ocnirs whm the heart is arrestsd. Maximum MVO, occurs shortly after weaning from

cardiopulm~nary bypass whm the heart is rrpaying the oxygm debt incumd during the aortic

cross clamp period. In a series of classical experiments, Buclrberg et al examined myocardial

oxygen consumption during d i f f m t amditions of myocardial activity; the empty beating

heart, the fibrillating hart and the arrested heart? Myocardial oxygen consumption was

greakst during normothermic (37°C) fibnuaton and least duMg hypothermie (22'C) arrest.

Hyperkaiemic arrest achieved a reduction in MVQ h m 5.6 f 1.95 mUlûûg/min to 1.1

+ 0.4 mL/lOOg/min. Hypothermia reduced MVO, to 2.9 f 0.9 mUlO@/min while the - . combination of potassium induced arrest and modesdte hypothermia reduced MVQ to 0.3 f

0.1 mVlûûg/min. (Figure 1)

Myocardial ischemia occurs when there is an imbalance of oxygen supply and

demand. This imbalance results in anaerobic myocardial metabolism. nie end-products of

anaerobic metabohm rapidty lead to acidosis, mitochondrial dysfunction and myocyte

The myocardium is remarkably adept at utilùing any amilable substr;ite for energy

production. -hydrates, fatty acids and amino acids can be utiüzed for the formafion of

8

aϔyl-CoA. In the notmal non-hhemic myocardium, fany acids are the predominant

substrate for enexgy prOaucti~n;~~-~ however, during and after ischemia myocardial glucose

metabolism is highiy upregulated.* Durhg ischania, anaerobic giycolysis predominatw and

results in the production of lactate and hydrogen ions. Unfortu~îeiy, despite apparently

adequate -cm, anaerobic myocardial meâabolism oui persist with the amtinued

producfion of iactatC and hydtogen ions." Teoh et al examincd perioperative rnyocudial fatty

acid metabolhm in 18 patients undergohg isoIated CABG? These investi- found that

"C-labelled pairnitate was extracted nOm the heart but was not oxiCfj7PR. In patients

reœiving pure crystaiïoid cardiople@ (n =7), there was no detedable fatty acid oxidation.

In the remaining 11 patients who received blood Cafdioplegia, htty acid oxidation was

minimal. Myocardial fatty acid accumulation without oxidation may be deleterious and

contribute to repemision injury. The inability of the hart to oxidhe exogenous fatty acîds

~eflects a delayed recovery of norrnal aerobic metabolism foiiowing cardiupfegic anest.

Persistent AMerobic MetaboliSm and 171e Recowry of Left Vemnrrr& Funcn'on

In a nxent study of 614 patients undergohg isoiated CABG, 523 patients (84%)

displayed net myocardial lactate release folIowing aortic aossclamp releaseY AAa five

minutes of repemision, JOO patients (64%) displayed pergstent anaerobic metabolkm and

continued to release lactate into the coronary sinus. Thirty six patients (5.8%) developed

postoperative low cardiac output syndrome (LDS)). The objective definition of low output

syndrome includes any patient who requires inotropic or in-c balloon pump support for

greata than thirty minutes to maintah adquate hemodynamics aftm ail b l d gas, electrolyte

and volume abnoRnalities are armded.* This definition requires the active intervention of

the attendipg surgeon or intensivist; therefore, the development of LOS represents deiayed

9

recovay of lef€ veatricular funciion and a faiure of periopaab:ve mya'ardial pmtection.

Figure 2 illustrates the relatiomhip betweai myocardial lactate release, oxygen

extracction and acid release and the developmmt of poBtopaative LOS. Myocarrlial kdate

reiwse at crossclamp ternoval was higher in patients who developed LOS (0.92I0.2 mmoi/L

vs 0.45&0.02, p<O.Ol). Similady, myocardial lirtitc release aRer five minutes of

repafusicm was higher in patients who develaped LOS (O.2Sf 0.07 mmoYL vs O.O6f 0.01

mmoyL, p<O.û!5). Myocardial oxygen extraction or acid release were not differept in

patients who developed postoperative LOS. Stepwise logistic ngressicm identifieci pasisteat

lactate release after five minutes of repemision to be the only predictor of postoperative low

output syndrome (odds ratio OR 5.85, 95% confidence interval CI 2.1-16.3).

In a diversifiai surgicai popiitation, poor preopemtive left ventricular function

(OR=5.7,95%CI 3.69.0), repeat operation (OR=4.4, 9596CI 3-3-59), urgent surgery for

unstabIe angina (OR=3.7, 95%CI 2.3-5.9), f e d e gender (OR=2.5, %%CI 2.0-3.2),

diabetes meIlitu (OR=1.59, 951CI 1.3-2.0), age>70 years (OR=1.5, 9546CI 1.1-1.8),

lefi main disease (OR=1.4,9596CI 1.1-1.8), preopefative myocardial &&on (OR=1.4,

95%CI 1.0-1.9) and triple vesseI corouary artery disease (OR=1.3, 9596CI 1.0-1.7) were

found to be the independerit predictors of postaperative low output syndrome.% None of

these clinical risk k t o r s predict the deve1opment of low output syndrome in the

homogeneous study populations often employed for clinical triais in myocardial protection.'

Although we found m y o d a l lactate release at five minutes of reperfusion to have poor

predictive capability (area under ROC =O.6328), it was the only signifiant ri& factor for the

development of postoperative low output syndrome. The poor predictive capability may be

due to the fgct that in some patients with adquate myocardial protection, postoperative LOS

10

may be a r&t of intaopaative technid problems. Ladate rdease during nprfhion may

be highiy piedictive of LOS in patients who had an uneventfiil intmqemtive course.

U n f i y , it is diffidt to distindistinguish between the effecrr of inrteqI1SltP- myocaFdial

protection and inhaoperative misadvmture as these two events may be highIy correiated.

Nevddess, the development of postopedve low output syndrome in a patient who had

an meventful in-e course is due in part to a Aelrryed fecovery of normaî aembic

myocardial metabolism. nierdore, f'acilitating the transition h m anaerobic to h i c

metabofim foilowing CaIrdiopIegic arrest should lead to an improved recovery of lefi

ventXi* function.

Aembic W. Rnaerobic Glucose MetaboliSm

Figure 3 iiiustxates the myocardial metabohm of gîucose. Aembic glu-

meotbolism involves the conversion of glucose to pyruvate which is then mverted into

-1-CoA by the pyruvate dehydrogenase (PDH) enzyme cornplex. Acetyl-CoA the0 mtap

mitochondria and is metabolised via the Krebs cycle into carbon dioxide and water. The net

result of giucose oxidation is the production of 36 moles of ATP for each mole of glucose.

Anaembic giucose metaboliSm involves the converSon of glucose to lactate. The initial

phosphorylation of glucose consumes 2 moles of ATP to form hctose-16-diphosphaîe

(FDP). The conversion of FDP to lactate and wakr involves the production of 4 moles of

ATP. nius, the net energy production of glycolysis is 2 moles of ATP for each mole of

glucose. Unfortunately , the metabolic end-products: dihydroniwtinaniide-aderiine

dinucleotide [NADHJ, lactate and hydnogen ions act to inhibit phosphofnictokinase and can

thereby inhibit furth- glycolysis. This inhibition cesults in continuhg energy consumption

M m the initial stages of glycolysis -out any energy production. Not only is anaembic

11

Iine&bolism a les efficient source of ATP production, but it is also associated with the

production of h y m e n ions. The resultant demaad intracellular pH has detrimental

collse~umces to membrane and mîtochondrial stability.- ImpaiRd fundon of the sodium-

potassium ATPase pump increases cell membrane permeabiiity to extraceiiuîar calcium

resuiting in an eldion of intracelluiar caicium. Impairment of the ATP-dependent calcium

pump resdts in deaeased calcium ion scqu*ltration and eveiitdy in organelle injury and

myofibriilar c~ntracbre.~*~'

The citrïc acïd (Kreb's) cycle utiüzes aœtyL-CoA to produce a nwnber of d u &

ad&e nucleutides including dihydronicotinamideadenlne dinucleotiide (NADH) and flavin

adenine dinucleotide (FADH3. These flavoproteias are then utilized by the eleztron transport

chah within the inner membrane (cristae) of the rnitochondrial wmplex for energy

production. Figure 4 summarizes the major complexes of the mitochondrial respiratory

chah. The niitochondrial respiratory chah consists of a series of coupled reactions involvhg

elecani transfers h m iron-contaïhg hemoproteins or cytochromes. In each step, Kon

fluchiates from the reduced ferrous (Fe?+) form to the oxidized f h c @@+) state.

NADH is oxicüzed by cornplex 1 and the electrons transferred to coenyme, a lipid

soluble quinone. Complex 2 utilizes FADH, produced fkom the conversion of succinate to

fumarate to reduœ cœnzyme-Q. C0e~zyme-Q thus transfas the electrons frorn complexes

1 and 2 to complex 3 (cybchrome C reductase). Complex 3 thai transfers reducing

quilavents to cumplex 4 (cytochrome c oxidase). Cytochrome c oxidase (COX) reaas

directiy with molecular oxygen to form hydmxyl ions (OH-) which react with the protons

produced by the complex 1 reaction to form water (m). For each NADH molede, three

molecules of ADP are phosphorylated to form ATP while two molecules of ATP are

l2

proâuced for each molecuie of FADK. Oadaton of one mole of glucose yields 10 moles

of NADH and 2 moles of FAD&. In ytnitiou, two moles of ATP are produced h m the

anvexsion of succinylCoA to Succinata T'us, each mole of gïucose provides the net en-

quivakat of 36 moles of ATP foUowing oxidatve phospharylaîion.

Glucose oxidation can be summarued by two redox reactions:

Tota oxidation of glucose to CO2 and &O yields 686 W m o l . Each mole of ATP

stores approxhakly 8 kcai of energy." Thus, anaerobic glycolysis recovers only 2% of the

potential energy stored in giucose compared to 4296 reooverecl by oxidative phosphorylation.

Therefm, rnaintaining normal aerobic rnetabolism duMg and a f k cardioplegic anest

maximues the efficiency of ATP production h m glycolysis. In arlniticm to the increased

efficiency of ATP production, oxidative phosphorylation may be " c o m p a r t m e n ~ "

intracellularly such that the eaergy stores are diahguished h m ATP produœd by anaerobic

glymly sis.

Weiss et al found that in i s o M rabbit hearts, ATP generated h m aerobic

rnetabolism was preferentially used for myocardial contractility while anaerobicaily pmduced

ATP was prirnarily utilized for intracellular repaire4 This difference is teIeoIogically sound

because the ischemic cell shouid ConSave energy for survival fwictions only. Weksler et al

also demonsûateâ a pivotal role of anaerobic metabolisni in the presemtion of membrane-

bound calcium c h a ~ e l s . ~ These resuits also expiain the slow recovery in hction seen after

cardioplegic arrest in acutely ischemic hearts. The initial periods of repemision clearly Save

13

to replenish cellular energy stores which are used primady for intradular repair. Once the

Sodi-Pollares was the first to use an inûavmous infusion of glucose and insulin to

treat the e l m i o g r a p b i c abnormalitiies of an acute myocardial inf.arCtioneu Seveaal

hvestigators have since attenipted to improve myocardial tolerance to ischemia by either

enhancing preoperative ai- storeda- or by stimiilating giywlysis during ischernia.-n

The d t s of these investigations appear to be amtradictory. The two kgest evaluatiioos

took phce over 20 years a g ~ . ~ * Mittra et al reported on a praspective series of 170 patients

who received either a glucoscinsuiin-potassium suppiement or placebo shortly after bang

admitted with a diagnosis of acute myocardial infarcton.n nie design of this trial was

quential in that the first 85 patients d v e d standard therapy while the remaining 85

patients received the GIK treatment. This study demonsbrated a significant reduction in

rnortaiity in patients who receieved GIK (11.7% vs. 28.296, p<O.05). The authons

athibuted the reduction in mortality to a decread p a l e n c e of pst-infarction anhythmiaas.

A subsequent prospective randomized study involving 840 patients and fimded by the British

Medical Research Council mncluâed that exogenous glucose-insulin-potassium (GIQ

solutions did not have a beneficial effed on mortaiity or morbidity foIIowing acute

myocardial infarctoa6" They found that the mortality in the G E p u p was 23.9% v e ~ s

25.3 96 in the control group @=O. 6). The stuây ' s authors specirlatEul that improvements in

the management of pst-infarcpon complications superceded any potential benefit of G M

treatment.

14

Majid and wUeagues prospectiveïy evaîuated the use of GIK solutions for the

treatment of mgestive heart A Statistically signincant, but clinicaiiy margiaal

benefit was obsaved in 6 patients who receieved GM soluti~ll~ compand to conml patients.

Of interest, in patients who only received a high glucose supplement no hemodynamîc benefit

was observeci. The authors concludcd that msulin treatment improved the ability of the

myocaniium to metabolize giucose.

Although stimulation of glycdysis wouid intuitively be beaeficial, rnany investigatms

reported a detrimental e H l t on both myocardiai viability and f w r ~ t i o n . ' ~ ~ H m et al

demonstrated that cardioplegic arrest with solutions containug high concentrations of g h ~

led to iarger infarcts and depressed functionai reco~ery.~ Sirnilar.1~. ûrita reported that

storage of isoiated rat cardiomyocytes in presgvaton solutions wntaining high glucose

concentrations reduced cellular viability." Both of these investigatm athibuted their findùigs

to an accumulation of me&bolic end-products, namely lactate and h y m e n ions, which led

to intracellular acidosis and cell death. In Hearse's study, isohkd rat hearts were given a

single cardioplepic infusion prior to pf01onged, global ischemia. In this model, i n d g

the glucose concentration was ddeterious and the addition of insulin exacerbateci the injury.

In c o n w , Steinberg and Doherty demonstrated a benefiaal e f f a of glucose and insulin

containhg solution^.^^ Th& model employed multiple infusions of cardioplegia which

prevented the accumulation of toxic metabolites and led to irnproved functional recovery.

Other investigators who have atternpted to stimulate glycolyis by providing gluwsehsuün

solutions during reperfusion have reported a beneficial effe~t=-~ Thus, it appear~ that

in order to achieve a beneficial effect of metabolic stimuiation, one must prevent the

accumulation of end-products by either providing intemittent or continuous pemision of the

myocardium.

QJM Ddïwy qf Gmtiopkgia

Cardioplegic amst was originaüy achkved by idhion of a hypothermie ( 1 0 , high

potassium (27 mEq/L) so1ution into the amtic r0a1' Surgeoas maintained cardioplegic anest

with intemitîent infisions of a low potassium (8 mEqn) solution evay 15 to 20 minutes

during the aortic mssciamp mod. Howevex, sevexai investigators demonstrated that ttiis

fonn of myocardial protection resulted in a delay of both me&bolic and functicmai

-W. 'ma Fnmes et al demonsbrated that mixing blood from the bypass circuit with tbis

"crystalloid" solution in a 2: 1 ratio improved the recovery of both metabolism and function

following CardiopIegic arrest." In a prospective, randornized trial these investigaîors showed

that blood cardioplegia enhanced aerobic myocardial metabolism during aortic aoss-

clamping, increased myocardial oxygen coasumption, reduced anaaobic lactate production

and presemed high energy phosphate stores. Blood cardioplegia improved both systolic and

diastolic fwiction foilowing surgery. Since the publication of that trial, ail surgeons at the

University of Toronto switched to blood cardiopiegia. A subsequetlt clinid trial in patients

undergohg urgent revasc-tion for unstable an* demonstrated that blood cardioplegia

nduced both morbidity and mortality following ~urgery.~ Most institutions now employ a

b1ood:crystalloid ratio of 4: 1 or greatcr in theh mutine cardiap1egic formulations.* Blood

cardiople@a has distinct advantages over crystalloid solutions in tams of oxygen d e l i ~ e r y , ~

buffering capacip and the abiiity to prevent ïnwersib1e ischemic i n j u ~ y . ~

Cardiopllegia is traditionaliy deIivered antegrade into the aortic mot, a technique still

empIoyed by many cardiac surgeons. The initial arresting dose of between 500 to 1OOO mL

of cardiopIegh is given immedxatdy after the aortic cross-clamp is applied. Although

16

cardiop1legia can be given contiauously in an antegrade fkhion, it usually d t s in flooding

of the operative fkid and may compromise the technical q d t y of the distaI anastomosis.

Therefixe, most surgeons continue to give "maiatenancem cardiaplegia in an intermitfeat

Won. Since the aortic mot is clamped and isoIaîed h m systemic pemisicm, intermptions

in cardiopllegic delivery resuit in myocardial ischemia, Furthmore, in patients with sevexe

proximal disease, coronary sten0~e3 =y d u c e cardioplegic delivery and produce

Wtmgde delivery of doplegia inm the coronary sinus was origirially desccibed

by Gott in 1957? This technique allows for continuous delivery of cardioplegia with less

fiooding than observed with contuiuous antegrade deiivexy. However, most surgeons h d

corniary anastomosis. Sinœ hypotfiermic eardioplegic amst was thought to aliow fm safe

interniptions of cafdioplegia for up to twenty minutes, rnost surgeons employed intermittent

antegrade m o n and avoided the use of coronary sinus cafheters.

As Buckberg demonstratcd, hypottieda does not reduce myocardiai oxygen

requirements much beyond the reduction achieved with hyperkalemic a r r e ~ t . ~ In addition,

hypothermic cardiop1egia results in delayed recovery of both myocardiai metabohm and

ventricular f u n c t i ~ n . ~ ~ " - ~ See et al demonsbrated a signifiant impairment of mitochondrial

structure and function in a canine mode1 of prolonged hypothermic storage." Rosenkranz a

ai hypothesized that the metabolic dysfiinction may be partially due to the washout of Krebs'

cycle intermediates such as giutamate and aspaaate and showed that a normothermic (3m

induction of glutamate and aspartate d c h e d cardioplegia improved metaboiic fecovezy in

energy depleted hear&s.- Teoh et al then demonstrated that a terminal infusion of warm

17

blood cardioplegia (a 'hot shot") k m e d h l y @or to crosscbp release resulted in a

prolangation of elecfrornechanil rarwt, improvement in eerobic metaboliSm and inaeased

diastolic cornphn~e.~ The bewficial e&d of the hot shot was thought to be due to eariy

temperature &pendent rnitochondrial rrspiration and ATP generation. Patients who reœived

a terimaal warm blood infusion had signincantly higher ATP and glycogm stores cornparrd

to patients who did not receive a tmninai 'hot shotu. Since this technique d t e d in a

prolongation of electromechanical arrest, the ATP produced was presurnably used for repair

of intracefluiar ischemic injury and -011 of depl@ energy stores.

Clinid data indicaîed that normothermic induction cornbinexi with normothermic

terminal blood infusion facilitaml early recovery of m y e metabolic function.

Lichtenstein extrapolaîed these findings and proposed that normothermic pemision throughout

the crossciamp period might be benefi~hl.~' However, the modest inæase in myocardial

normothermic rnyocardial ischemia. Thus, Saiemo and coUeagues renewed the interest in

retrograde cardioplegia by delivering warm blood cardoplegia continuously hto the wronary

sinus.D1 A prospective. randomiÿed trial in 1732 patients revealed that normothermic blood

CardiopIegia giveii either aategra.de or retrograde Sgnificantly reduced the incidence of

postope&ve low output syndrome, but Med to show any ciifferences in perioperative

myocardial infkrctim @y ECG criteria) or m~rta l i ty .~

Although continuous retrograde delivery of wann blood cardiaplegia appead

d i v e , many investigators found that this technique provideci inadequate protection to the

right ventricle and posterior interventricular septum.- Since this region of rnyocafdiwn

remained normothermic, ischemic injury was potentiaily exaœrbated. Maatsura et al

Therefm? in an attempt to opîimjze myocardial pemision surgeons began to employ a

technique of combined antegrade and retmgde d e l i ~ e r y . ~ - ~ ~ In addition, Hayashida and

colleagues htmduced the concept of 'tepida carüiopiegia (29C) which was a natumi

compromise between h y p o t h d c (Io@) and normothemnic (37°C) extremes." These

investigators found that tepid blood cardioplegia reduceâ the anaaobic production of ladate

seem with normothermic m o n , but also prevented the delay in functional recovery

observed with hypothermie cardi~plegia.~ The same investigators then demonstrated that

providing tepid blood perfusion in a combined antegraddretrograde fàshion optUnized both

the temperature and delivery of cardioplesia,'a'w

GLUCOSE-INSULIN-POTASSIUM

The impvements in the delivery of cardioplegic solutions was paraiieled by

investigations into s e v d cardioplegic additives. The use of giucose and insulin enhanced

solutions for cardiac surgery mis unfortunately evaluated in an era of hypothennic,

crystalloid cardiopllegia. The mults of these investigations were controversiai and were

d e s c n i eariïer (page 13). The benefits of tepid blood cardioplegia erihanced with insulin

and delivered in a marner to optimize myocardial pemision have not been adequately

asessed." In addition, the management of systemic body temperatures duruig

cardiopulmonary bypass has also changed with the. Most surgeons now allow body

tempadhnes to "driftw to approximate1y 30-32°C. Previously, body tempemues were

19

actively cooled to below 2&C. Kuntschea et ai foMd that systemic hypothennia signifïcantly

impaireci the hepatic and pancreatic responses to a dextrose 1oad.l In particular, they f o d

hypothdc CaTdiopuimonary bypass and pcrsisied hto the eariy postopaative @ad.

Diabetic patients undesgohg nm- cardiopulmonary bypass displayed a simüar

enhanced cardioplegia would most W y demonstrate a benefit in the modern era of cardïac

surgery employing relativdy normothennic myocardial and systemic pemision.

Further evidence to support the narmothermic use of insuiin cornes h m severai

reports of its efficacy in the treatment of Iow cardiac output syndrome folIowing surgery.lOH1O

Of interest, Svedjeholm dernonstrated that high dose insulin potentiated the effects of

dopamine in the early postoperative period?' ûther investigators have also reported both a

VaSOdiIafOryfP and positive inotropicxn effect foIiowing insulin administraton. These reports

a h g with cLinical anecdotes support the need for a formal prospective re-evatuation of

glucose-insulin solutions in cardiac surgefy.

GLUTAMATE- ASPARTATE

Teoh et al used a glutamate and aspartate enriched cardoplegic solution in patients

undergohg isolated coronafy bypass surgery.lu Although low-ri& elective patients did not

benefit, glutamate-aspartate provided a slight benefit to those patients undergohg urgent

surgery for unstable angina. Similarly, Rosenkranz et ai showed a baiefit of glutamate

enriched blood dopleg ia in high risk patients with cardiogeaic shock? These authors

hypothesized that patients who present for surgery shortly after an ischemic event have a

deplebion of Krebs' cycle intermediates such as giutamate and aspartate. They suggested that

phosphates. However, the myocyte i s remrkably adept at utiüziag any available substrate.

Thus, in the piresence of oxygai, a norrnally hindionhg mitochondrial system should be able

to meet the mergy requirements of the cdl. The mle of Krebs' cycle intermediates may k

W y understood. A clarificafion of the mitochondrial rnetabolic defecf~ caused by ischemia

and npemision may p d t the development of a more site-speafic compound aimed at

restoring norrnal aerobic metahlism.

COENZYME Q

Ubiquinone, altanrativdy hown as coaizyme Q,, is a iiaturally arwring compownt

of the mitochondrial nspiratory chain.lM As discussed previously (page Il) , coenyzme Q

functions to transfer electrons h m complexes 1 and II to cornplex III. Several reports have

suggested that the endogemous leveis of coenyme QI,, are reduced in a wide varïety of

cardiac disorders, including patients with coronaf~ artery disease undergobg

fevaSc-tiOn. 11~11s Chen et al randomized 22 patients W ~ O undment cor~imry bypass

surgery to d v e Q, (1 1 patients) or placebo (n = 1 l ) . I u Aithough there were statistidy

signifiant improvements in mitochondrial ultrastnicture observed in biopsy specimens, ttiere

were no appreciable hemodynamic benefits. The Iole of coenzyme Q , as a cardioplegic

additive thus remains unclear.

DICHLOROACETATE

The stirniilntoiy efféct ~f dichloroaicetate (DCA) on pyruvate dehydrogenase acfivity

was originally described by Wtehoue and Randle in 1973.ll9 As d d b e d earlier, PDH

21

ngulates the amvemïon of pynmite to acetyl-CoA. Thus, stimuIation of this eazyme

cornplex may lead to incmued substrate lnnls for the -sr cycle. Substrate drivetn

stimuiatkm of oxidaîive phospharylaticm may lead to improved toIerance and recovery nOm

ischemia. DCA has beai used as a cardioplegic additive with variable effect. Wahr et al

demonsûated that DCA imp~oved giucose oxidation and led to better functional recovery

f o U d g ischemia.la In an aâcütional report, tbe same authors found that myocardial aiagy

charge was better preserved in DCA treated hearts and that DCA resuited in a reducticm in

both myocardial lactate release and NADH accumuiati~n.'~ niese authors concluded that

DCA resdted in improved f i r n c t i d recovery folïOmng global ischemia by stimulating

PDH. In contrast, Mazer et ai found that DCA did not improve rnyocardial sysblic function

dcspite a stimulation of aerobic carbohydrate metaboii~rn.'~ Thus, a cornpoumi which can

mimic DCA's stimulatory e f f î on PDH and also lead to impved functional nmvery

would be a usefiil additive to çardioplegic formulations. In orda to iden- such a

cornpond, a more detailed understanding of the ppvate dehydrogenase cornplex is

required.

1.2. THE PYRWATE DEElYDROGENASE COMPIZX

Sm<cnuc Ond Ration of the PDR Cornplex

Figure 3 demonstrates that pyruvate is an intermediary product of glycolysis. In

humans, ppvate is metabolized via four major eqmatic pauiways. Lactate dehydrogenase

WH) conveits pynivate to iactic acid as the final step in anaembic giyoolysis. q.niVate

carboxylase converts pyruvate to oxaioacetate, an intermediary Krebs' cycle component. In

the liva, alanine aminotransferase (ALT) regulates the reversible conversion of pyruvate to

the amino acid alanine. Pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase

22

Pm*

The pynnrate dehydrogemse complex is a large macnm~oleaie (8500 kDa) which was

fust described by Reed and coileauges h 1 % P This complex teSides in the inna

membrane of the mitochoIlclria and is composed of sevexai distinct sub-units. The El subimit

is a tebramer of 2 or and B mmponmts each. This subunit acts to decarboxy1aî.e pynivate to

form CQ and thiamine pyrophospbate CrpP-El). The E2 subunit transfers the acetyl- gmup

h m TPP to a lipoy1 cofactor and then subseqwtly to cofactor A, forming acetyl-CoA.

Protein X also functions as an acyltransfaase, but its unique role in the complex is unc1ear.

The E3 subunit is a flavin requiring enynae which o x i a the iipoyl cofaaar h m E2 by

tmsferring electrons to NAD+. The wmplex is regulated by two additional enzymes: PDH

kinase and PDH ph~sphatase.~~ The PDH complur is illustrated in Figure 5.

Pynivaîe dehydrogemue kinase cataiyzes the phospharyWon of Saine residues in at

least two positions on the E l a subunit, raidexhg the complex inactive.'* It is postulated

that phosphorylation of a third serine residue is irreversible and renders the complex

insensitive to PDH ph~sphatase.'~ PDH kinase is inhi'bited by high oon~trations of

p p a t e and ADP. DichiorOQcefate is thought to act synergistically with ADP to inhibit the

PDR k h s , but the exact mechaniSm remains unclear." The PDH lanase is stimulated by

the end-products of PDH metabofism, niimely NADH and acetyl-CoA. mus, when the

mitochondrial NADH/NAD and acetyI-CoA/CoA ratios are high, the kinase is -Y

s t i r n a . Ravindran et al demonstrated that PDH kinase is regdatai by the redox state of

a lipoyi domain on the E2 subunit[= Ushg isoiateû PDH complex fnnn bovine kidney, they

found that compkte eaymatic delipoylation of the E2 subunit prevented NADH and acetyl-

CoA medi;ated stimulation of PDH kinase. Their pmposed mode1 suggests that acetyïation

23

of the L2 lipoyl domain of the E2 subunit produces a OOnfORnafiOnaf chauge tbat mhanc*i

biDdiDg to the PDH kinase. Restmhg the U Qmam to an oxidized form prevents PDH

kinase biading and subsequently preveats thePDII cornplex h m becoming phosphorylatpd.

AIthough each El-a subunit in the El tetramer h identicai, neariy complete iirnr?tivaficm of

the PDH compkx occrus if even one subunit is phosphorylsteA.'*

Pynivate dehydrogenase phosphatase rewses the E l u phosphorylation and retrans

the complex to the active state. Calcium and rnagaesium are re~uired by this enzyme f a

maximal activity.lB LUey et al found in bovine heart mitochondria that insulin pref-My

stimulates the PDH phosphafase.lm Larner et al fomd that incubation of epididymai fat with

insulin increased the sensitivity of PDH phosphatase to magnesium.*' Similarly, Newman

and colleagues found that isolatPri PDH phosphatase muid k stirnulated by incubation with

an insulin 'mediatora.lP In laboratory eovircmments, the PDH phosphatase is readily

inhiiited by sodium fluoride.

ïk Effect of Ischemia on PDH A m .

Mochizuki, and Neely demOIlSfrafed in 1980 that the recovery of normal giuaxe

oxidation in isolat& rat hearts subjected to global ischemia was delayed for over 20 minutes

foilowing normothermic reperfus~n.~ In this study, and in a preyious study by Liedtke and

N e l l i ~ , ~ ~ the infusion of pynivate during repahision substantiaüy irnproved the recovery of

mechanical function. Neely postulated that high cytosolic and rnitochondrial leveis of W H

and açylCoA following ischemia rnay act to inhibit the mitochondrial PDH cornplex?

Concomitant infusion of pyruvak prevented the inhibition of PDH and led to impved

fwictional recovery foïiowing ischemia.

24

gobayashi and Neely inv- the e&a of short tenn isdiemia and repemisiOn

on PDH activity in isoiated rat hearts subjcded to tm minutes of global ischemhul T h e

authors found that in hearts pemised with 11 mmoVL of giucose abne, ten minutes of

ischemia did not depress the acfivity of PDH. Appfolgmately 80% of the complex remaineci

in the active form in both control and ischemic h m . However, foîlowing 2 minutes of

repafuson only 45% of the enzyme d e d in the active fona. The addition of 10

mmoYL of pyruvate to the qafimte pmented the iaactivatcm of PDH.

The authon found that ischemia resuited in a 260-fold increase in the mitochondrial

NADWNAD ratio and a 43 96 reduction in myocardial ATP stores. Repfwion with glucuse

alone restored the rnitochondrial NADWAD ratio to near normal lm& and ATP stores to

72% of non-ischemic controls within two minutes. Repedhion with glucose and pyruvate

resulted in a more rapid resbration of mitochondrial NADWNAD ratios with a simiîar

r e c ~ ~ e r y of ATP stores.

Righ pst-ischemic NADHINAD ratios would be to stimulate the PDH

khi& and result in inactivation of the complex. However, the low mitochondriai ATP lm&

rnay have beea insuffiCient to allow for phosphorylation of the E-la subunit. As the celfulat

ATP stores became resiored following npemision, PDH kinase regain& the ability to

phosphorylate and inactivate the complex. In addition, the high leveis of ADP following

ischemia may inhibit the PDH kinase alIowing PDH to remain in its active fonn during

i s c h d Again, as mitochondriai ATPfADP stores are restored during reperfusion this

inhibition of PDH 14Mse would be lost, uniess high ambient concentrations of ppvate are

maintained with exogenous administration. Aitematively, the intraceUular acidosis which

accompanies ischemia may lead to activation of the PDH phosphatase which has a pH

25

optimum betweea 6.7 and 7.1 in the pregare of miignesi~m.~

Anadditidstudy by VaryetaP alsoJhowtdthPPDHactivity~asstuniilntPAby

no fiow ischemia. In thar isolateci rat kart model, these &ors found that pxeishemic

pemision with insuiin (20 IUL) causcd a f k t k bxease in PDH activity following 30

minutes of ischemia. Unforhiaately, they did not assess PDH activity during repafusim.

Howevex, k i r resuits suggest that pre-ischemic intemation an affect PDH d M t y durhg

ischemia. To date, the d t s of pn-ischemic interventions on the pst-ischernic activity of

PDH are unknown.

Lewandowski and White performed important eqexhents in isolatPA rabbit hearts

to detemiine the relationship between PDH inactivation and pst-ischemic fuactional

recoveqm In their experimental protocol, they stimuiaîed post-ischemic PDH actiyity by

the addition of 5 mmoYL dichloroace&te (DCA) to the repemiSare. In normal non-ischemic

hearts, DCA did not affect left ventri& function despite a stimuiation of PDH activity.

However, in pst-ischernic h m the rate-pressure-produe i@~antly from

83ûû*1800 to 21300*2400 when PDH acbivity was stimuiated by DCA treatnient during

repemision. Postischemic hearts displayed reduced pyruvate oxidation compared to non-

ischernic conbrois. DCA stimulated hearts increased the oxidation of pyruvafe without an

attendant in- in the by-products of glycolysis. Therefore, these authors concludeci that

countezacting depmsed PDH activity in the postischemic rnyocafdium preveated contrade

dysfunction. Furthemore, the imprwed cardiac @ormance did not d t h m 7 nor

require, hcreased glycolysis. Restaing carbon flux thrwgh PDH alone was suffiCient to

Unprove mechanid work by post-ischemic hearts.

26

McVeigh and Lopaschuk have s u g g d th& PDH acîiviîy is inhibiteci by fatty acid

fmm Qrculatirig fhe fatty acid me&boIism act to stimiilate_ the PDH kinase and thereby

"hibit giucose oxidation. In several reports, Lapaschuk has demonstrated that inhibithg

faüy acid metabolian by inhibition of d t i n e paimitoyltransfeaase (CPT) improves post-

Shortly afta the discovery of the pyruvate dehydrogenase complex in 1969," it was

show that insulin was capable of reguïating this enzyme. '&le in a review article published

in 1989, Weiland defined the characterisiic ffeatures of insulin's regdation of the pyxuvate

dehydrogenase complex in adipose tissue.'*

Insulùi activates PDH at physiological concentrations (K.: 10 mUL) Activation i s rqid and occurs in minutes. Insuiin activates via ppvate dehydrogenase phosphatas activation. Activation of the PDH phosphatase requires the presence of a metaboiizable sugar. Insulin does not change the rnitochondrial ATPIADP ratio. The activation pemists in mitochondria isoiated h m insulin-treated fat ce&. Cyclic AMP is nat responsible for the efféct of insulin on PDH. Insulin cannot be demonstrated to stimulate PDH in isolated fkt c d mitochondria, Insuiin activation of PDH has been dernonstrated in white and b r m adipcytes, fibroblasts, myocytes and lactating mammary giand, but not in liver*

Several of these statemmts require furth- discussion.

The dose-respcmse relationship betweai insulin and PDH bas beai extensively

evaîuated in adipose tissue and has km confirmed to'axw at physio1ogic concentrations (1-

100 mU/L,).161n However, in the sethg of acute ischemia, many investigators have

employed a much higher concentration of insulin (10-20 Un) in thar GIK fomuiatim.

27

The possible dmmguMhg eff8Ct of high iiwulin concentrations has not beai adeqiiately

daemhed. High doses of insulin may be rrquind to Jtimulate other protective &ectS, such

as reducing fatty acid oxidation. incfeasing glucose transport and reducing afterload.

Therefm, it is important t~ detamine if high doses of insulin continue to stimulate PDH

nie t h e course of the stimulatory &éct of insulin suggests Uiat it works on a post-

translational basis. That is, insulin does not require the synthesis of novd mRNA or proteins

to aext its action cm PDH activity. In seüings of impending ischemia, such as d o p l e g i c

arrest, it would be important to exert a stimulus which acts hmediately and persists during

the repemision phase. As mentioned previously, it is unclear if pm-ischemic insuiin treatment

GUI iesult in pst-ischemic PDH stimulation. Shuües on mitochondria isolated from fat d k

exposed to insulin suggest that the insuiin &ect can pasist for up to 30 minutes." Randle's

p u p have also examined a possible long-terni regulatory effect of PDH activity.ja

Conditions of chronic hypoxia, such as wngenital cyanotic heart disease, may be ameaable

to long-km PDH stimuIarion by pharmacoiogic additives. Recent work by Meranfe1#

mgge~ts thaî the aictivity of mitochondrial PDH and cytochrome oxidase is inhibited rapidly

foI.lowiag exposure to low partial pressures of oxygen @4 = 40 m d g ) .

Insulin has been demonstratsd to stimulate bah a metai dependent and independent

PDH phosphatase by Lanier's group."'' Lamer initially demonstrated that insulin acted by

reducing the PDH phosphatase's requirernerit for divalent cations (calcium and magnesium).

In a subsequent investigation of isolatpA bovine mitochondria, Lamer's group detected a

metal independent PDH phospiqtase which was not inhibited by okadaic acid, not stimulated

by spamine and not YnmunopreQpitated by antiphosphatase 2A anh'body.lD This novd

28

phoephatase demcmstrated a ttveefoId ina*ue in activity foliowing insului exposure.

Thcabilityofinsulin toregulatcthcPDHkhaseisiike1ydependmtuponchangesin

the mitochondrial ATPIADP and NADHINAD ratios. Howeva, Denton's group

demonstrated in isotateA white fat mitocbondria that iasuün treatmmt did not resuit in any

changes in ATP, ADP, NAD, NADH, acetyl-CoA or CoA which would result in inbrl'bitim

of PDH tjlia.Pe,.Ia In contrast, a shidy by Hughes a al demoastrated ùumzsed W4-

incofporatiion in mitochondria exposed to insuiin, suggesting incrrased activity of the Ianase

suôunitJB The authcm rationalized the apparent contradiction betweai increased PDB kinase

activity and the ovedi increase in PDH activity by claiming that insuiin acts to stimulate the

dephosphorylation of the cornplex which in tum leads to an inaeased turnover of the PDH

phosphoryhtiondephosphorylation cycle. If insuiin acts by inhibithg the kinase subunit,

then an ovedl decrase in tumova would have been expected.

Ihe Rolc of a Second Messenger

Much interest has centred on a possible second messetlger system responsible for

rndatbg insulin's effm on PDH."-1~'3'*14'a1s1*18 GottschaUc demonstirated in two sepiiiate

cell lines, rat embryonic fibroblasts and chinese hamster ovas, cells, that insulin' s stimulaMy

effect on PDH activity bypasses the insulin receptor tyrosine kinase? In œlls made

deficient of the insuiin receptor, PDH activity was StimulatPrl to a smilar extent as œlls

which overexpressed the insulin m t o r . However, Gottschalk employed transfection

vectors expressing normal human insului teceptors or receptors with inactivated tyrosine

kinase domains. Native insulin r e c e p a activity rnay have sti l i accounted for the stimukt~ry

e f f e on PDH activity. In ceb which overexpresseù the human insulin reœptor, saturation

of a second messenger system may have limiteci the stimulatory effect of insulin e n p o s ~ ~ ~

29

Discovery of a putative .insulin mediatar" bas thus iàr eluded investigators. Saltiel

proposed that these mediator molecules may be inosif01 phosphate glycans. One of these

phoephotidyl inosito1 glycans has bem s h m to d t in & lu^> synthesis of dpcvlgîymal

@AG).m DAG is a hown stimulator of praein kinase C, a cornmon second messeaga

presait in most œiî typeda S e and coUeagues Qnonstrated that insulin stimuiated

phoBphatidylinosito1 giycan hydrolysis with subsequent & mw syathesis of DAO and PKC

activaiion in BC3-HI myOCYfeS.m However, inhi'bition of G-protein activation by pertussis

toxin did not inhicbit insulin's ability to increase DAG concentrations and stimulate PKC.

These authm concludeâ that novel phosphatidylcholiae hydro1ysis was responsible for DAG

synthesis and subsequent PKC transidon and activation. Craven and DeRuberth aiso

demonstrated that exposure to high glucose amœntration results in & now synthesis of DAG

and incteased PKC activity.la

Benelli et al demonsûated in CUItured 2ajdela hepatoma tells that insulin activated

PDH via a PKCdependent pathway.la In this model, insulin was found to stimulate PDH

within five minutes of incubation and was maximal (70% increase over baseline) at 7.5

minutes. In the presence of the phorbol ester PMA (4fbphorbol 12B-myriState 13a-acetate),

PDH activity was increased within 3û seconds, achieved maximal activity (90% wer

basehe) at 5 minutes and was no longer detectable at ten minutes. In addition, incubation

with insulin and the PKC inhibitors staufospo~e and sphingosine wmpletely blocked the

stimulatory effect of insulin.

However, the d e of protein kinase C in the insulin signaUing pathway continues to

be c o n t r o v d . DowmguMion of PKC with chronic phorbol esta exposure does not

inhibit insqlin-induced glucose transport in B3CHl rny~cytes.'~ Similarly, KEp and Rarnlal

30

demOQlStrafedthatpn)teinlMaseCwasnatrequirrdfmkosetransportincultiaedslteleéal

muscle cells.la Stumpo and BiacWear demcmstrated that c-far expression in 3T3-L1

fibroblastswasina*isedafkinsutinaposun, eveniftheœiisweremadeprofeinkirÿise

deficiat.lm Both cfos and c-myc are eariy respaue peptides which have kai show11 to be

stimiitatPA by PKC.lQ The rde of protan kinase C in the insulin signalhg pathway

wntinues to be inMstigated predominantiy in adipose tisoue or non-cardiailc myocytesm.

Establishing the relationship between PKC and insulin in ischemic mya'ardial tissue is of

paramount importance in understanding the mechaniSm of the cardiop~otective e f h t of

insulin.

1.3. THE ROLE OF PROTEIN KINASE C

Signal Tzcurrducn'on P d m q s

Sutherland first proposed the model of a second messeriger system in 1972.1a In this

model, extracellular signais termed ligands either petrate the ceii membrane or bind to

membrane bound reqtors. Activatexi feceptOrs with or without an associated membraneœ

bound transducer (such as a G protein) trantranslate the 1igand-reçeptor binding signal into an

intracellular chemicai message. Most o b , this involves changing &ha the distribution or

the ancentration of " second messenger" compounds.

The insulin signalling pathway has been one of the most actively studied rnammalian

systems since Banting first described the hypogiyœmic effécts of this purified pr0tein.I" The

bovine and porcine hormones were sequenced by SangeP and cowmkers in 1955 and in

1963, Katsoyannis d d b e d the fgst chernid synthesis of purified insulin.In As M e r

information about the structure and function of human insulin became avaüable, many

investigaiors simultaneously concluded that insulin's inûaœiiuiar effezt involved the

Figure 6 ciescri- the struchin of the human iasulin LeceptoT. The insulin reaptor

cornpiex is a tetrameric transmembme giycoprntein consisbing of two 120 kDa and two

90 kDa B subunits.lp The two a-subunits are h k m i by disutphide bonds, are entirely

extraceiluiar and amtain the insuiin binding sites. Each &subuait is linlred to an a-subunit

by a disiilphide bond, crosses the piasma membrane and amtains a tyrosine kinase domain

in its intracellular portion. nie association of insulin protein with its ~~cep tor stimulates the

tyrosine kinase activity of the Bsubunit and d t s in autophosphorylation of the second &

subunit Autophosphorylation of the &subunit stimulates the kinase activity of the receptor

towards 0 t h substrate pk ins , including insulin receptor subsûate 1 @tS-l).m The insulin

receptm is capable of binding 1 or 2 molecules of insullli.Is The unoccupied a-subunit

exerts an inhibitory effed on the tyrosine kinase activity of its amespondkg Bs~bunit.

Proteolytic cleavage of the a-subunits relieves this inhibition and resuits in a stimWon of

kinase actiVity.lB

IRS-1 is a highly conserved Cytosolic protein with multiple serine/threonhe d d u e s .

Interestingly, IRS-1 contaias over 30 potential serine/threonine phosphoryîatim sites which

have homologies to protein kinase C. ln Prior to insuiin activation, IRS-1 is highly serine

phosphorylated and weakly threonine phosph~ryhted.'~ Foilowing insulin-reœptor bindùig,

there is an increase in both serine and Uinonine phosphorylation." Sinœ IRS-1 shares

homologous domains with a wide variety of intraceUular messagers, including PKC, it is

likely thaî insulin stimulation of PKC is associated with the phosphorylation of IRS-l.In

However, this association has not yet been demollsfrated.

Req tm mediattd hydrolysis of membrane-bound phosphatidyluiositol gïycans is now

realird to be a cornmon signal transduction mec)iiuii.(wn in mimy ceil types and organ

systerns-" S tnh and colleauges demOllSbafed that Mtol - l , 4 ,5 triphosphate p3) is reieased

into the cytaphm following hydrolysis of membrane bmmd phosphatidyl4,5-biphosphate

(PIPa and ùiduœs the mobibatiion of caicim fiom inbraicellular stores.1x 'Lbaeafter, Takai

repmted Uiat anothex product of PIP, hydrolysis, diacylglyœml, initïates the activation of a

s p a h h d protein Linase.lW This kinase had been previously ideiitified by the same group

in 1977'- to be protein kinase C and has since been show to be present in most cell types

and organ systems. '"MI

In its W v e f m , protein kinase C is widely dispased in the cytopIasm.

Stimulation of PKC resuits in its transiOcati011 to peMucIear and ceU membraneP

Translucaîion of PKC is associateci with binding to Receptor for Activated C-Kinase Subtype

(RACKS) which is present on cytoskeietal elements and the ceil membrane-lm Referatiai

binding to RACKS is thought to fiditate the isoforni-specifïc functions of PKC for dinerent

proteins." Although translocation of PKC from the cytosol to the plasma membrane is often

associated with stimitlation of the enzyme, it may not necessarily result in an increase in

kinase activity."

Isofom Speciic Pr~pem'es of Protein Kinase C

There have been numerous isofanns of protein kinase C identifd.'" AU represent

kndthreonine kinases with a similar molecular weight (68-83 kDa), however each may

play a different d e in the ceiluiar response to extenial ~tirnuli.'~ lbgoyevitch and

coUeagues descfibed the expression of numemus PKC isofoms in adult rat hart and

33

m e d that by fâr the most abundant was the eIsooform.m The otha isofarms preseat

in heart include the B, a, 8, eta and zeti isofann~.~*

NishUulrals Onginal description of the m e s of PKC suggested that it was

dependent. Based on these differe31t isofmm-speafic characterista, a ciassification system

for the PKC i s o f m is now commonly used in the 1iterature.l" The calcium dependent

isofarms or conventional P K ' s (cPKC) include the a, 8 and gamma subtypes and are

adivated by calcium, phosphatidylserine, dkylglycerol and phorbol esters such as PMA.

A s g ~ d group is comprised of four novei PKC's (nPKC), 6, E, eta ande. This group is

actiMtcd by phosphatidylsaine, diacy1glyerol and phorbol esters, but not calcium. A third

group includes two atypical PKC hforms (aPKC) zeia and lambda. These isoforms are

actiMted by phosphatidylserine, but not by diacyigiycerol, phorbol esters or calcium. The

zeta f m rnay exert a constant low level of activator-independent kinase activit~.'~ In

addition to their different responses to extemal and intanal stimuli. these isoforms may be

responsible for differential effects within the œil.In One manifestaiion of this property was

descnbed by Banerjee and colleagises who provideci evidenœ that a-adrenergic stimulation

d t e d in translocation of the a, 6, eta and zeta isofoms while the 8 and c isoforms were

unaffected.ta Stimulation with a DAG d o g u e resulted in a translocation of the a, 6, eta

and zeta isofoms with the r i s o f ~ ~ n translocafing to the perinuclear membrane. However,

a-adrmergic stimulation proîected against ischernic acidosis in a PlCCdependent manner

while exposure to the DAG analogue did not prevent acidosis. Thus, it appears that

inhibition of the E-isoform resuited in protection against acidosis, but this inhibition was

dependent upon stimulation of the a, 6, eîa and zeta isoforms.

whose &ty was stimuhd by the hyQolysis of membranebound phosphatidylinosito1

g l y c a n ~ . ~ ~ The naturally OcCuring by-product of PIP, hydroIysis, diaçylglycerol @AG),

is a potent stimiilatnr of p t e i n Linase C and has given rise to several synthetic analogues

such as l-oleoyl-2-~1glyycerol (OAG), l , 2 d i ~ o g i y c e r o I (DûG) and stearoy1-

arachidmoyl giyceml (S AG). la

Tumm pmmoting phab01 estas such as 4BphoroOl 12Brnyristate 13~-acetite

(PMA), have a structure which is very similar to that of DAG and also activafe PKC.'?

Howmr, uniike the rapidly metabolized diacylgiycerols, the phorbol esters are slowly

degradeci and can resdt in eventuai downregulaton of PKC activity with prolongeci

exposure. lalm

Inh'bition of PKC can be accomplished by a variety of biochemicai mechanisms.

Staurosporine and H-7 both inhibit PKC by intaference with the ATP-bùiding site of the

enyme.lW Unfortunately, these compounds iack specificity for PKC and can also interfere

with the ATP binding sites of other kinases such as protein kinase A, tyrosine Liaases and

calmoduiin-depaident kuiase. However, che1erythMe qmsents a PKC specific inhibitor

which aiso works by blodang the ATP binding site of the enzyme.'"

As mentioned previously, transiocafion of PKC is thought to muire stnictural

cytoskeletal elements such as micro tubule^.'^ Therefore, agents such as colchicine which

inhibits microtubule synthesis may be usai as a relatively non-spxiiic P K antagonist.

However, other cellular enzymps requiring cytoskeletal association may a h be inhibited by

colchicine. Interference with the lipid cofactor binding sites of PKC urn produce highly

In 1986, Miary, Jamings and Ramt published a landmark shuly in which they

reportcd that the magaitude of canine myocardial hhction ptoduced by a 40 minute

circumnex coroiiary artery occlusion was sipifkmtiy r e d d whm the myocardium at rislr

had previousiy been subjeded to four cycles of 5 minute wronary occlusion and 5 minutes

of qxxfhi~n.~* This phenornenon was termed "ischemic preconditioningW and may

represent the most potent form of endogrnous myocardial protection discovered to date.

Preconditioning has now been d & M in a wide variety of organs and multip1e

species.'" The preconditicming ~esponse ha9 been reported to occur in human

r n y o c a r d i ~ r n . ' ~ ~ ~ konomidis and coileauges have demonstrated that isolatPA cultures of

human ventricuiar cardiomyacytes can be protected from 90 minutes of prolonged ischemia

if they are first subjeaed to twenty minutes of brief ischemia followed by twenty minutes of

reperfusim. loner and coUeagues reviewed the d t s h m the TIMI 4 trial. IS In

patients who were admitted with a diagnosis of acute myocardial irkction, an anginal aîtack

in the preceduig 48 hours prior to admission led to a decreased incidence of in hospitai &th,

severe congestive heart Mure or shock. In addition, these patients exhibited las myocardial

damage as evidmced by CPK levels and eleçtrocardiographic abnodties.

Yelion and coïkagues reported the first experience of pfeconditioning for cardiac

~urgery.'~ In this report, brief aortic occlusion and tel- pior to prolonged aartic

crosscîamping led to irnpfoved preservation of high energy phosphates in atrial trabecuiae.

However, this fonn of myocardial protection is unlike1y to be adopted by many surgeons due

36

to the additid risk of multipIe aortic manipulatons and the poBsibüity of atheroscleriotic

embli." Eauciclating the mechaniSm of kichemk prrconditioning has ken the focus of

group in CofOziadO bave also demonsüated that PKC is required for ischemic preconditioning

of the rat heart.'n.mn Ikonomidis and coileagues have dernollstrafed that preconditicming

of isolated human ventricular cardiomyocytes is aiso dependent upon protan kinase C and

is mediated by the Aease of aden0sine.l

To date, a number of compounds have been demonstrated to rnimic the effect of

"ischemic" prec~~lditioning including adenosine,- bradykininm and a-adreaergic

agents. 'W.'" Each of these stimuli, in tum, bas been shown to be dependent upon PKC

stimiilati0n.'- The end efféctor of PKC stimulation is stül disputecl. However, besed upm

the many rne&hlic effeçts of premnditioning, it is reasonable to poshilate that PKC

stimulation may d t in a regdation of cellular metabofism.

Miyamae et al demonstrated in a porcine mode1 that ischemic preconditioning resdted

in a better preservation of rnyocardial ATP, phosphocreatine and intracellular pKn

However, this beneficial metaboiic eR't did not translate into protection against stunning.

Similarly, Ryzkledc and Kloner's group demonstrated in a canine mode1 of myocardial

stunning that ischemic preconditioning was unable to exert a beneficial functional e f f a m

Banerjeets group feported preiiminary data suggesting that ischemic preconditioning

may act to p e n t cytochrome induced œiiuiar ~x ida t ion .~ A report by van Wylm

indicated Wt ischemic pmconditioning aîteniiatprl the accumulation of intracelluiar iactate and

37

intedtial purine metabolites during wheqmt prolmged ischemhaO Wolfe and colleagua

providsa evideace that suggesfed that pncaditioning d t e d in pischemic giycogea

depletion which penteci subsequent intraceiiular acidosis during plonged i s c h d m

Hcwever, this d t is in contradiction to the results obtaïned h m Taegtmeyer's groiip who

demoiistrated that preischemic giycogen depletion did na improve functionai recovery a€kr

hypothdc ischemic amst in a rabbit h a u t m

U n f e y , the end-effectar mecbanism fm the ischemic preconditicming

phenornenon remains unknown. However, the attenuation of intraceUular acidosis with a

reduction in lactate accumulation and pRsenraton of myocardial ATP strongiy suggest that

preconditioning ex- a beneficial effect on myocardial metabolism. Due to the questionable

abiiity of ischemic preconditioning to faalitate the fecovery of left ventricuiar fiinction, it is

likely that the preservation in ATP emanates from an aMerobic source. As a coroiiary to

Weiss' theory on the funaionai c o m p a r t m e n ~ o n of ATP prod~ction,~ it appem that

anaerobic ATP presenration h m ischemic preconditioning is preferentially used for ceiluiar

homeostasis. The degree of aembic ATP production is probably insuffiCient to allow for

functional benefit. Therefm, metabolic stimulation of aerobic myocardial ATP synthesis

in combination with a preconditioning stimulus may be an ideal combination to pro-

against both myocardial infarcton and stunning.

1 4 SUMlMARY OF PROPOSED INVESTIGATIONS

Contemporary techniques of perioperative myocardial protection continue to d t in

aelayed recovery of normal aerobic rneotboli~rn.~ Pmnous studies evaiuating the effed of

giucose and insulin soIutions for penoperabve myocardial protection have been

~ntroversial.~ ui addition, these stuclies were performed in an ad of hypothennic

38

m y d and systmiic m o n which may have attmuated the benef~ciai e&cts of inSulni

treatment. Many gtuciies ernployed intravenous gIu00seinsulin-potassium sotutions and did

not examine the &8d of dind myocardial pemisicm with insulin-enhanced cardioplegia

delivemi at normothermic or tepid (29°C) temperatines.

Myocardial PDH ha3 bem demoIlSfrated to be inhibitecl during eady rrpafusion

foliowiag brief ischemia in isoIntprl nit h m r n The &ecfS of ischemia and repafusion on

hrmimr myocardial PDH remain undetermbed. Insulin has been shown to stimulate

mitochondrial PDH in adipose tiss~e,~'*'~ however the ability of insulia to stimulate

myOCOrdiQl PDH remains undetermineci.

The proposed investigations are designed to:

1. Evaiuate the effed of ischemia and npafusion on human myocardia pynivafe

dehydrogenase activity.

2. Evaiuate the effects of pre-ischernic insuiin exposure on pst-ischemic myocardial

3. Detennine the mechanisrn of the insulin effect on pst-ischernic myacardial

4. Evaiuate the diffefential effects of pischemic insulin exposure on hurnan ventricular

cardiomyocytes and human saphenou vein aidothehi celis.

We hypothesize that ischemia and repemision wiii inhibit the activity of myocardial

pyruvate dehydrogenase. We believe that pischemic exposure to insulin wiU preverit the

inhibition of PDH by a protein kinase C dependent mechanism. An improved transition from

anaerobic to aerobic metabolism foilowing prolongeci ischemia is believed to improve cellular

tolerance to ischemia. Furthemore, the metabolic effects of insuiin expomre are

To f'acilitate these investigations, we propose to employ cuitulres of isoIsitpA human

ventricufar carriiomyocytes and saphenous vein enQthelinl cells. The d t s of thest

isvestigations wiU provide important new informaficm &out the metabolic abnofmalities

associated with myocardial iscbemia In addition, the in vino evaluation of a simple, safe

rnetaboiic intervention would provide jdcaf icm fa UI viw investigations which uitimately

may lead to supgior myocardial toleranœ to ischemia and improved clinicai oufcofnes

following cardiac Surgery.

Figure 1: Myocardial oxygen wnsumption (MV03 in the empty beating, fibrillating, and a& heart at normothermia (37'C) and moderate hypothennia (22'C). M V Q is markedIy reduced following cardiopIegic*arrest with a further minor reduction associated with hypothefmia. (A@fredfrom Buckberg et al.')

GLUCOSE

2 ADP

LACTIC ACID

36 ATP

FATTY ACIDS

Figure 3: Myocard'i glucose metabolism.

KREB'S CYCLE

FAD

+ H+ Nm3 ADP f i

Figure 4: The mitochondriai electron transport (respiratory) chain. NADH is oxidized by complex 1 and coenzyme-Q, a lipid soluble quinone. Complex II wnsists of

I succinate C reductase and coenzyme-Q reductase and transfers reducing quilavents fkom FADE& to complex III (cytochrome c reductase). Complex III I

then transfm electrons to complex IV, cytochrome c oxidase (COX). COX then reacts directiy with molecular oxygen ta form hydroxyl ions (OH-) which react with the protons produad by the complex 1 reaction to form water (H,O). For each NADH molecule, Uuee molecules of ADP are phosphorylated to form ATP, while two molecules of ATP are produced for each molecule of FADH,.

PDH PHOSPHA'IASE - PDH KINASE

Figure 5: The pyruvate dehydrogenase cornplex. The El subunit acts to decarboxylate pynivate to form CO, and thiamire pymphosphate (TFP-El). The E2 subunit transfers the acetyl- group h m TPP to a iipoyI cofhcto~ and then subsequentiy to cofactor A, f d g acetyl-CoA. The E3 subunit is a flavin re~uuing enyzme which oxidizes the lipoyl cofâctor h m E2 by t ransfdg electrons to NAD+. The complex is regulated by two additional enzymes: PDH kinase and PDH phosphatase.

+ Phosphoryhtion of Intracellulrir Peptides

Figure 6: The insulin receptor wmplex. The insulin reœptor cornplex is a tetrameric transmembrane glycoprotein consisting of two 120 kDa a and two 90 kDa 8 subunits. The two a-subunits an linlred by disulphide bonds, are entirely extraceiiuiar and contain the insuiin binding sites. Each Bsubunit is linked to an a-subunit by a disuiphide bond, msses the plasma membrane and contains a tyrosine kinase domain in its intracelluiar portion. nie assoaation of insulin protein with its receptor stimuiates the tyrosine W d v i t y of the &subunit and resuits in autophosphorylation of the second Bsubwilt. Autuphosphorylation of the Bsubunit stllnuiates the kinase activity of the receptar towards other substrate proteins, including insuiin receptor substratel m-1)-

CHAPTER 'Iwo INSULIN STIlMULATlES MYOCARDIAL PYRUVATE DEKYDROGENASE

AND PROTECTS ISOLATED HUIMAN VENTRIcnn,aR CARDIOMYOCYTES FROM SIMULATED BCHElWIA

However, the effect of insuiin cm human myocardizl PIH remains mdetermineû.

Furthermore, the effed of ischemia and repafusion on the activity of human myocardial

PDH rernains unlaiown. Kobayashi and N d y m demanstrated that npahision following

brkf ischemia resuited in a 55% inhiiitim of rat myocardial PDH. This inhibition was

thought to be due to stimulation of PDH Iànase by accumutated NADH and acety1-CoA in

the presence of adequate intracelluiar ATP stores. LRwandowsLi and WhiteU provided

&dence in an isolaîed rabbit heart mode1 that the recovery of PDH activity dinectiy

correlated with the rrcovery of ventricular fiuiction. Therefore, attempts to stimulate PDH

acfivity and improve the transition from aMerobic to aembic metabolism foliowing ischemia

should improve œiluiar tolerance to ischemia and result in enhanaxi functional fecovery.

To investigaîe the abiüty of insulin to stimulate human myocardial pynivate

dehydrogenase, we employed a mode1 of simulateci ischemia and reperfiision using primary

culaires of isolated human right ventricular cardiomyocytes. This model has several

advantages for the investigation of cellular responses to ischemia and feperfusion. Firstly,

the effect of other ceii types such as endotheliai d s , fibroblasts, smmth muscle cek and

migating neutruphils are absent in this model. The effect of other organ systems such as the

liver, pancreas, brain and pezipheral vascular system are aiso absent. In addition, the

extraceiluiar environment can be carefully controiied to eliminaîe the effects of altemative

substmtes such as fatty acidsJ" and to inüwiuce metabolic interventions such as insulin or

glucose. The nature of the cuihired cardiomyocytes has been extensiveiy reviewed in

cardiomyocytes, these tells retain many charaderistics of àr vnio rnyocardium and provide

a useful model for the investigation of injury due to ischemia and repafusion. However, the

d t s re~uire confirmaton in a whoie organ or m I&O model.

2.2. METHODS

Hrcnion VmniaJor Ccvdiomyayte aclavc

Rimary cultures of human ventricular cardiomyocytes were established as previously

d e ~ c r i b e d . ~ ~ ~ ~ Brkfiy, 20 mg biopsies were obtained h m the nght ventricle of patients

undergohg caective repair of tetralogy of Faiiot (Courtesy of Dr. W.G. Wilüams .-

Hospital for Sick C h i l a , Toronto). The myocardial biapsies were washed in phosphaîe-

buffered saline without calcium (PBS: NaCI 136.9 mM, KCl2.7 mM, Na?Hp04 8.1 mM,

lZ&P04 1.5 mM). AAcr removing amnective tissue, the myocytes were separated using

aizymatic digestion with a mixture of 0.2% trypsin (Difco Laboratories; Detroit, MI) and

O. 1 96 collagenase (Worthington Biochernicai Corp. ; F d o l d , NJ) . The isolated d i s were

culturecl at 37T in 5% C 4 and 95% air in Imve's modified Dulbecco's medium (GIBCO

laboratories; Grand Island, NY) containing 10% fetal boWK senim, 100 U/mL pallcillin,

100 pg/mL streptornycin and 0.1 m M &mercaptoethanol. Purification was achiwed using

the dilution cloning technique. Enzymatically isoiated cells were seeded at a low density ( S e

100 d i s per 9 cm diameter culture dish). At this low density, individual cardiomyocytes

were distinguished morphologically by their rectangular shape and large 9ze (40x80 cm)

h m con taminating œlls such as fibroblasts and endothelid œlls. AU cells on the plate were

aUowed to divide and at 7 days cardiomyocyte, fibroblast and endothelial colonies formed.

Single cardiomyocyte colonies were thm transfernd usuig a Pasteur pipette to another cuiture

49

dish. Cell cuitures were uupeded daiiy and any conbmbatd cuiture dishes w a e discarded.

Culture purity gnater than 95% was afta the third cell passage with fi-t

monocIanal ant i i iy stainïng for human ventricular myosin heavy chain (Rougier BieTech

M.; Montreal, QUE). Ce& passage! 2 to 7 tims with a time h m primary cuiture of less

than 60 days were used for this shdy. Figure 7 ïiiustcates the phenotypic appearance of these

cardiomyocytes at 2ûûx magnincation.

SmuJcltcd Isahcma anà Repctjksbn

The cardiomyocytes were studied in PBS with rnagnesium and calcium WgC&

0.49rnM, CaCI, 0.69m.M). The addition of these divatent cations ensured th& PDH

phosphatase activity wouid not be inhibiteci (see page 23). In addition, œiis exposai to PBS

in the abserice of calcium lose their ability to adhere to culture dishes after approximately 30

minutes. Our in vin0 technique to simUfilfP. 'ischemia' and "repemisona has been

previously described in detail." Briefly, cells were stabikd for 30 minutes in 10 mL of

normoxic @O2= 150 mmHg) PBS. *Ischemiaw was simuLateci by placing the ceils into a

d e d plexigiass chamber fiushed with 100% nitrogen and exposing the celis to a low volume

(1.5 mL) of deoxygenated PM. Deoxygaiated PBS was prepared by degassing norrnoxic

PBS with 95% N, and 5 % CO, until the rneasured p Q reached O mmHg. 'RepemiSon" was

accomplished by exposure to 10 mL of normoxic PBS for 30 minutes. A srnail sarnple of

deoxygenated PBS (2 mL) was placed into a centre di& in the chamber to rnonitor

temperature and to con- the absence of oxygen at the end of the "ischemic" period. The

temperaaire was maintained at 37C throughout aU experimental pmtocols. The osmolarity

of the degassed PBS solutions was maintained between 280 and 320 mOsm/L using sodium

chloride or water as required. The pH of aU PBS solutions was mâintained between 7.35 and

50

7.45 with 1M hydrochloric acid or 1M Wum hydroxide as quired. Figure 8 is a schematic

mpmeatation of the equipmeat requind to simulate ischmlla and npezfkion. Nimgen gas

is bubbled through two oxygen trapa pria to flushing the seaied plexigiass chamber. A

singie port on the chamber is lefi opm to vent the c h b e r . The plaiglas cbambex is

phcd on an eiectric heating pad which raciiitaks the mainteriance of normothermic

te-

EqerUnental Pr0ta:oIs

During the minute stabïüzation period, ceils were exposai to varying

co~lcentratious of glucose and insulin. FoiIowing stabitiÿaton, each di plate was washed

with normoxic PBS without glucose or insulin prior to the ninety minute ischemic exposure.

Similarly, following ischemia d plates were washed and repemised for thi.rty minutes in

normoxic PBS wïthout any additives. Fo11owing a dose response analysis for both gîucose

and insulin cm cellular survival, we chose to cumpare four groups. A physiologie glu-

concentration of 5 m M was compared to a glucose concentration of 100 mM. The latter

concentration agproximates the glucose concentration in the cardioplegic solution currently

employed at The Toronto Hospital. nie effects of 10 IU/L of insulin was then evaiuated at

both leweIs of glucose. (Gmup I : 5mM gluc~se; G m q 2: 5mM glucose and 10 I U L insulin;

Grwp 3: 1 0 mM glucose; Group 4: 100 rnM glucose and 10 IüL insulin).

Assessntenr of C e Z I ~ l .

Cellular injury was assessed using nonanfluent cultures of cardiomyocytes

(approximately 337,000 œlls per 9 cm diameter culture dish) cuitured for 4 to 5 &YS aAa

the iast passage. Following the intervention of interest, ceil plates were incubated with 0.4%

trypan blue dye (Sigma Chernical Company; St. Louis, MO), dissolved in normal saline and

Mississauga, ON) at 2OX magnification. I n j d tells were unable to exclude the large

molecular weight dye and stained blue. The aumber of blue stained ce& was counted fiwi

five srandard locations on each plate and expres& as a perœntage of the total number of

cells. AU cuunts w a e p a f d by a single obsedver who was blinded to the intervention.

Fi- 9 displays nepresaitative microgrciphs (Zûûx mgnScaticm) asseshg cellular injury

foliowing normoxic stabili7ation and afta ninety minutes of ischemia and thirty minutes of

repemision.

BiOchemicd M m -

Connuent cuitures of cardiomyocytes (approximately 600,000 ceils per culture dish)

culaired for 5 to 10 days from the last passage w a e used for biochemical d y s i s . The

activity of pyruvate dehydrogenase (PDH) was rneasured afkr each intervention of interest

using the modifications describeci by Robinson et aim of the method of Sheu et al? Ce11

extracts were aliquoted into separate Eppendorf tubes containing PBS. Foilowing incubation

for 10 minutes, ceils were treated with a buffkr containkg 25 mM sodium fluoride and snap

fkozen in Iiquid nitmgen. Ceil extracts were then reacted with a '%-pyruvafe containhg

b a e r in an open Eppendorf insert and piaced in Sealeci containers containhg benu,thonium

hydroxide to trap "COz. The reaction was stopped with 10% trichloroacetic acid and "CO,

wllected for one hour. Following "C4 wilection, the 4 inserts were removed and

scintilfation fluid added to the exposed bnizothonium hydroxide. The co1Iected I4CQ was

then counted in a Bcounter. PDH activity was calcuiated a f k correction for protein content

and expressxi as nmol of pyruvate oxidized per mg of protein per minute.

52

In a dupliciaîe series of experimeats, hfmcdlular and exfmdlular lactate

accumulation was rneasured using an m c me&d (Stat-Pack rapid lactate test kit,

Behriag DiagnoiCS; La Job, CA). Briefly, lactate is converted to pyrwate with an

equimok reducfion of NAD to NAM. Pynivatc is trapped by conversion to L-alanine by

alanine aminotransferase. The amount of lactate in the sample is directly Praportional to the

amount of NAD r e d d to NADH. The concentration of NAD is ckrmined by meaJuDng

spectrophotornetric absorbante at 34û nm. htracellular and extracellular lactate

concentrations were thai corredcd for the DNA content of each plate.

In a separate Senes of experiments, extraœiluiar lactate and pynivate were measured

in tandem in the supernatant of each plate. Supeniatants were wiiected and treated with a

measured volume of 6% perchloric acid to inhibit emymaîic degradation. Lactate

concentrations were determineci as d e s c r i i above. Pyruvate concentrations were

determinexi using a lactate dehydrogenase (LDH) couplad remion. Briefly, ppvate content

was determined fluofornetncally by measuring the change in NADH as LDH converted

pynivate to lactate. The lactate/py~vate ratio (L/P d o ) was thai calciilatprl for each plate.

Lactate and pyruvate concentrations were coneded for the protein content of each plate using

the Bradford spectrophotometric dye &on with bovine semm albumin as the standard?

The concentration of hydrogen ion w] in the extraceilular fluid was determined by

mnverting the pH value measured using a b l d gas anaiyzer (1312 Blood Gas Manager,

Instmmmtation Laboratory; Milano, Italy) tu [H+ J by the formula= [R+] = antilog (-pH).

Confluait plates of cardiomyocytes were used to determine cellular adesine nucleotide

contents a f k the intervention of interest. The specimens were fiash frozen in liquid nitrogen

and thai fheaAried. Specimens were anal@ by high performance iiquid chromatography

53

with the modifications described by W U et P of the step gradient technique &elopcd

by Hull-Ryde et al.= Using thh me2had, we demmined myocardial ccmcmtrations of

ademshe triphosphate (ATP), aderioshe diphosphate (ADP) and adenosine monophosphate

(AMP). W e also measured creatine phosphte (8) and the metabolites adenosine (ADO),

inosine (INO), hypoxanthine and xanthine 0. Total adenine nucleutides wete

de&mhed as the sum of ATP, ADP and AMP. Total Qsradation producfs P P ) wexe

calcuîated as the sum of ADû, INO, HXN and XAN. Energy charge (EC) repre~ents the

utilizable high energy phosphate pool and was calculated as:

EC = ( A n +O.SADP)l(ATP+ ADP+ AMP).

The DNA in the cell ex- was recovered in 5% perchloric acid and quantitated

using a spectrophotomebnc, diphenylamine &or &on with calf thymus DNA as the

standard.= Hydrogen ion and adenine nucleotide values were then correctal for DNA content

from each plate.

Non-ischemic control &ornyocytes were subjected to similar protocols employing

nommxic PBS @4= 150 mrnHg) for equivalent the perïods as their ischernic counterparts.

Baseline biochemicai measurements were made after removing the culture media and washing

the cells with normoxic PBS. In a separate Sefies of experiments designed to * . a

glucose effect, cells were arposed to 5 or 100 mmoi/L of mannitol with or without insului.

StcuLtical

The SAS Statistical Package (SAS hstitute, Cary, NC) was employed for analysis of

a l i data. Raw data are expressed as the mean f standard exmr with eight plates per mup,

dess othenivise specifîed. Biochemical md-points are expressed as a percentage of the value

obtained h m non-ischemic anitrol plates exposed to an equiva2ent volume of normoxic PBS

54

for the equivaimt period of tirne. Analysis ofvPiimce (ANOVA) was used to s i a i u i t a n d y

compsnmcansbetwemgraips. Wbenstatisbc4U m . y signin*mt difféxences wexe found, they

were speafied by the method of least sigmficant differerirts. Statistïcaï signifïcance was

assumed at pCO.05.

23. RESULIS

A s s c S m e n t @ G ? ~ I *

The e f f e of insulin was asses& using two lewls of ambient glucose concentrations,

a physioIogic lwel of 5 mmol/L and a cardioplegic level of 100 mmoVL. At physiologie

giucose amcentrations, insulin fWed to produce a statistically signficant reduction in cellular

injury (F=0.27, p=0.84 by ANOVA). However, at an ambient glucose concentration of

100 mmoüL Iowa ceiiuiar injury was observexi foUowing exposure to 10 lU/L of humuiin

R (F-4.48, p=0.01 by ANOVA). No firrther protection could be demonstrated by

increesing the insulin concentration to 100 IUlL (Figure 10).

The effect of glucose was asessed with and without 10 IU/L of insulin. Higher ceii

injury was observed as the glucose concentration increased @y two-way ANOVA, glurse

@a F=6.48, p < 0.0001). The addition of 10 NL of insulin reduced cellular injury

(insuün @ect Ft24.26, p <0.0001). Thexe were no statisticaUy signifiant interactive

effects between gluwse concentration and insuiin treatment (gIucose*Ursulin Mecf F =2.05,

p =O.O8). Duncan's multiple range test specitied differences between insulin and non-insulin

treated groups at glucose concentrations of 50 and 100 mmoYL. (Figure 11)

l d n Effect on Pyruw~te Dehydrogenase Acti* .

Figure 12 demonsnates pynivate dehydrogenase activity foIIowing stabilhation,

ischemia and mpfkion. Insulin exposure increased PDH d v i t y at both l d s of giucose.

SS

High glucose amceatraticms a h incnased PDH activity compared to 5 rnmoVL glucose with

or without insuiin. PDH activity was inhibited foilowing ischemia in the gmups with 5

mmoi/L g i m , while those œlis expoBed to 100 moUL of giucose with or without insulia

retained PDH activities near control leveis. FoUowing npanision, only those tells acposed

to 100 mmoYL of glucose and insulin retained near nomai PDH activities while a i I other

groups demonstrated intiiited activity.

To ehmate . . a glucose eff-, cells wexe subjeded to 5 or 100 mmoYL of mannitol

with or without 10 IUlL of in&. Figure 13 illustrades the ability of insulin to stimulate

pyrwate dehydrogenase independent of glucose.

Inrulin Wea on Zntennediate Merabolites

Figure 14 displays the inhra,cellular lactate accumulation which was highest in tells

arposed to 100 rnmoi/L of glucose and insulin during stabilintim and m o n . However,

after 90 minutes of ischemia, intracellukir lacgte accumulation in œ U s exposed to 100

mmoIlL of glucose was lower when insului was added. Ischernia and repemiSion increased

intracellukir lactate in all groups.

Figure 15 iliustrates extracellular lactate release measured in the supernaDuit over each

plate. Ischernia resulted in a large increase in lactate release in al l groups. Extracellukir

lactate release femained eleMted foilowing repemision in di groups, suggesting persistent

anaerobic metabolism. hctate release was Iowa in the insulin treated ce&.

SimiiarIy, extracellular pyruvate concentrations were lower in the insulùi treated œlls.

The lactate/pyruvate ratios were higher in the cells exposed to high concentrations of glucose.

The raw biochemicai data wi@ rneasuremmts of iactate, pyruvate, pH and PDH activity

following each intervention are aven in Appendix 1.

56

l i ~ o n A d Q i i n c N U c l e 0 ~

Figure 16 illustrates the fidi in ATP in aJl groups foUowing ischemia. haaiceuular

ATP was best preserved in the group eqosed to 100 mmol/L glucose and insuiin comparsd

to the groups with 5 mmoYL giucose. Repemision resulted in only a s d increase in

innra~efluiar ATP with aiï four groups remainiag w d below non-ischemic amtrol values.

SimiMy, ischemia resuited in a signifiaint fdi in total adenine nuc lddes in aîi

groups (Figure 17); however, they were best presewed alls exposed to 100 rnmol/L ghcose

and insulin. Foliowing m o n , total adaiine nuc1eotides w a e higher in the 100 rnmoYL

glucose groups but remained below 50% of cuntml values. The raw biochemicai data with

measurements of intraceUuiar high energy phosphates and thev metabolites are given in

Appendix 2.

2.4. . CONCLUSIONS

This study examined the role of insulin and pyruvate dehyâmgenase activity in

primary cultures of human cardiomyocytes subjected to simulatprl ischemia and m o n .

W e have dernonshated that pre-ischemic exposure to insulin improves cellular tolerance to

Srnulateci "ischemia" and "repenUsionm in isolated human ventrïcular cardiomyocytes. The

activity of pyruvate dehydrogenase was shown to be inhibited foUowing ischemia and

remained depnssed foliowing thirty minutes of repemision. Insulin was shown to M y

stimulate pynivate dehydrogenase activity before ischemia and partially prevented the

inhibition of PDH activity during repemision. Insulin treatment also significantly reduced

intraceiiuiar lactate accumulation and extraœilular lactate release during ischemia.

Intraœllular high energy phosphate levels were better pfesemed in tells exposed to hi&

giucose and insulin.

57

The resuits of these inves@ticms suggest that insulia treatment can enhance PDH

surgery. Howeva, the mechaniSm by which insUün stimiilstris pyruvate dehydmgemse

actinty requim further investigation. Denniag the mchanism of insulin's effect on

myocardial PDH may aIlow for phmnacdogic enhancement of the sthuiatary response.

Figure 7: Representative photomicrographs of primary cultures of hurnan pediatric (A) and adult (B) ventricular cardiomyocytes. (200~ magnification, reprinted from Li et a1219)

'Ikp #1 h p #2 Humidifier 37'C 4'C 37' C

Figure 8: Schematic diagram of the equipment required to simulate "ischemia" and I

reperfllsion. 100% nitmgen gas (NJ is bubbled through two oxygen traps prior t

to flushing a d e d plexiglass chamber. Four plates of cultured ceUs can be placed in each chamber which is equipped with a central sarnpling dish to ensure the absence of oxygen and to monitor temperature. (Reprinted from Tumiati et alm)

Figure 9: Representative photomicrographs of human venfricular cardiomyocytes following assessrnent for cellular viability using 0.4% trypan blue dye. In control non- ischemic cells (far left panel) no cellular injury is visible. In contrat, the ceils in the far right panel have undergone 90 minutes of ischemia and 30 minutes of repemision resulting in approxirnately 50% cellular injury.

Figure 10: Assessrnent of cellular injury following ischemia and repemision. At an ambient I

glucose concentration of 5 mmoIL, there was no signifiant effect of insulin I treatment. However, exposure to 10 IU/L of insulin at an ambient glucose concentration of 100 mmol/L reduced cellular inj ury with no further protection demonstrateci as the insulin concentration increased to 100 TUIL.

GLUCOSE CONCENTRATION (mmoVL)

Figure 11: Assessrnent of cellular injury foUowing ischemia and reperfusion. Higher celi injury was observed as the glucose concentration increased @y tweway ANOVA, glucose qect F ~ 6 . 4 8 , p < 0.0001). The addition of 10 IUL of insulin reduced cellular injury (insulin effect F = 24.26, p < 0.000 1). Duncan ' s mu1 tip1e range test specified differences between insulin and non-insulin treated groups at glucose concentrations of 50 and 100 rnmoVL.

GLUCOSE CONCENTRATION (mrnoV1L) CONTROL

F i 2 : qmvate dehydrogenase (PDH) artiviry fofiûwing thirty minutes of a p u r e gIucose and insulin (STABILEATION); ninety minutes of ISCHEMIA and thirty minutes of REPERFUSION. Insulin exposure increased PDH activity at both levels of glucose @or to ischemia and prevented PDH inactivation during reperfusion. (Resuits compared to non-ischemic control values obtained at baseline or from ceiis exposed to equivalent volumes of normoxic PBS for equivalent time periods-)

'p<O.OS vs W NO INSULIN O INSULIN h.0 ~ N ~ U L W

MANNITOL CONCENTRATION (mmoVL)

Figure 13: Pynivate deh ydrogenase (PDH) ac tivity following thirty minutes of exposure to mannitol (5 or 100 mmol/L) and insulin (O or 10 IUIL). Insulin resulted in sirnilar PDH stimulation at both concentrations of mannitol, indicating that the stimulatory effect of insulin is independent of glucose.

w L

s + fi nos-

Y + I a * e -; 1;1 3 a J aol - W u ,: 2 REPER FUSION + m ; z i1

Q05 - * i

GLUCOSE CONCETJTRAnON (xnmoVL) CONTROL

Figure 14: IntraceUuiar lactate accumulation following thirty minutes of stabifi7iition, ninety minutes of ischemia and thirty minutes of reperfusion. Lactate extraction during stabihation and reperfusion was highest in celis exposed to 100 m M and insulin. However, insulin reduced lactate accumulation following ischemia in =Us exposed to a glucose concentration of 100 mM. Intracellular lactate increased in a l i groups following ninety minutes of ischemia. (Results compared to non-ischernic contrd values obtaùied at baseline or fkom cells exposed to quivalent volumes of

, normoxic PBS for equivalent time wods.)

S IO0 NON-ISCHEMIC GLUCOSE CONCENTRATION (mmoVL) CONTROL

Figure 15: ExtraceUuIar lactate release into the supernatant over each plate. Lactate release increased significantly with ischemia and remained elevated during reperfusion, suggesting persistent anaerobic metabolism. Insulin treatment reduced lactate reiease at both glucose concentrations. (Results wmpared to non-ischernic control values obtained at basehe or from c d s exposed to equivalent volumes of normoxic PBS for equivalent time penods.)

2 "1 F.li 44

REPERFUSION

5 IO0 NON-ISCHEM IC GLUCOSE CONCENTUTION (mmoYL) CONTROL

Figure 16: Intraceiiular adenosine triphosphate (ATP) levels following stabihation, after ninety minutes of ischemia and a f k thirty minutes of reperfusïon. ATP fell significantly in ai l groups, but was bew preserved in cells exposai to LOO a glucose and 10 IU/L insulin. (Results wmpared to non-ischemic anitrol values obtained at baseline or h m cells exposed to equivalent volumes of normoxic PBS for equivalent time periods.)

5 100 NON-ISCHEMIC

GLUCOSE CONCENTRATION (mmoVL) CONTROL

Figure 17: htraceiïular total adenine nudeotide U A . ) levels following stabiiization, aRer ninety minutes of ischemia and afkr thirty minutes of reperfiision. TAN fell significantly in ai l groups, but was better presewed in celis exposed to high glucose and insulin. (Results compared to non-ischemic wntrol values obtained at basehe or from ceils utposed to equivalent volumes of nonoxic PBS for equivalent time periods.)

INSULIN STIMULATES PDH PHOSPHATASE BY A PROTEiN KINASE C-a DElPENDENT

70

3.1. INTRODUCTION

We have demOIIStrafed that msulin nposiue pria to ischernia riesults in human

myocardial pyrwate dehydrogenase stimulation and partially prevents the inhibition of PDH

activity foUowing ischernia and repafuson. The pmsmt Series of investigations were

designed to derennine the mechaaiSm of the stimulatory e&ct of insulin,

Several investigatm have nported tbat irisuün's intraalluiar effects are mediatcd by

protein kinase C.U1alQsl*lm*l-m Howeva, both IClipHl and Blackshea? have shown that

insulùi mediated glucose transport in skieletal muscle das not require protein kinase C

activafion. In addition, Jarett and wIIeagues demonstrated that insulin 'mediatas' h m rat

skeletal muscle have diffefent effects on insulin-sensitive metaboiic pathways in isolated

adipoc~tes.'~ T'us, the mechanisms of insulin signalliag present in adipocytes, hepatocytes

or deletal muscle are like1y very diffefent h m the paîhways which exist in human

myocardium. Most investigators have employed isolated adipocytesin.J61*11"DD or

hepatocytes151a6 to explore the mechanisms of insuiin-mediated glucose transport or PDH

stimulation. U n f m I y , the intracellular effects of insulin on cardiac muscle are poorly

described.

The foliowing studies addressed two hypotheses:

1. Insulin exposure results in protein kinase C (PKC) activation in isolated hurnan

ventricular cardiornyocytes.

2. Insulin mediated stimulation of the pynivafe dehydrogenase cornplex is dependent

upon PKC activation.

To inv- these hypotheses, we eniphyed primary cultures of human vmtricuIat

cardiomyocytes as describeci in aiaptrr two. We assesai the ability of insului to stimuhte

protein kinase C by Ob-g the branslocatiion of PKC h m the cytosol to the perinuclear

and sarcollannal membranes. We d u a t d PKC translocafion by two methods: fiwrescent

in-situ immunohistochemistry b &mamate PK- translocation h m the cytosol to

the membrane; isoform specific quantification of PKC transIOcation using dot blot anatysis

and computerized densitometry. Since PKC translocation does not necessarily nsult in an

hcrease in the kinase activity" of the T e , we deve1oped an in virro assay to measure

the ability of PKC to phosphorylate an erizymeqxcific peptide (a &due of the epidermal

growth factor receptor, RKRT3LRRL).

To detamuie the effeds of PKC modulntion on myocardial pynivate dehydrogenase

activïty, we employed the phorbol ester PMA (4i3-phorbol 12Bmyristate 13a-acetate) as a

PKC agonist. In addition, we employed PKC antagonists which act by either inhibition of

the ATP binding site (CHEL: chelaythrine) or by interference of the lipid cofkctor bïnding

site of PKC ( C U : caiphostin C).

Dichioracetate was employed to inhibit the PDH kinase subunit and sodium fluonde

used to inhibit the PDH phosphatase subunit to M e r spenfy the mechanism of insulin's

stimulatory effect.

W e believe that determination of the intracellular mechanisms of insulin's effixt on

myocardiai ppvafe dehydrogemase may allow for augmentation of the stimulatory response

and lead to impmved cellular tolemce to ischemia,

Primary cultures of human ventricuiar cardiomyocytes were estabïished as previousiy

describeci in chapter two. Confluent culaires of cardiomyocytes (approximately 600,000 alls

pex dture di&) cuitured for 5 to 10 days from the last passage were employed for these

sbdies. InnJin qeéa on mm KIlrrrse C

Immunofluarescent staining ushg a monoclonal anisaisus antibody to protein kinase

C (PKC) was employed following exposure to insulin and/or high glucose in order to obsave

the patteni of intradlular PKC distniution. Foiiowing the intervention of interest, the

cardiomyocytes were fixeci in 100% methanol for 20 minutes then washed three times in

phosphate buffered saline. The œlis wexe thai s&ed with a 1:40 dilution of rabbit a&

human anti-protein kinase C IgG antiWy (Calbiochem Laboratones, La Jolla, CA) at

37°C for 45 minutes. The antibody was then removed with three 15 minute washings of PBS

in a gentle shaker at room temperaftire. The celis were then exposed to a 1:20 dilution of

fluoresah isothiocyanate-labelled goat anti-abbit IgG antibody (ICN Biochemicais,

MisSssauga, ONT) at 3743 for 45 minutes. Each plate was then Wasfied until background

staining in a control plate with no primary antibody was barely detectable. The plates were

then viewed and photographed under ultraviolet tght using an epinuorescent microscope with

a blue filter (Kikon Canada, Mississauga, ONT) at 200x magnincaîion. Positive coatmls

were obtained a h cells were exposecl to 10 nmollL of PMA (48-phorbol 12-myristate 13-

acetate), a PKC stimuiating phorbol ester, for Uiirty minutes. This concentration of PMA

was show to stimulate PDH activity in isoiated hepatoma tells.'" PMA was first dissolved

in dimethyIsulfoXde (DMSO) prior to dilution in PM. The final concentration of DMSO

was mainmed below 0.05% which does not produœ injury in human cardi~myocytes.~

73

In orda to detect isofonn spccinc transIocation of PKC. we pedOfmed dot blot

analyses on cytosoiic and membrane ~ 0 0 s . FoUowing the intervention of intaest, ceiis

were wahed, scraped and resuspended in 50 p L of 50 mmoYL TRIS-buffered saline. Cells

w e n thai sonicatcd and centrihiged at 14 000 rpm for 5 minutes. Foiiowing rem& of the

soluble cytosoiic supenÿitant Won, the plla was resuspeaded in 50 p L of TRIS-bufked

saline to yie1d the membrane enricheci fracton. Both fractons were then divideci inta equal

aliquots of 25 p L each. Che aliquot was employed for determination of protein concentration

and then the equivaient of 20 pg of protein for each sample was placed in the dot blot

apparatus. Foliowing protein transfer to nitroceUuiose, each blot was reacted with an isoform

specific a n t i i y for PKC-E. The same blot was then pmbed with an antiibody specifïc to

PKCu (AU antibodies courtesy of PIERCE Biotechnology; RocHord, IL.). Western blot

analysis using cherniiuminesœnt M o n demonstmted that each isoform-qecific antibody

was specinc for PKC with no evidence of non-specific background staining. Slot blots were

then iclwined using a commercially available soAware program (Molecular Images;

Mississauga, ONT) and each band assessed daisitometridy. For these studies, positive

controls were obtained foiiowing exposure to 10 nmoYL of PMA (as above) and aAer thirty

minute exposure to 50 and 100 pmoyL of adenosine. Adenosine has k e n show by

Ikonomidis et al to stimulate PKC translocation at these ~onœntrations.~ In addition, cells

w a e exposed to 50 pmoYL of ademosine in the -ce of the adenosine receptor antagonist

8-@suiphopfienyl)theophyiiine (SPT. courtesy of Research Biochemicals Intemalional.

Natick, MA). SPT is a non-selective adenosine receptor antagonist and has previously been

shown to inhibit the e f fm of adenosine at a concentration of 100 pmol/L." Both adenosine

and Sm weE &y dissolved in PBS to achieve the final desired concentration.

74

Pmain kinase C activity was measmeci using a modification of a pmiousfy report&

assay." Connuent CUItures of cardiomyocytes were expwed to the breatmmt of intexest for

thirîy minutes. nie tells were thca washed, saaped and resuspended in 50 pL of 50

mmoUt TRIS-buffered saline (150 mmoYL NaQ in 50% giycaol, pH 7.2). FoUowing

scmicaîion, 10 pi, of each ceiI extract was addeti to 15 pL of mon buffet for 60 minutes.

The r d 0 1 1 b s e r amsisted of quai concentratio~ls of a lissamine rhodamine Blabelled

peptide containhg a PKC-specifïc phosphorylatim site (epidennal growth factor receptor,

RKRTLRRL), an activating solution (Phosphatidyl-L-serine, lm*) and a b t d k

containing 10 mmollL ATP, 50 mmoi/L M g Q , 0.5 mmoVL CaCl2, 0.01 % Triton X-100

and 100 mmol/L Tris-(hydroxymethy1)-amino methane, pH 7.4. (AU reagmts h m PIERCE

Biotechn010gy; Rockford, IL.)

Foliowing incubation, the &on mixture was fiactionated through a DEAE-

sepharose column equilibrated with 20 rnmoYL HEPES @H 7.9 @4"c), 20% giycerol and

lmmollL EDTA. Phosphofylated peptide bound to the positively chargeci m1umn and was

then'eluted with a 2 m o n NaCl, H E P E S - b f l i saline solution. The absorbante of the

eluted fluid was then meanired using a spectmphotorneter (Beckmann Ltd; Fullerton, CA)

at 570 nm. Reaction baer that had not been exposed to any ceii extracts was also run

t h u g h the column and the eluate sewed as a negative control. CeU extracts exposed to 10

nmoUL PMA were employed as positive controls. Non-specific absorbante was adjusted

using the negative control and the finai absorbante correcfed for protein content and

expressed in relative units of absorbance/mg protein.

75

P p w m Dchydmgenase Aahiity Followùrg PKC Morhhtion

The activity of pyruvaYe dehydrogenase (PDH) was measimd a b each intervention

of interest using the modincafions desaibtd by Robinson et a P of the method of Shai et

aim as desc r i i in chapter m.

To as~ess the mle of PKC stimulation on PDH activity, ceils w a e exposed to 10

mi/L of PMA for tbirty minutes. Bendli and colieagues bave prwioudy shown in isolateci

hepatoma ceils that PMA results in PDH activafion within t a minutes of exp~sure.~'

Cardiomyocytes were then exposed to 100 mmoUL of glucose and 10 IU/L of insulin in the

presence of the PKC inhicbitors chelayllirine (CHEL: 1 firnoVL; Calbiochem Biochemicals,

La Joiia, CA) and calphosth C (CALC: 2 0 nmoyL; Calbiochem Biochernicals, La Jolla,

CA). As descnbed in chapter one, CHEL inhibits the ATP binding site of the m e .

CaEL is isolated h m the leaves and stems of Macleaya cordatu and ~~ and has

been shown to inhibit PKC at a concentration of 500 nrnoVL.IPr CALC is ~ecovered h m

Cladospon'm ~Iodoporioides and inhibits PKC by intrrfkzhg with the lipid conldor binding

site.'" CALC was found to inbiit PMA induced cardiomyocyte preconditioning at a dose

of 200 nmoVL." We therefore employed a CEEL dose which was approximately two thes

the published effective inhibitory dose (Calbiochem) and a CALC dose which was previously

shown in our lab to inhibit the functional properties of PKC.*

An additional expairnent involved cardiornyocyte exposure to 100 mmoYL of glucose

and 10 Iü/L insulin in the presence of the PDH phosphatase inhibitoi sodium fluoride (NaR

25 rnmol/L, Sigma Chemicals). Insulin has beai s h o w to stimulate the phosphaîase subunit

of the PDH complex in isolated bwine heart mitoch~ndria.'~ Ceil extracts were also

expoBed to the PDH kinase inhibitor dichicmacetate (DCA: 5 mmoyL) in the presence of

100 mmoi/L gîucuse and 10 NL insului.

. *

The SAS Statistical Package (SAS Institue, Cary, NC) was employed for analysis of

a l i daot Raw &ta are expressed as the mean f standard aror with eight plates per group,

uniess othemk specified. Anaiysis of variance (ANOVA) was used to simultaneously

compare means betweea groups. When statisticaily signifiant ciifferences were found, they

were specifîed by DUNCAN'S muttiple range test. Statisticat signifïcance was assumed at

p<o.05.

3.3. RESULTS

Ine Effetz of Z d n on ProteUr Kinase C

Fluorescent in-süu immunohistochemistry demonshated haî thirty minute exposure

to insulin resutted in a translocation of PKC h m the cytoplasm to the membrane

(Figure 18). A more modest trandocafion was observed in c a s exposed to 100 mmoi/L

glucose &ne. Figure 19 displays a simiiar immunohistochernicai study in cells exposed to

the phorbol ester PMA (1 pnolL) and adenosine (50 pmol/L), both of which have been

shown to stimulate PKC translOcafion.m In panel D, PKC translocation is obsemd in

cardiom yocytes which have undergone ischernic preconditioning .

Figure 20 displays a representative dot blot analysis which shows an isofm specinc

translocation of PKC in c a s exposed to insulin. Densitometric analyses mealed no e f fd

on PKC-é distributions with either insulin, the phorbol ester PMA (10 nmolL), 50 or 100

p m o n of adenosine or with the non-specifïc adenosine receptor antagonist Sm. However,

there was a marked cytosolic to membrane translocation of P K C a in ceUs exposed to insulin,

PMA and adenosine. This translocation was inhibiteci with SPT. Digitalized densitometry

77

revealed the foïlowhg membrane:cytosolic ratio5 for each group: 5 mM Glucose 1.43; 5 mM

Gluase and insiiün 7.75; 100 mM giucose 5.44; 100 mM giucose and insuiin 3.%; PMA

4.93; 50 pM adenosine 6.4; 50 adenosine and 1 0 f l SPT 2.27.

Figure 21 illustrates the effwt of insulin, glucose and PMA (10 nmoYL) cm total PKC

activity. A signincant interactive &kt was fouad betweea glucose and insulin with respect

to total PKC actmty (glucaree*innJ @kt, F=5.14, p=0.035). Insulin stimiilatprf PKC

activity with a higher effax seen at an arnbient glucose conceritration of 100 mmoYL. PMA

was a potent stimulaior of PKC activity wiîh an efféct almost double h a î of high glucose and

insulùi (O.58f 0.05 vs 0.29I0.04, p<O.OS by DUNCAN'S multiple range test).

P y m e Dehydmgenase Activity F o l M g Pn,eiin Kitcase C Modulan'on

Figure 22 displays the activity of rnitochonckhî ppvate dehydrogenase (PDH)

following exposure to 10 nmoi/L PMA, 200 nmoYL CALC, 1 pmoK CHEL and 25

mmoYL NaF. Cells exposed to CALC, CHEL and NaF were also exposed to 100 rnmoVL

glucose and 10 IUL insulin. PMA stirndated PDH activity to a level si& to that of 100

mmoi/L @ucose and insuiin while C U , CHEL and NaF prevented PDH stimulation.

3.4. CONCLUSIONS

In these Sefies of experiments we have deiemllned that insulin exposure results in

translocation of PKC-o h m the cytosd to the membrane. Translocation of PKC-a resuited

in an inaease in the kinase activity of the enzyme. Pharmacologie stimiilsticm of PKC with

the phorbol ester PMA resulted in a stimulation of myocardial PDH activity to a levd simüar

to that achiwed with 100 mmol/L glucose and insulin. Cells exposed to 100 mrnoVL glucose

and insulin in the presence of the PKC inhibitors C U or CEEL did not exhibit an

increase in PDH activity. Similarly, ce& exposeû to high giucose and insulin in the presence

78

of the PDH phosphatase inhibitor sodium flwride did not display a stimulation of PDH

activity.

niese studies demonsûate that in isoiated human ventricular cardiomyocytes, insuün-

mediated stllnitlation of pynivate dehydrogeriase activity is &pendent upun protein kinase C-

a stimufiition of the phosphatase subunit of the PDH complex.

Figure 18: In-siru fluorescent imrnunohistochernistry demonstrating the distribution of protein kinase C using a 1:40 dilution of rabbit anti-human anti-protein kinase C- IgG antibody. The left panel demonstrates generalizad cytoplasmic st&g in ceils exposeû to 5 mmoUL glucose alone. Ceils exposed to 10 IU/L of insulin with 100 mmoVL glucose (right panel) display a redistribution of PKC staining to the sarw1ernna.i membranes.

Figure 19: In-situ fluorescent immunohistochernistry demonstrating the distribution of protein kinase C using a 1:40 dilution of rabbit anti-human anti-protein kinase Cc- IgG antibody. Panel A dernonstrates generalized cytoplasmic staining in cells exposed to 5 m M glucose alone. Cells exposed to the phorbol ester PMA (1 &; panel B) or adenosine (50 pM, panel C) display a redistribution of PKC to the perinuclear and sarcolemnal membrane. Ischemic preconditioning (panel D) results in a similar redistribution of PKC staining. (Reprinted fkom Ikonomidis et aizod)

10 nM PMA

50 uM Adenosine

100 uM Adenosine

50 uM Adenosine + SPT

-71IpI-'

PKC - E PKC -

Figure 20: Slot blot analysis demonstrating insulin's effect on protein kinase C (PKC) transiocation. Each lane is blotted with 20 pg of protein. The left panel shows no effect of insulin, PMA or adenosine on PKC-e distribution. The right panel demonstrates that insulin, PMA and adenosine cause a translocation of PKC-at to the membrane fraction. The adenosine mediated PKC-û! translocation was inhibited by the adenosine receptor antagonist SPT.

"p4.05 different from 5 1

Figure 21: Measurement of total protein kinase C (PKC) activity using an NI vitro phosphorylation assay. The phosphorylation of a PKC specific peptide (epidennd growth factor receptor, RKRTLRRL) is measured colorimetrically as spectrophotomemc absorbame at 570 nm and corrected for protein content. The phorbol ester PMA resulted in a significant stimulation of PKC activity compared to 5 a glucose alone. A significant interactive effect between insulin and glucose and total PKC activity was found (glucose*imulin, F-5.14, p=0.035 by rm-ANOVA). Insulin stimulated PKC activity with a greater effect observed at an arnbient glucose concentration of 100 mM.

100 PMA -INSULIN

CALC L NaF 1Oo+rNsULm

Figure 22: The activity of rnitochondnal pyruvate dehydrogenase (PDH) activity following protein kinase C (PKC) modulation. The phorbol ester PMA stimulated PDH activity to a similar extent as high glucose and insulin. Cells exposed to high glucose and insulin in the presence of the PKC antagonists calphostin C (CALC) and chelyrthnne (CHEL) demonstrateci PDH activities similar to control valws (5 rnM glucose).

CBAPTIERFOUR

ADDITIONAL INVESTIGATIONS

85

4.1. INTRODUCTION

In chapm two and k, insulin was found to stimulate ppvate dehydrogenase in

isolatecl human veatticuiar &omyOCyfeS. Although vmtricuiaf dysfiuiction is clearly due

to cardiomyocyte injury, there is inæasing &darce that endothelial cell dysfuoction

contributes signifïcantly to ischernic npxfkion injury.-* EndothW ceils produce a

potent Msoactive compound now idmtified as nitric mide." Although nitnc oxide itsdf is

a negative inotrop,- preservatian of endothelisl fimction has been show to imprwe

ventricular function f0Uomng ~ o p 1 e g i c alTest."

The effect of metabolic intervention on endothelisl celi tolerance to ischemia is

unknown, The effect of altering the aembic to anaerobic ATP ratio in nonumtractile œk,

such as endothelium, is also unknown. Ischemic injury involves a c o m p l i W interaction

between coronafy endothelial œ i l s and cardiomyocytes. Howwer, in order to distinguish

differeritial responses between these two ceii types, an isoiated cell culture mode1 may be

preferred.

For example, Shirai and coiieagues demonstrakd that preconditioning was ineffective

in protectuig endothelial cells h m ischemic injury but was extrernely protective in

cardiomyocytes."' To detennine the effects of insulin exposue on endothehi ceii

metabolism we employed isolated cultures of human saphenous vein endothelial cells. These

cells have previously been show to respund in a si* manna to coronary and aortic

endothelium.-

W e origindy hypothesized that stimiilation of myocardial PDH by insulin would

improve the fecoveq of aerobic metabolism and lead to be$ter huictional recovery fohvhg

cafdioplegic arrest, Since the isolaîed cardiomyocytes employed in these sîudie~ w m

86

quiescent, we were unabIe to perfarm hmdicmai analyses in chapters two and three. We

thdore developed a porcine model of orthoOopic cardiac ttansplantation to evaluat~, the use

of insuiin-enhanced doplegia on the recovery of myoardial fùnction foilowing four houn

of hypothermie cardiop1egic arrest.

4.2. MmHODS

lih&hew Gdl snuty

Human saphmous vein endothelid tells were cuitureû as previously describeci.-

Briefly, segments of vein were tirst fiushed wiüi PBS and then an enzymatic solution

containhg 0.2% trypmn and 0.1 96 coiiapenase. The effluent was then collected and

incubated in medium-199 (GIBCO laboratores; Grand Island, NY) containing 20% f d

bovine senun, 100 UlmL penicillin and 100 IrglmL streptomycin. Endothelid œiis were

distinpuished M m fibroôlasts and smooth muscle celis by morphologid criteria. CeiI cuiture

pwity was greater than 95% and was confirmed after the third passage by

immunofiuorescenct staining with a monocIonal antibody against faicbr eight antigen. Cells

passaged 4 to 7 times and aged less Uian 75 &YS wexe used for this study.

Endothehl c d s were subjected to a similar protocol of ischemia and repemision as

descnibed in chapter two. Foliowing thirty minutes of reperfusion, ceUs were incubated for

24 hours in semm free medium. Non-viable cells lost th& adherence to the celi culture plate

and fimted M y into the supernatant. In mtrast, viable cells were firmly adherent and

required trypsinization in order to harvest. Each fraction (supernatant and trypsinized) was

then counted in a coulter counter which measured for particles above 10 microns in size.

chapter two.

We have pmiously d e s c n i a p r c k mode1 of orthotopic transplantation to

evaiuate the effects of l m fiow pemiSicm during hypothermie ( 4 0 storage.= We employed

a simplifed perfusion apparatus using donor blood harvesîed fiom the chest at the time of

orgao procurement to amtinuously p v i d e low flow (15 mllmin) pemiSion during three

hours of &mage. To evaluate the hemodynamic ened of insolin, 10 IU/L of hum& R was

addeci to the initial cardioplegic solution, to the donor b l d pafiisate and to the blood

cardiuplegic solution.

The nperimentai protoc01 was approved by our institutional aaimal care cornmittee

and was in accordance to the Gÿide for the Eore Md use of labormory Mimalr (NM

publiCCLtiOn no 862.3, r&ed 1985). Thuty two fernale Yorkshire pigs (50-6û kg) were used

to perform sixteen orthotopic cardiac transplants employing either continuous donor blood

pemi9on (BIXH)D, n=8) or WC storage in a crystalloid preservation solution

(CONTROL, n=8). In addition, 7 transplants were perf'ed employing wntinuous

pemison with donor blood supplemented with 10 IUL of humulin R (INSUUN). The size

mismatch between donor and recipient was les than 5% of body weight in all experiments.

Donor Operation

The animals were anesthetized with intrmuscular ketamine (30 mgkg) and

isoflurane, inhibaM and venfilated with 100% oxygen to maintain normocarbia, Following

stexnotomy, the heart and great vessels wexe exposexi. Umbilical tapes were placed around

the supCnor and inferior vena cava to p d t adjusbnent of cardiac preload by cavai swing.

Systemic anticoagulation was whieved with the intravenous injection of 10 000 units of

heparin.

A Millar niic~omanorneter was insated via the apa of the left vmtricle to permit on-

line measuremats of heart rate, lefi ventricular Sysblic aad diastolic pressures as well as the

maximum positive dezivative of the systolic pressme curm

Badine measuements of ali hemodyaamic pmme&m were made at a left ventriculaf

enddiastolic pressure (LVEDP) of 10 mmtIg and after bicavai saaring to achieve an LVEDP

of 2 mmFIg. A third measurement was made release of the b i c a a mares. Donor

hearts that failed to recover a left vmtricuiar developed pressure of 80 mmHg foliowing

release of the snares were discarded,

A pursestring suture was p l d in the asceadhg aorta to permit placement of a

cardioplegic cannula. Arterial and cozonafy sinus blood samples were obtained just prior to

aoaic crossclarnping and then one litre of a hypothennic (4Q, hyperkalemic crystalloid

solution (composition in m 1 L : Na+ 127, K+ 20, M e 6, Ci- 7, SO: 6, Pis-hydroxymethyl

aminomethane 0 4, Dextrose 135) was infused into the aortic mot to achieve

cardioplegic ;urest, Following cardioplegic infusion, the donor heart was extracted, placed

in a bag containhg 300mL of hypothermie cardioplegia and stored on ice. The d o p I e g i c

carmula and aortic crossclamp was left in place to permit perfùsion during storage.

In the BLOOD and INSULIN groups, donor blood was harvested from the chest

a f k organ extraction. Following Ntration for particdate matter and the addition of 10 000

units of heparin, the blocui was sfored in sîandard transfusion bags (lhzvenol, Baxta

Healtficare Corp, D d e i d , Ill.). in the INSULIN group, both the crystalloid cardiople@

and the donor blood p h s a t e were supplemented with 10 IU/L of h u m a R. Blood

perfusion was initiatexi within ten minutes of cardioplegic amst and was delivered at m m

temperature (2m) at a vertical height of 100 cm (to correspond to a perfusion pressure of

$9

60 mmsg) employing a standard intravmous bransfusicm apparatus ( F d ,

Heaithcare Corp). Blood pafusion was mhtained thn,ughout the storage period while the

donor kart was on iœ. A myocardial probe was insertcd into the apex of the left veatricle

in aii hearts to monitor temperaairit duriag m e . Coraiary flow rate was de&mhed as

the total volume of perfbak deliverd corrcted for storage tirne in minutes.

Recipient 0ptrmanon

Pnoperative sedation and anaesthesia was similar to the donor protocol. In addition,

a marginal ear vein was used for intravenous access and kept opai with a 50 mLlhour 5%

dexttose infusion. Continuous electrocardiographic monitoring was employed and a camtid

artery line was inserted to measure arterial pressure.

Foilowing stemotomy, the kart and gnat vesseis were exposeci. Umbrlid tapes

were placed around the superior and infaior vena cava. Systemic anticoagulation was

achieved by injacting heparin into the pump prime (10 000 U) in addition to an intrawnous

dose of 10 000 U. Ascending aortic and bicaval cannulation wae used to place the recipient

on cardiopulmonary bypass. Flow rates were adjusted to maintah systemic perfusion

pressures above 50 mmHg. No vasoactive medications were a d m i n i s ~ during

cardiopulmonary bypass. Systemic -sion was maintaineci at 37C

After the aorta was crossclamped, the rtxipient hem was extracted maintaining a cuff

of nght and lefi atrium. nie left hemiazygous vein was figatexl at its insertion into the

coronary sinus. The anastomotic rnargins were then inspected and trirnmed in pfeparation for

orthotopic transpiantation.

Donor blood pemigon of the allog& was stopped and an initial 350mL blood

cardioplegic dose infùsed in all groups at a flow rate of 100 W m i n . Cardiaplegic

90

probctioa consisted of a 2: 1 mixture of b1wd:aystalloid and was Uvered at 1VC foïïowing

the completion of each anasfornosis. Foliowing completion of the puimonary artaial

anastomosis, 350 rnL of blood cardioplegia was delive& at 37°C. Artaial and coronary

sinus blocxi samples were obtained at erh cardiop1egic infusion, at the time of cn>ssckmp

r e m d and e v q fiffeen minutes during r e p h i o n . If ventricular BrilIation ocaured

during repafusion, thne attempts wae made to defibrillate the heart. if unsuccesshil, 100

mg lidocaine was delivered intraveriously and defibnuation attempted once again.

At the compktion of forty-five minutes of m o n , hemodynamic mea~urements

were obtained. Preioad was adjusted by transient venous occlusion. FoIIowing completion

of aU measufements, the recipient was weaned off bypass if possible. If indicated, Ig

calcium chloride was given and if additionai inotropic support was required an isoproterenol

drip was esGrblished (4 mg/L at 100 mUmin). Thirty minutes foilowing discontinuafion of

cardiopulrn~~liiry bypass, decannulaîion was pexfbrmed and the anima sacrificeci by

intravenous potassium chloride injection. If the animal was not successfiil in maintainhg a

mean art& pressure above 60 mmHg for thirty minutes despite inotropic support, weaning

was deemed to be unsuccessful. Ventrïculat pacing was employed if necessary to mainiain

the heart rate over 80 beats per minute.

Biochemical Memr-

A r t . and coronary sinus blood sampIes were m y e d for the pamal pressure of

oxygen @Q3, carbon dioxide @CO& pH, hemoglobin concentration (Hb) and oxygen

saturation (SaW. ûxygen content (Q,Con) was calculated from the formula. @Con = 1.39

Hb*Sa02 + 0.003* p02. Blood samples for iactate determination were mixeci with a

measured volume of 6% perchloric acid. Lactate concentration mis measured in the protein-

91

fkee qematant with a mmmexcially available assay (R@d Lacrate Stit Pack, Caîbiochem-

Behring, Ia Jolla, W.)

Starisn'COIA~&

Statistid anaïysis was p a f d using the SAS statisticat software program (SAS

Institute; Cary, NC). Categorical data were anaiyzed by chi-squared or twetailed Fier's

exact test whexe appropMte. Continwws data are exprrssed as the meankstandard M m

and were analyzed by tweway Analysis of Variance (ANOVA) evaluating the main effects

of gmzp and rime as weU as the interactive (gmup*tac). Duncan's multiple range

Endothelid cells were highly susceptible to ischernic injury compared to human

ventri& cardiomyocytes. Following ninety minutes of ischemia and overnight incubation

in sexum-free medium, oniy 15 % of the endothehl ceus at risk remained compared io over

40% of the cardiomyocytes (Figure 23). Insulin treatment impmved cardiomyocyte survivai

but did not affect endothelial ceil tolerance to ischemia. However, insulin did stimulate

endothelial ceîi PDH activity and reduced extrace1lula.r Iactate release (Figure 24).

UnfortuMtey, this effect was ody obsaved prior to ischemia with no evidence of insuiin-

medïated stimuiation occiirring aRer ischemia and repemision. In addition, endothelid 4 s

did not increase extracellular lactate release in rrsponse to ischemia. In amtrast,

cardiomyocytes reacted to ischemia 6 t h an approximate two-fold incfea~e in iactate release

(Figure 15).

92

Po& shu€y

Figure 25 cietnonstrates the mefabOIÏc &cts of umthuous low fiow âcmor bLood

m o n dwing tkee hours of hypothermic storage. Dowr blood pafusim pamitted

limited myocardial metaboliSm and despite predomhmtïy anaerobic metaboïism led to a

signifiant improvemmt in functional recovery (Figure 26). The addition of insulia to the

pemisate and to the cardioplegic soluticms d f e d in simüar h a t e and acid rdease as in

the blood group. Howwer, ininsulin treated hearts displayed improved recovery of aerobic

metaboliSm which led to a m e r recovery of left ventncuiar function (Figure 27).

4.4. DISCUSSION

These additionai shidies provide evidence that inmlin treatment can affect endothem

c d metabolism. Exposure to insulin resufted in higher PDH activity and lower lactate

producfion. However, insulin treaûnent was unable to pmtect saphenous vein endothehi

celis from ischernic injury. It is possible that 90 minutes of ischemia was too severe an insult

for adotiiefial d i s and that insulin treatment rnay be protective against a less severe insult.

In the porcine model of orthotopic transplantation insulin treatment increased

myocardial oxygen extraction d d g organ implantation and resuited in irnpmed recovery

of left ventricuiar funaion. SUrpnsingly, there was no efféct of insului treatment on lactate

or acid release. This may be due to the fdd that hypothermic storage was employed which

may have a t t e n d any me&bolic stimulation. The metabolic différences observed occurred

foilowing the completion of the pulmonary arterial anastamosis at which the a i i hearts

received normothermic blood cardiopIegia. It is possible that maintahg normotherrnic

temperatures durhg storage rnay augment the metabolic eff- of insulin.

*p<0.05 vs CONTROL

*

Figure 23: Twenty-four hour sumival foilowing ninety minutes of ischemia and reperfusion. Endothelia1 œiis (EC) displayed increased sensitivity to ischemia comparrd to cardiomyocytes (CM). Insulin treatment conferred protection to cardiomyocytes, , but not to endothehl A s .

I

PDH ACTIVITY (nmollmg) O P P g i : a h 0 0 k b

O P g m g LACTATE RELEASE (mol/@

BLOOD CONTROL -e-

Figure 25: Effm of continuous low flow donor blood perfusion during three hours of hypothermie storage. Measurements were obtained pnor to organ procurement (PRE); after the initial cardioplegic infusion (PLEG); afkr the cornpletion of the left atriai (LA), right atrial (RA) and pulmonary artenal (PA) anastomoses; after , removal of the aortic crosscIamp (XCL) and every 15 minutes during reperfiision. Hearts in the BLOOD perfused group displayed persistent anaerobic metabolism with greater lactate and acid release during cardioplegic amest. There were no diffmces d u ~ g the reperfusion period.

V

2mmHg 10mmHg LVEDP 2 m m H g 10mmHg PRE POST

Figure 26: EEect of conthuous low flow donor blood pemision during three hours of , hypothemic storage. Perfûsion of donor blood (BLOOD) led to an improved recovery of left ventricular developed pressure compared to non-pemised control hearts (CONT) .

OXYGEN EXTRACXION (mUdL) LV DEVELOPED PRESSURE (mmHg)

-FIVE

DISCUSSION

99

5.1. INTRODUCTION

CardiOvaScular disease continues to be the mosf signincant source of morbidity and

morhlity in Canada.' Although many patients with angina pedoris are appropriately

managed with medical therapy, connary artery bypasp sufgety (CABG) remains a proven and

effdve treatment for athemsc1erOfic commry artay disme- Severai large, prospective

randomized trials have demonshated the supaiority of CABG wer medical treatmmt in

patients with lefi main disease,'6 were triple vessel coronary artery disease,' uris&b1e

anginat7 and left ventncular d~sfiuiction.~

Coronary artery bypass grafting was initiatly assoaated with signifiant morbidity and

mor&lity.ls However, advances in surgical techniques and perioperative myocardial

protection have led to impved surgical outcornes despite an increasingiy high ri& patient

pop~lation.'~~~~ Unfortunately , several subgroups of patients continue to be at high risL for

perioperative complications. These include patients with poor preopaative lefi ventricular

fiiricti~n~~ and those who require urgent revaSculafizaticm for unstable angina? Further

improvements in periaperative myocardial protection are r e q d to irnpmve the resuîts of

surgery in these precariously high risk patients.

Postoperative low cardiac output syndrome has been objedvely defineci as the need

for inotropic or intraaortic balloon pump support for longer than thirty minutes in the

intensive are unit UCU) despite o p W volume and electrolyte The development

of low output syndrome results in prolonged lengths of ICU and hospital stay and is

associated with a higher pfevalence of pexioperative myocardial infanction and operative

mortdity. Interventions designed to reduœ the incidence of postaperative low cardiac output

syndrome may have a subsîantid clinid and eccmomic impact on the delivery of essential

in a recent miew of 623 patiaits who wae enroIled in prospedive cliaical triais

assessiag periopaative myocardial rnetabolism, the development of low output syndrome was

found to be associated with persistent iinaerobic metaboliSm during early repahisi~n.~

The prevaience of low output syndrome was 13% in patients whose net myocardial lactate

release after five minutes of repafuscm was greater than 0.4 mm01 per Litre of blood

compared to 5 % in patients who released les than 0.4 mrnoLn of lactate @ < 0.M). In

patients who displayed net myocardial lactate extrocrion the prwalence of low output

syndrome was d y 3%. Therefore, improving the transition h m anaerobic to aaobic

metabolism foiiowing cardioplegic arrest may prevent the development of low output

syndrome.

Several clinid trials have demonsuami a delayed recovery of normal aerobic

myocardial metabolism foUowing cardioplegic a x r e ~ t . ~ ~ ~ * ~ ' - ~ Anaerobic glycolysis

produces only 2 moles of ATP for every mol of glucose utiiized compared to 36 moles of

ATP produced by oxidative phosphorylationen In addition, Weiss and colleagues

demonstrated that aaobic ATP prpduction is preferentially utilized for contractile functio~,~

whereas ATP produceci by aaaerobic giycolysis was utitized to maintain cellular integxity and

homeostasis. Thus, fàcilitating the reawery of normal oxidative phosphorylation following

cardioplegic arrest should resuit in impmed contrade function foiiowing surgery.

The pyruvate dehydrogetlslse (PDH) cornplex regulates the conversion of ppvate to

acetyl-CoA which in turn is utüued by the Krebs' cycle. When PDH is inhibited, pynivate

is converted to lactate by the enzyme lactate dehydrogenase WH). The latter reaction is

reversible and lactate can be exûacted by the heart and converted to pyruvate. Kobayashi

101

and Nedy demonstrated thaî the actîvity of rat myocardial PDH was significantly inhibifed

foUOwiag brief ischemia a d repafusi~n.~ Inhibition of myocardial PDH leads to persismt

of iasulin exposure on the activity of myocatdial pyruvate dehydrogenase was previously

unknown. Furthermore, the effects of pre-ischemic insulin treatmmt on the posr-ischemic

activity of pynivate dehydrogenase have not previousïy been defined.

W e hypothegzed that pischemic insulin treatment wouid prevmt the pst-ischemic

inhibition of PDH and faciltate the recovery of n o d myacardial m b i c metabolism. W e

empIoyed primary cuitures of isolated human venbricuiar cardiomyocytes to aramine the

metabolic response to Smulated cardiopIegic arrest (ischemia) and repemision. In addition,

we investigaied the role of protein khw C as a mediator of insulin's intracelluiar effects on

the myocardial pyruvate dehydrogemase cornplex.

Our investigations dernonstratexi that insulin was capable of stimulating human

myocardial PDH @or to ischemia and thatpre-ischemic insulin treatment partially prevented

the pst-ischemic inhibition of PDH. Furthermore, we found that the mechanism of the

beneficial effect of insuiin involved a PKC-a dependent stimulation of the PDH phosphatase

subunit. The results of these investigations suggest that pre-ischemic insulin treatment may

fiditate the recovery of normal aerobic metabolism foiiowing cardioplegic anteSf. We

beliewe that an earlier recovery of aerobic metaboihm may lead to impved myocardiai

function. Howwer, the in vifm results ob&ined h m these investigation require M e r

cOilfirmafion in a whole mgan or in viw model.

5.2. CZUCULTURE MODEL

Riaon Vaibicularcmdq.aytes

The cardiomyocytes employed in these studies have been exfensively evaiiiatpA in

prwious reports. 1*3739a The tells used in these studies w a e passaged 2-7 tirnes and were

cuitured for up to 60 days h m the tirne of piimary dture. These domyocytes recain

many characteristics of freshly isolaîecl cells, but have distinct differedlœs. These celis

becorne quiescent following enymatic digestion and passaging. Other investigators who have

successfully cultumi aduit cardiomyocytes have reported that the ceb lose theh ability to

con-- Despite an abundant suppIy of mitocfionciria and contractile proteins, the

sarcomeres becume disrupted d u ~ g division and do not reestablish their characteristic

functional format.

The cardiomyocytes in culhue are easily differentïated from other cell types.

EndothW œlis are oval shaped (15x20 cm) and fibroblasts are spincile-shaped (4x80 cm),

cornparrd to the redangular and much iarger cardiomyocytes (40x80 pm). in addition,

endothelid cells grow pooriy in the medium used for cardiomyocytes whereas fibroblasts

have a much fdster doubling time in culture and are easily ideritified as a spindle-shaped

contaminant.

The quiescait nature of the cardiomyocytes is W y a resuït of trypsin digestion whkh

causes a breakdom of myofibrillar mganbation. These œk may simulate the

cardioplegidy arrested heart encountaed duMg cardiac surgery. The œilular

concentrations of troponin 1, troponin T and the MB isoform of creatuie kinase are similar

to that seen in viw."' The ability to pmcondition these ceUs against prolonged ischemia is

membrane is similar to that fouad in the r n y o c a r d i ~ m . ~ ~ ~ ~ The activities of supaolnde

dismutase, catahe and glutathicme pemxidase are also simüar to those found in cardiac

biopsies.= In addition, giutathione peroxidase gene expression is simiiar to that seen in

whole h e a r t ~ . * ~

Therefm, despite th& quiescent state!, we believe that these œlls are phenotypidy

cardiomyocytes and provide a unique oppmtdty to d u a t e the cellular response to ischemia

and repafusion and the effects of metabolic interventions such as insuün. However, th&

inability to contract during 'nperfusion" may affect the interpretsition of our metabolic

resuits. The NI vivo contracting hem may s e e r h m a greater lactic acïdosis and a greater

depletion of high energy phosphates than seen in this study. However, a recmt clinid

h e w demonstrated that the myocardial be ls of high energy phosphates in the region

supplied by the left anterior descendhg artery fell by only 22% following cardioplegic arrest

and ten minutes of @sion." In contrast, the experiments described in chapter two

produced a greater than 50% fall in ATP and total a c i d e nucleotides.

SUnulared nI~chemia' and "Rep&sionn

Our technique of simuiated 'ischemia" and repemiSon has beai previousiy described

in detail? W e are able to produce a d e o x y g e d PBS sohtion with a m e a s d p4 of O

mmHg. This solution has a stable pH and can be storab in seaied containers for severai

weeks without an appreciable increase in oxygen tension. Exposure of this solution to a

normoc atmosphere results in a p Q of approximately 150 mmHg within 10 minutes. Brief

104

eqosms to normOlLia oess than 15 seconds) are required to adjust pH, osmolality a to

withdraw aliquots and do not resuit in incxieased oxygai tension.

Enposing cardiomyocytes to 90 minutes of "ischieniia" resuits in significant cellular

as assesed by tfypan blue uptake?" The amamt of cellular injury followiag

minutes of ischemia and thirty minutes of repafuscm is remarkably reproduci'ble and

avemges 45% of the cells at ri& Reducing the volume of solution mer the cclls h m 10

m . to 1.5 mL resulted in an accumulation of the products of anaerobic metabolisrn and a

marked reduction in the extràceluiar pH. Therefore, this modd is Smilar to the e f f e of

global ischemia on the heart. Unfornurately, the volume overlying the ceils rernains greater

thsn the solution to which the d i s are exposed during giobal ischemia and may actuaily

represent a form of low-flow ischernia analagous to limitai cardioplegic pemision.

We befieve that our mode1 qmsents low-flow ischemicr verms simple hypoxia due

to the effects of the volume reduction. Whai œils are exposed to 90 minutes of hyparicr

with IO nrL of deoxygenated PBS, the biochemicai abnoRnatities are much less severe and

celiular injury is only 25030% cumpared to approximately 45% with 1.5 mL of anoxic

PBS.' Therefore, the volume reduction over the culture dishes resuits in a greater ischemic

insult than occurs with simple hypoxia and reoxygenafion, likely due to the higher

concentrations of glycolytic metabolites.

In previous studies, ischernic injury was distinguished h m injury due to reperfusion

by assessing trypan blue exclusion immediately following the ischemic phase of the

experimedW- lschemia was found to injur approximate1y 30-3596 of the cek at ri&

with an additional 10% injury occurring following reperfùsion for thirty minutes. In these

Senes of expeximents, we did not assess injury foliowing ischemia alone nor did we assess

las

the effects of insulia exposure dimng isctimiia Heame dernonstrated that metaboiic

stimufstjon duriag ischemia was detrhmtd to the recovery of left ventricuiar functio~t.~

Thus, we chose to adrmrilster . . insulin prier to ischemia. To assess the role of insulin in

protecting agahst nperfusion injury, we nposed œ h to 5 or 100 mmoyL of glucose with

or without insuiin (10 IUL) during reperfirsion. In addition, we evafiratprl the ability of

insuiin to protect caniiomyocytes fiom exogmous in via0 &t injury. Insullli was found

to reduœ repemision injury and provide an antioxidant defmse. However, this protection,

whiie StatiSticaUy si-canî, was minimal compareci to the protection affimiexi by

conventional antioxidants such as Trolox or ascorbic acid." Tagami and colleagues have

demons&aed that insulin partially reversed the inhibition of antioxidant enzyme activities in

aortic endotheliai a î i s from diabetic rabbitS.= Our experiments were limited to the

assessment of glucose and pyruvate metabohm in cardiomyocytes subjected to simulated

ischemia and repemision. Further investigation is required to dua ie the ability of insulin

to stimulate native antioxidant defenses.

5.3. ' INSULIN MEDIATED CARDIOPROTECTION

Redudon of Cellular I i j . y

Pre-ischemic exposure to insului resdted in protection against injury foiiowing ninety

minutes of ischemia and thirty minutes of feperfusion. In cornparison to other interventions

such as ischemic preconditioning'" or adenosine pretteatment,' the magnitude of the

prokaive effect of insulin was small. Both preconditioning and adenosine reduced cellular

injury h m approximately 45% to 25%. whereas insulin only reduœd injury to

app~oYtimately 35%.

106

The magniû.uk of the protective e&ct of ihsulin may have be!en limiteci by our modd

of low-fiow ischernia which was uscd to simuiate cardioplegic amst with iïmited pahuion.

An earlier study by Hearse in an isolated rat heart modei demonstrated that giycoIytic

stimiilatim following high gluccse expo~lire was &rhw&d and led to depressed riecovery

of fiindion foiiowing ischemia? Mannitol treatment aïme, as an osmotic wntrol, a b

fesulted in depnsd recovery compared to amtrol. Howevex, in& m e n t erocerhed

the injury in high glucose tmatexi hearts whiie insuiin crnenuaied the injury in the niannit01

treated hearts, suggesting that the injury sustained f b m high glucose was both metabolic and

osmotic in nature. Hearse's mode1 employed a single cardioplegic dose to achieve arrest and

then hearts wexe s u b j d to hypothermie global ischemùz for seventy minutes without any

cardioplegic pem>sion. In contrast, Doherty and mileagues perfonned experiments in a

simüar isolateci rat heart model with the important addition of inteminent cardïopIegic

infusions evay 15 minutes during two hours of hypothdc In Doherty's -y,

high glucose pemisates ditring doplegic arrest protected against ischernic injury and

resulted in superior recovery of function. W e believe that it is potentially detrimental to

atternpt metaboiic stimulation during low flow or global ischemia. Thus, we chose to

administer insulin prier to ischernia to prevent the irreversiible inactivation of the PDH

cornpiex during early repemision.

Several investigators have evafuated glucose-insulin so1utions in a wide vafiety of

animai rn~dels.~~~-~-~- Unforhwtey, these studies have yie1ded COIlflicting

resdts and the benefits of glucose andfor insulin in the setting of myocardial ischemia

remains controv&. Similarly, several clhicai studies evaluating the use of glucose-insulin

solutions have failed to cansistently demonstrate a protective effect.Y41-nsnx1m*1mf1~

doplegic arrest. In addition, îhese studies involved eittiea global ischemia or intermittent

CatdiopIegic pemisicm during the iscbemic paid. As Neely and Grotyohann d e m o n M ,

accumuiation of metabolic end-products such as lactate and acid can signifïcantly exacabate

ischemic injury."' Contemporary techniques of blood cafdioplegic protection offer continuous

norrnothermic or tepid ( 2 W ) penu9on combinai with the superior buffering qdities of

blood pemision comparexi to the crystdloid sofutions used by most in~estigators.~

Insulin and glucose enhanced cardioplegic solutions require a re-evaiuation with

cantemporasr techniques of blood cardiopkgk protection. In addition, the moleailar and

cellular effects of p i s c h e m i c insulin exposm nequire definition. Clarifcation of the effkct

of insulin on pst-ischemic myocardial metabolkm may af6-d an oppominity to

pharmacologically ahance and specify insului's actions while simultaneoudy preventing any

detrimental effects due to glycoIytic stimulation d u ~ g ischemia.

M ' f i e Effem of l d i n Epsure

Insulin treatment was found to d u c e extracellular lactate release, i n c ~ e a ~ e ktaîe

extraction and impmve the preservation of high energy phosphates following ischemia and

repemision. In addition. insulin was found to stimulate the activity of rnyocardial pyruvate

dehydrogenase and partiaüy prevented the inhibition of PDH activity following ischemia and

repemision. The magnitude of the effect of insulin on PDH activity appears small compared

to the large reduction in extracellular ladate release. However, the energy quivalent of

1041

oxidative phosphorylaticm of one mole of glueose would require the production of 18 moles

of laaate by glycolysis. Thus, a small increase in PDH activity can account for a large

nducticm in lactate production with quivalent eaergy production by the d.

Despite insuiin treatment, extraœUular laciate &ase remaided persistesitly el&

cornparrd to non-ischemic cuntrol cells. Similariy, high en- phosphate levels mmained

demonstrated that in conditions of zero or low flow ischemia, the accumiitation of lactate,

NADH and hydrogen d t s in signifcant myocardial damage mlated to the los of tissue

adeaine n~cleotides.~ Of interest, they found that the recweq of ventricular function

independent of the tissue adenine nucleotide levels, but was i n v d y related to the tissue

lactate content. niese findings aiso support our hypothesis that the ratio of aerobic to

Hearse's early study demonstrated that insulin aECeriuaîed the pst-ischemic depression

in left ventricular funcbion due to to p i s c h e m i c mannitol infusion.' However, insulin

aacerboted the depression in LV function when administered in cardioplegic s01utions with

high gïucose concenîrat%ms. Rearse aüributed this exacerbation to glycolytic stimulation

during global ischemia leading to an accumulation of toxic metabolites. These results have

been confïmmi by other investigat~rs.~ However, insulin treatment in the -ce of low

flow cardioplegic -sion may have beneficial hemodynamic effeds.

in a canine model, Lucchesi demonstrated a direct inotropic effect of insului

treatrnenP2 W e developed a porcine model of orthotopic transplantation ta wduate the

effm of low flow m o n during hypothennic (4C) storage? Using donor blood

109

hamsisd h m the chest at the thne of organ pmmmmnt, we found that continuous low

flow (15 &min) pafusicm d e g thrre hours of storage perrnitted Iimited myocardial

metabolism. (Figure 25) Despite predomjnantly ariaerobic me&bolism, low flow penusim

led to a signifiant impvement in f i m c t i d recmery. (Figure 26) Insului was alen addeci

to the initial cardiop1egic solution, to the donor b l d pemirrate and 0 the blood dap1egic

solution at a amtinuous concentration of 10 NIL. Insdin treatment Ynproved the recovefy

of aerobic metaboiism and led to a further recovery of left ventricular fùnction. (Figure 27)

required intraaortic M o o n pump (IABP) support foilowing coronary bypass surgery and

found an Unprovernent in cardiac fiuiction.'" Svedjehohn et al used a glucose, insulin and

giutamate solution for patients who couid not be weaned b m cardiopulm~nary bypass?

These authors identifiai sixteen patients who were unable to wean fmm bypass and gave

these patients subsequentiy required the IABP to be weaned h m bypass and al i three died

of irreversible cardiac injury. The authors concluded that administrah . .

'on of gIutamate-GI

sohtion may potentially reverse transient myocardial dysfiinction and avoid the use of

mechanical support. Unforhsnately, these studies were non-randomized and did not d u a i e

In a preliminary prospective randomized chicai trial, we evaliiatPA the effects of

insulin enhanced cardioplegia in patients undergoing isolated coronary bypass surgery." We

found that patients who received insulin enhanced cdioplegia (10 WL) displayed an

improved transition h m anaerobic to aerobic metaboiism. Cornpared to persistent lactate

110

feiease in the plaîebo p u p , patients in the ioJuliD gmup w a e found to extrad ladate

îmmxhtdy aAer crossclamp reïease. (Fm 28) The dif'fém~ces in lactate flux disQwed

a f k five minutes of repafusion; bowenr, at two hairs following crossclamp r e m d

insulin mrdiuplegia Ied to an improved rramry of left ventriah fiinction. Om previous

findings (Figure 2) also demonshrated that a transient mefaboiic derangement in the earfy

repahision peziod predicted the developmerrt of postoperative low cardiac output syndrome.

Therefare, the transient effect of insulin during early qexfbsion led to enhanced left

ventricular hction duMg a time @od at which patients are most at risk for suffeming

from Iow output syndrome. A iarper, muhicenter prosptive randomized trial is curzentiy

underway to d u a t e the ability of insulin enhanced cardioplegia to d u c e the incidence of

low cardiac output syndrome foUowing isolarPI1 coronary bypass surgexy.

Mechmbm of tire Znsuün mect

These studies demollstrated that insulin rnediated stimuiation of rnyocardial pyruvate

dehydrogenase led to improved metabolic mmvery foliowing ischernia and repfbsion. In

our cellular studies we found that this improved metaboIic status conferred protection against

cellular injury as assessed by trypan blue uptab. W e extrapokîed our cellular findings to

a porcine mode1 of prolonged hypothennic storage as weii as a clinid trial ewluating insulin

enhanaxi cardioplegia. In these in vivo studies, insulin resuited in rnetaboiic benefits which

translated into irnproved fiuictional recovery foliowing cardiopllegic arrest. UnforhinateIy,

in each study there was a delayed movefy of myocardial metaboliSm a n d h left ventricular

fwicîïon compared to pre-ischemic values. Exploiîing the mechanism of the insuiin e f f i

may allow for site-+c enhancement of the stimulatory eff- and further improvements

in both m-lic and fiinctional recovery.

Itis unclearifinsuîin m~prOteinLinaîcCadivityorifproteinkinaseC modulaus the

cellular q m s e to insulin. For example, Standaat and coiieagues demoastrated an

asSo&ion kianea insulin-mediated gbcOse tmqort ard imeased diacy1glyceroI l m l s in

BC3H-1 rnyocyte~.~~ The higha DAG 1evelS also led to activation of protein kinase C. The

sarne investigators also demonstrated a relationship between insulin-mediaîed giucose

transport and the DAGlPKC signalling pathway in rat Meta1 muscles.m However, Klip

demonstrated that insulin-mediated hexose transport in skeletal muscle was not dependent on

protein kinase C Insulin was also found to stimuiate both the N a + pimp

and the N a + antiporter by protein kinase C dependent pathways?" To M e r evaluate

the relationship between insulin and the protein kinase C Sgnalling pathway, Blackshear and

coileagues evaluated the phosphorylation of a major htraçellular substrate for PKC,

MARCKS (rnyristoylated, alanine-rich C hase s~bstrate).~ In Uiese elegant studies, the

authors employed fibrobiast ce11 lines that overexpressed normal human hsulin receptors.

These authors found that insulin activateci PKC to a minor extent in ceil lines that M y

overexpress insulin receptors, but cuncluded that this stimulatory effect yas iiiUely not of

physio1ogic importance. However, these studies did not incorporate models of hypoxia or

ischemia, Hypoxia has been shown to stimulate several insuiin-sensitive proteins such as the

GLUT4 glucose transporter.- In addition, hypoxic andlor ischemic stimuli have been

shown ta stimulate protein kinase C a~tivity.~'-

1x2

The stimiilatim of protein kinase C activity foUowing a brief ischemic insult bas bea

shown to induce aie ischemic pmcmdïtioning phenorne~~l.'- This efkct, Onginaiïy

d e s c r ï ï by Murry and cu11eagues in 1986,1m is the most powerful endogenous form of

myocardhi protection cüswvered to date. Several stimuli have been show to mimic the

preconditiothg &kt including but n d timited to a d e n ~ s i n e , ~ a-adrenergic

agents,ln- bradykini.n302 and endotbelin-1? In our cellular studies, we found that insulin

exposure resulted in a &indocation of protein kinase C-a from the cytopiasm to the

membrane (Figures 20 and 21). A similar translocaiion was observecl following a tweaty

minute ischernic preconditimhg stimulus and exposure to the phorbol ester PMA?

However, the in vitro kinase activity of PKC w a ~ found to be only miaimally stimulated

foiiowing insulin and high giucose expure. Ln contrast, both ischemic preconditioning and

PMA exposure d t e d in a substantial increase in kinase activity. Simüarly, compared to

either ischemic pfeconditioning or PMA exposure, insulin treatrnent resuited in only a minor

reduction in œUular injury. However, the ability of ischemic preconditioning to privent

myo*irdial dysfunction is q u e s t i o ~ b 1 e . ~ ~ In contrast, insulin has been demonstrated to

i m p e fiinctional recovery foliowing The differeaces may be due

to differential isofonn-specific translocation and stimulation of protein kinase C.

Banerjeets group dernonstrated in isolated rat heart that both transient ischemia and

phenylephrine treatment resuited in a translocation of PKCd and PKC-dm Interestingly,

these authors found that PKC-b branslocated to the sarcoIemnai membrane whereas PKC-r

was observed to enter the myocyte nucleü. These studies employed direct in-situ

immunofluorescent staining gmilar to our techniques employed in chapter three., however,

they were unable to detect a redistribution in the cr or B isofoms. in contras& we found that

dkmibuticm was observeci with the eiJofOrm. We cantirmed our h m u n o f l u ~ t and

dot blot analyses with an in vin0 asay to m*rcnue the kinase activity of PKC and found that

insulin d t e d in a stimulation of PKC activity. Uafortuaately, cornparrd to the results

obtained with either ischernic pmxmditiOnjII~ or PMA exposure, insulin treatment yielded

only a modest stimuiatim of PKC d v i t y . However, PMA -sure d t e d in a simiiar

stimiilstion of pyruvate dehydrogenase d v i t y as thaî observeci with high giucose and insuiin.

Therefore, it is W y that a Sngie PKC-isoform, probably PKC-CY, stimulated by both PMA

and insulin is respcmsiiIe for the stimulation of myocardial PDH. Supporthg this hypothesis

are the findings of M i e and Van Wylaiw who found that ischemic preconditioning

impmved the preservation of myocardial high energy phosphates and reduced myacardial

iacîate production. Additional isoform stimulation by PMA andfor ischemic preconditioning

may be responsible for the added protection not seen by insului hreatment alone.

Our results are in contrast to those obtained by Banerjee's group and is iikeIy due to

species-specific signalling pathways. Importantly , Banerjee's studies demonstrated that PKC

inhibition in the contml condition did not exacerbate ischemic injury. Furtheniore, PKC

translocation occurred rapidly within minutes a& the brief ischemic insuit. Thus, PKC must

reguiate a positive protective mechanism foiIowing Qther a brief ischemic or pharmacologie

stimulus and is capable of affording protection against subsequent prolonged ischemia.

The time course of the preconditioning e f f a is also important. Kloner's p u p

demonmatai in rats that repemision perïods for greater than one hour foilowing the

preconditioning stimulus abolished the protective effects. * However , a second " whdow"

of protection appears after approximately Wenty-four heurs.= DDumably, ably second

114

window e&d is a d t of altered gme ~ 0 0 in the pfec~aditioned myocardium.

Unfbrûmkiy, the chatzicteristics aad mechanisms of the sa'oad window e&d are vexy

pwriy undezstood. We f m d that insulin nposure for thirty minutes d k m û protedion

agaïmt ischemia and was also sufncieat to cause PDH and PKC stimulaiion. We did not

stimuiaiion by in& acts via a similnr mediam'am to ischemic preconditioning, it aui be

postuiated that protection against ischemia may be COIlfenied for as long as twenty four hours

afkr insulin treatment. If this if found to be true, then patients who present for

m e o n can be treated with insulin on the day @or to su~gery.

The relationship between PKC-a st imWon and PDH phosphatase stimulation

=mains w1cIear. In our studies, we found that insulin mediateci PDH stimulaîicm was

abolished by the PKC antagonists chelaythrine and calphostk C. Furthermore, exposure to

the phorbol ester PMA stimulated PDH activity to a si& extait as observeci following high

glucose and insulin treatment. Exposure to high glucose and insulin in the presence of the

PDH phosphatase inhibitor, sodium fluoride, aiso prevented PDH stimulation. T h , we

conclude that the effect of insului on myocardial PDH is dependent upon PKC mediated

stimulation of the PDH phosphatase. Lamer's group has also anived at a similar conclusion

in both bovine heart mitochondriaUD and adipose tissue.u1

The PDH phosphatase is comprised of two distinct subunits and is the most abundant

mitochondrial phosphoprotein ph~sphatase.'~ The catalytic activity is associated with the

srnalier subunit (50 kDa), while the larger subunit (97kDa) is a f1avoproki.n of unlaiown

fimction. nie phosphatase nequires magnesium and at saairating concentrations (10 mmol),

is capable of ten-fold stimulation by minomolar conœntratims of caici~rn.~ Binding studies

115

have demonstrated that the phosphatase binds to the E2 sabunit of the cornplex provided

calcium is pmmt? One caicium ion is reqwred far eech 147kDa unit of the phospm.

However, when the phosphatase binds to the E2 subunit, a second calcium binding site

becames appamt. It is betieved that the fint calcium bindirig site plays a pivotal d e as a

bridging ligand between the phosphatase and the E2 subunit while the second 'nding site

altefs the seasitivity of the the E2-p- mmpk to magnesiun, thefeby increasing

PDH phospharase activity with migomolar hcrements in caicium concentration^.^

The ability of insulin to Stimulate PDH phosphatase in permeabiihd mitochouciria

argued agahst the d e of an intennediary moiecule. However, when mitochomiria are

disnipted, the isoiated PDH phosphatase no longer remains sensitive to in~uiin.~' This

evidence, combined with the o b d o n s of a membrane bound phosphatidyl inositol glycan

menistm'56, suggests that a second messenger, k l y bond to the mitochondrial b e r

membrane is required to mediate the stimulatory e f f i of insulin on the PDH phosphatase.

W e hypothesize that PKCa stimWon resuits in the release or alteration of this

rnitochondrial membrane phospholipid which d t s in an hcrease in mitochondrial caicium

concentrations leaduig to enhanceci PDH phosphatase activity. This hypothesis would explain

why isolated mitochondria from insulin treated tissues retain stimdated PDH activity and

calcium sensitivityu' while completely extracted mitochondria do not demonstrate a sensitivity

to in~ulin.~ There is iïkely a cornpkx signalhg pathway between PKCo stimulation and

the modulation of the membrane bound phospholipid which may prevent dinct PDH

stimulation by PKC agonists in isolated mitochondria.

5.4. ADDITIONAL EFFEcTs OF INSuIm ExmmRE

The studies reported in this îksis fociucd on the efféct of insulin cm myocaiidial

pyrwate dehydmgenase. By utiliPag an iso- d dture model, we were able to m . elunuiate the effects of other cell types and organ systems. In addition, this model allows

for the careful c o ~ o l of extracellular conditions. Although useful for investigating a spedî~c

metabolic pathway, our model may not accuately represent in vbv conditions.

Insu& and Fmty AM MeÉQbOlism

Circulatingfreefattyacidslevelsa~animporiantfactorinischemiaandrepemisim.

Lopeschuk and colleauges have demonstrated that under aerobic amditions fatty aads are the

preferred substiate for energy production by the heart while during ischemia anaaobic

glycolysis pred~minates.'~ However, in an isolatPA rat heart model they found that pasistent

fany acid oxidatim during repemision inhibited glucose oxidation and depressed functicmai

fecovery. Stvnulating giucose oxidation during repafusion was found to irnpme mechanical

rrwvery. Similarly, using a camitine paimitoyhransfease 1 inhibitor to v e n t fatty acid

oxidation, these investigators demonstmed that glucose oxidation was improved leading to

enhanced recovesy of ventricular function."' In a cIinical trial in patients undergohg isolated

coronary bypass surgery, Teoh and colieagues demonstrated thaî "42 W e d pairnitate

extraction was increased during cardioplegic arrest, but that B-oxïdation of this fàtty acid was

inhibited.P Myocardial fàüy acid accumulstjon wïthout oxidation was found to be deleterious

and may reflect a derangement of normal myocardiat metabohm during repemision. Otha

agents employed to stimulate glucose oxidaîion during reperfùsion such as dichlofoacetafe

'- and molapneaio have resulted in improved functionai recovery foilowing ischemia.

Both of these agents act to stimulate mitochondrial ppvate dehydrogenase. Futha evidence

117

of suppressed gïtmse oxidation was pvided by Liedthe's group in a porcine mode1 of

regional ischemia and npafusion.' These investigators found that suppressing fatty acid

unda conditions of either physiologie or high ciratlating levels of fatty acids, glucose

oxidation during reperfusion was inhibited.

To date, th= have been no repas of direct measwements of PDH activity in

human rnyocardium subjected to ischemia and repemision. The in vivo e f f e of insulin on

hurnan rnyocardial PDH also r e d unknown. Thus, the beneficial hemodynamic effects

of insulin may by unreiated to a stimulation of PDH activity. Most investigators measure the

relative rates of 14-C lactate and tntiated water production as a surzogate assessrnent of PDH

activity?- However, Lewandowski and White perfonned direct measurements of PDH

acfivity in rabbit hearts using NMR spectrosoapy to assess "C ppvate oxidati~n.~~ These

authors fouad that dichloroacdate did not improve the function of normal hearts, but did

prevent functional impainnent in hearts that sUnered 10 minutes of global ischemia. PDH

acîinty was increased

in the hearts that received dichloroacefate and correhted directly with the recovery of pst-

Insuiin has bem show by many investigators to alter myocardial fn+ faay acid

me&bolism.- Early studies by Roger!? and McDanielm demonstrated that glucose-

insulin-potassium solutions effectively decreased circriliiting ke fatty acid Iwels and reduced

my<x#rdial fatty acid uptake by up to 75%. A recent study by Knuutim employed positron

emission tomography to evaluate myocardial giucose uptake in patients wïth coronary artay

disease. ?me investigators found that myocardial gïucose uptake was increased to a similar

118

extent by both insuiin and acipox, an antilipolytic drug which inhibits fatty acid synthesis.

However, myocardial giuawe uptake was not related to saum insulin leveis, but rather to

cirmlating fkee fâtty acid levels which were mdcdly duceci during insuiin admkkmicm.

T'us, an a d d i t i d beneficial e f h t of iasulin ûeaûnent may be to redwe free fktty

acid syathesis and b reduœ the accumulation of potentially toxic fatty acids in the

myocardium. Since our mode1 does not accomt for tfus efkct, the in MW response to msulia

treatmerit may be greater than the in vin0 response observed in these studies.

ZnnJul ami rhc Sodium-Hjdmgm Exdanger

The sodium-hydrogen antipt is a plasma membrane protein pce~ent in most ceil

types, but most notably in cardiomyocytes and renal tubular tells. During ischemia, this

protein exchanges potentiaiiy toxic hydmgen ions for sodium.m Unf-ly, this resuits

in an accumiIlation of inbracellular sodium which is removed by either the sodium-potassium

ATP dependent pump or the sodium4cium exchanger. Due to the low intradiuiar leveis

of AIT during ischemia, the sodium-potassium pump is ineffective and thus intraceuuiar

sodium is repiaced by dcium. Further increases in in trac el lu la^ calcium stores may occur

during reperfusion leading to mitochondrial swebg and ceU death, a phenornenon termesi

"The calcium p a r a d ~ x . " ~ ~ WMe the calcium paradox is not present in these studies due

to the presence of calcium in the phosphate buffered saline, the effects of caicium overlœd

due ta other pathologie mechanisms may exist.

Several investigators have suggested that inhibition of the Na-H antiport wodd reduce

the accumulation of intracellular sodium and thereby p e n t calcium-induced inju~y . -~

Several inhibitors of the Na-H+ pump inc1uding amiloride and HOE-642 have k e n show

to pmtect against both infarctm and stunning foiiowing is~hemia.-~'"

119

This membrane protan quires phospharylaticm to kanne activated. Xncerpi and

coileagues danonstrated in nit hepatocytes that insulia stimulated the Na-H antiport SlCtiYity

by a protan kinase C depndent rnecbism." Howmr, inhibiton of protein kinase C

aüemaks preamditioning and dœs not provide protection by blocking the Na-H+ a n t i m L

Furthermare, inhibition of the Na-H+ antiport pmte!cts against ischemic injury even in the

presenœ of PKC antagOIilSfS?

It appears counterintuitive thaî insulin stimulates the Na43 antiport while at the same

t h e deniRg protection against ischemia. This apparent contradiction rnay be zes01ved by

examinhg the inüaceliuiar high energy phosphate data obtained in chapter two. Insulin was

found to preserve ATP lewels follmwing ischemia It is poss'bte that insuiin provides sufficient

ATP presentation to allow for sodium-potassium exchange to occur via the ATP-dependent

pump. In this way, inûaceiiular sodium is removed without a quisite increase in calcium

concentrations. Opie's group arrived at si& mnclusions in an isolatprl rat heart

investigation? These authors found that lactate-pemised hearts displayed greater injury than

pynnrate pafused hearts despite a less severe acidosis. However, in the pyrwate perîûsed

hearts, greater ATP IeveIs were thought to aüow for adequate N a 4 exchange preventing

sodium-calcium exchange. In con-, the Na-K pump was thought to be inhibited in &aîe

pemised hearts due to low intraceiiular ATP lewels and thus inmiiuiar sodium was

exchanged for calcium, leading to ceii injury.

Sampson and coileagues also reported a d i n a stimulatnry effect of insulin on the Na-

K pump in skeletal m~scle.~' Of interest, this stimltlltory effect wwas also mediated by

protein kinase C. Thus, insulin may act to increase H+ efflux by stimulation of the Na-H+

antiport while at the same time preventing sodium (and calcium) accumulation by stirnWon

120

of the Ni& purnp. In these saies of e q e h m , we did net directy measme hydrogm

enlux but did notice a protective e 8 4 of insulti agaiiist extraœUuïar acidosis. Direct

mawnments of Na43 activity a d o r sodium-po-um uchange during ischemia and

repafusion would provide important dditioiial infbmmion about the intracelluiar e f f i of

insulin.

53. -c EFFECIS OFlNsuIm

Although insulin receptars are present on alrnost aU cell s d à œ s , différent organs and

tissues may have different respcmses to hormonal exposure. In partic&, the metabolic

response of endothelhl ceiis may be rnarlϟly different from that observed with ventncuiar

cardiomyocytes. For example, Piper's grwp demonstrated that coronary endothehi tells

rely predominantly on anaerobic glycolysis for energy producfion in anitnast to culturied rat

cardiomyocytes which continue to oxidize fatty acids.- Of note, endotheiial celi lactate

production remained constant even as the atmosphezic pO, dropped h m 100 mmHg to 3

mmHg." M o w 3 mmHg, iactate production increaseû invefsely as the pO, dropped to O. 1

m g . This 'Pasteur effect' is markedly diminished compareû to UI vivo cardiomyocytes

which begin to produœ large amounts of lactate at much higher oxygen tensions. Piper

suggests that coronary endothelial cells dispiay the 'Crabtree" effect. This phenornenon,

originaUy described in 1929 in tumour cells, refers to persistent anaerobic glycolysis despite

high oxygen ten~ions.~ Due to th& anatomic lucation, coroaary endothelial ceiis are W y

not affecfed by endogenous ïactate production which is released into the circukion uniike

cardiomyocytes which are exposed to the lactate and hydrogen ions which they p d u c e .

Since conniary endothW ceîi metabolism is highly resistant to the ambient oxygen tension,

it is unclear if a metaboiic intervention such as insulin would provide benefit.

121

Although vmtricular dysfiuicticm is ciearly due to cardiomyocyte injury, thae is

incieaSiLlg evibce that endothelia1 ceil dysfuncticm contributes signifïcantly to ischemic

npafusim injury- Endothelhl alls produce a potait vasoaceive compound now

idmtified as nitric oxide? Although nittic oxide itself is a negative isotrope,-

preservation of endotheIial fwiction has ken shown to imprwe veatricuiar function foilowing

Cardioplegic aIrest,"

Scharer and coUeagues demonstrated in hurnans that the vasculâr &kds of insulin

are mediated by nitric oxide release. If insuiin exposure can increase aidotheliaî QU nitric

oxi& production, it is possible that insulin may protect endothelid ds against ischemia.

To invdgate this hypothesis we perfôrmed additional expaiments employing primary

cultiaes of human saphenous vein endothelial ds.- Endothefial ceLls were found to be

highly susceptible to ischemic injury compared to hwnan ventricular cardiornyocytes.

FoIiowing ninety minutes of ischernia and ovemight incubation in serum-free medium, oniy

15% of the endothehl ceUs at ri& remaineci cornparrd to over 40% of the cardiomyocytes

(Figure 23). Insulin treatment improved cardiomyocyte survival but did not affect endothelial

ceil tolerance to ischernia, Howeva, insuiin did stimulate endothelial ceïi PDH activity and

reduced extraduiar lactate re1ease (Figure 24). It is therrfore possible that in& may

pmtect endothelial cells against a less severe ischemic insult.

5.6. RJMMARY OF INVESi'IGAATIONS AND ORIGINAL CONTRIBUTIONS

These investigations evalUiltPA the rnetabolic response of isolated human ventriadar

cardiomyocytes to simulated ischemia and w o n . Insuiin treatmmt was found to . stimulate aerobic metabiism and protect cardiomyocytes against ischemic injury. The nsuIts

of these investigations have yidded the follohg original contributions to the literature:

l22

Human myacardial pyruvate dehydrogaiarre is inhr'bited following simutnteA isc-

and repemisi011.

Insulin exposure results in a sîimWoa of human myocardial pynniate

dehydrog-.

The stimcilaiDly dfEa of insulin is mediated by increased PDH phosphatase activity

and is profeia kinase C s riepaiQent,

The stimiilatory efféct of insulin is not depaideat on ambient glucose concentrations.

FUTURE AREAS OF RESEARCH

These studies provide a rnetabolic ratide for administering insulin prkx to an

anticipated ischemic insult. Cardiopllegc amst is a unique situation in which ischemia cm

be anticipated and interventions such as insulin can be employed to irnprove myocardial

metabolic and fiinctional recovery. 'Ibo cluiical trials are cumntly evaluating the ability of

insului enhancexi cardioplegia to stimulate aerobic metabolism and impme venbricuiar

function following cardioplegic arrest. In the fint trial, insulin cardioplegia was found to

stimulate ktate extraction following aortic crossclamp removai. This impmved m v e r y of

aerobic metaboihm was fond to pnserve lefi ventncular fiuiction a k two hours of

repafusion (Figure 28). The second trial is a larger, multiceriter trial designed to assess the

ability of insulin cardioplegia to reduœ the incidence of perïoperative myocardial i.nfiu&on

and postopefative low output syndrome. The results of this trial will be available in

approxirnately two years.

Stimulation of aembic metabolism by other pharmacologie agents, such as

dicblatodcetafe, has been found to irnprove myocardial hinctional recovery following

is~hemia.'~~~-"'~ Dichloroacetate has not been e v a l d clinically as a cardiaplegic

123

additive. A d d i t i d clinical trials are requireû to as ses^ the beaefit of dichlmmcetak with

or without insulin.

Pafusing cardiac aiiograftp d m stmage with donor blood erihanced with insulia

was found to improve f u n c t i d t i ~ c o v q h11owing OZttlotopic transplantation. The

hemodynamic &ectS of insulin may be ben- in the &y pst-txansplant period if one

requires a positive inotmpe which a h red- systedc niscular r e s i s t a n ~ e . ' ~ ~ ~ ~ Further

investigations are required to evaluak the clinid baielits of insulin treatment either before,

duriag or affa cardiac transplantation.

The e f f e of insulin were demOIlStrated to be mediateci by PDH phosphatase

stimufation via a PKC-a dependent pathway. New phamiacologic agents designed

specificaily to enhance and augment this response may prove to be beneficial as dopllegic

additives. The additional molecuiar and biochemical eff- of insulin enposure, such as

sodium-hydrogen exhange and sodium-potassium exchange, require m e r investigation.

These investigations have provided additional evidence üiat stimulation of aerobic

metabolism following ischemia protects against cellular injury and may enhance functional

recovery. Additional methods of resuscitaîing the ischemic myocardium are required as the

proportion of high risk patients presenting for comnary bypass surgery inaeases.

Furthemore, enhancing myocardial tolerance to ischemia may aUow for an expanded use of

donor organs which are currently deerned unsuitable for ûansphntation. Cardiovasaikr

disease remains the predominant cause of m d t y in North Amedca. Coronary bypass

s u r g q is an important intervention for the treatment of atherosc1erotic heart disease. Novel

techniques of myocardial protection are required to reduce the risks of surgery in an

increasingly high risk patient population.

-1.2 I

PRE XCL XCL ON OFF

LVEDP (mmng) :

Figure 28: Upper Panel: Myocardial lactate flux during and after cardioplegic mest. Patients 1

who received insulin enhanced cardioplegia displayed lactate extraction , immediately afkr crossclamp removal compared to persistent lactate! release in the placebo group. Lower Panel: Left ventricular function was better preserved in the insulin cardioplegia group after two hours of reperfusion.

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Appnâix 1: Extraccllulu Lactate and Ppvete Muclion

STABJWZATION Lacdnte (inoUmin/g)

&mate (moUnrln/g) Laclole/4)mwte Rudo

sxhocell&r pH PDH adv& (lvllourng)

ISCHEMIA hcdate (nwUmIn/g)

&mute (moUmtnlg) t O c ~ e / 9 ( ~ ~ u u i l ~ Ratio

&tmceUulor pH P M adv@ (moumg) +0.62 (.04)

REPBRFUSION tacdPh (nioVmldg) + 1.3 (0.4)

&m~ate (moUmln/g) +41 (6) & c ~ e / ~ m v o t e Ratio + 32 (5)

ajetmceluar pH 7.24 (0.04) PDH activity (nmol/mg) 1 + 1.01 (0.11)

Mean (SEM)

1.8 (0.2) 30 (6) 63 (6)

7.19 (0.05) m.96 (O. 13)

e1.2 (0.2) m 9 (2)

65 (9) 7.21 (0.04) + @+1.26 (O. 12)

CONTROL

0.4 (O. 1) 61 (5) 9 (1)

7.27 (0.03)

ontm ml plab r<sas#l atbudine (slcrbl&t&n)or iTta W ( i ~ ~ a ~ m t a ) or 150 (mperf'on) minutes of incubation in nonnoxic phoephate butked dine.

- - Oh^^^^ , P P P P r N N N N P O V w w w w w

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