The Role and Cellular Mechanism of Protein Kinase C in Ischemic Preconditioning of Porcine

Skeletal Muscle Against Infarction

Richard Alan Hopper

A thesis submitted in conformity with the requirements for the degree of Master's of Science

Graduate Department of The Institute of Medical Science University of Toronto

a Copynght by Richard Alan Hopper 1997

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The Role and Cellular Mechanism of Protein firiase C in lschemic Preconditioning of Porcine Skeletal Muscle Against Infarction

Master of Science 1997 Richard Alan Hopper

Ins ti tu te of Medical Science University of Toronto

ABSTRACT

The objective of this thesis was to investigate the role and cellular mechanism of protein

kinase C (PKC) in ischemic preconditioning (IPC) of skeletal muscle against infarction.

using the pig latissirnus dorsi (LD) muscle flap model. It was observed that the muscle

sustained 43 2 5% infarction when subjected to 4 h of global ischemia and 24 h of

reperfusion. PC wiih three cycles of 10 min ischemia / reperfusion reduced the infarction

to 25 2 3% (pd.05). This anti-infarction effect of IPC was mimicked by PKC activators

and blocked by PKC inhibitors (pcû.05) adrninistered pnor to the sustained ischemia. In

addition, the anti-infarction effect of PKC activators was blocked by an ATP-sensitive

K+ (Km) channel antagonist (pc0.05). Lmmunoblot analysis of muscle biopsies

demonstrated presence of nine PKC isoforms, with ~ P K C E alone undergoing cytosol to

membrane translocation dunng isc hrmia following IPC. Funhennore. P C and

preischemic, but not postischemic. PKC activation reduced ( ~ ~ 0 . 0 5 ) muscle

myeloperoxidase activity during reperfusion compared to ischcmic controls. Taken

together, these observations indicate that PKC plays a central role in the anti-infarction

effect of IPC. likely through a PKC-K,,.,,, channel-linked signal tranduction pathway.

Acknowledgernents

Completion of diis Masters thesis would not have been possible without the

contributions of a number of individuals and organizations. Foremost. 1 would like to

thank my supervisors. Dr. Chnstopher Fo'orrest and Dr. Cho Pang. Their countless hours

of proof-reading, instruction. and support taught me the basic skills required before

considering an academic career. More important. however. their exemplary professional

character and conduct introduced me to the personal qudities necessary to excel in this

field. 1 would like to thank Dr. Anguo Zhong and Dr. We He for their expertise and

assistance dunng the animal surgery, Ms. Huai Xu for her technical work with the

myeloperoxidase assay, Ms. Shemlyn Hubbard for her introduction to the technique of

western blotting, and Dr. Jim Rutka for his unconditional welcome to the facilities and

hospitality of the Brain Tumour Research Laboratory. Without the vision and efforts of

Dr. Peter Neligan, Chair of the Division of Plastic Surgery, and Dr. Ralph Manktelow.

past Chair, the supponive research environment available to plastic surgery residents in

Toronto would not exist. 1 also wish to acknowledge the Hospital for Sick Children

Reseach Training Committee (RESTRACOM) for theii- financial support, as well as the

Medical Resèarch Council of Canada (MRC) for operating gant MT 1248 and a p s t -

doctoral fellowship. Finally, for their support. encouragement and understanding

throughout rny academic training, 1 can only continue to thank humbly and inadequately

my wife, Genilyn. and my parents.

... I I I

Table of Contents

Page

Introduction ................................................................................................. 1

3 Overview of the Presen t Research Projeci ......................................................... - The Clinical Problem of Skrletal Muscle Ischemic Injury in Surgery ............. 3

Pathogenesis of Skeletal Muscle Ischemic Injury ...................................... 4 . . .

1 .3.1 Isc hemc injury ....................................................................................... 5

1.3.2 Ischernia-induced reprfusion injury ....................................................... 7

........ 1.3.3 Potential methods of minimizing skeletai muscle ischemic injury 10 . . . ......................................................................... isc hernic Preconditioning ( PC) 12

1.4.1 Mec hanism of ischemic preconditioning ................................................. 13

1 .4.2 W indows of ischemic preconditioning ................................................ 19

1.4.3 Ischemic preconditioning of skeletal muscle and the potential

.......................... ............. role of PKC ......................................... 30

Protein Kinase C (PKC) ..................... .. ........................................................ 21

1 .5.1 Historical review of piotein kinase C .................. .... ................. 21 ............................. .......................*.............. 1.5.2 Protein Kinase C isoforms ... 22

1.5.3 Translocation and activation of protein kinase C isoforms .................... 23

1 S.4 Protein kinase C inhibitors and activators ............................................... 25

1 .5.5 Measurement and detection of protein kinase C ..................................... 78 . .

Objective ............ .... .................................................................................... 30

Hypothesis ....................................................................................................... 30

.................................................................................................... Specific Aims - 30

............................................................................ 2.0 Materials and Methods 32

......................................................................................... 2.1 Exprimentai Animal 33

....... ...............................*................................. 3.2 AnirnalCare ................ .. ,.. 33

Page

2.3 OperativeProcedure ......................................................................................... 34

.............. 2.3.1 Justification for the use of the porcine LD muscle flap model 34

............................................................................... 2.3.2 Anesthetic technique 35

............................................................... 2.3 -3 Creation of the LD muscle flap 36

2.3.4 Global ischernia of the LD muscle tlap ................................................. 37

.......................................................................................... 2 - 3 3 Drug delivery 39

2.3.6 Muscle sampling ..................................................................................... 40

.............................................................................. 2.4 Muscle Analysis ............ .. 41 . . .

2.4.1 Assessrnent of muscle viability ............................................................. 41

7-42 Radioactive microsphere technique for assessrnent of muscle

........................................................................................... blood tlow 42

3-43 Myeloperoxidase (MPO) assay ....................... .. ................................... 44

2.4.4 Protein kinase C translocation detection ............................................... 45

2.5 Biochemicals .......................................................................... .-.. ...................... 48

2.6 ExperimentalProtocols ..................................................................................... 49

Aim 1: To investigate the effect of PKC inhibitors on the muscle

infarct protective effect of IPC and adenosine in LD

muscle flaps subjected to 4 h ischrmia and 24 h reperfusion ................ 49

Aim 11: To investigaie the effect of IPC. adenosine. a PKC

activator and a PKC inhibitor on muscle blood flow in

......................................................... LD muscle tlaps ..................... .... 50

Aim III: To idcntify PKC isoforms in pig LD muscle and to

investigate the effect of IPC on intracellular distribution of

PKC in pip LD muscle at various time-points during sustained

................................................................................................ ischemia 50

Page

7.6.4 Aim IV: To invcstigate the effect of a Km channel blocker on the

muscle anti-infarct effect of PC and PKC activator treatment ......-....,... 53

7.6.5 Aim V: To invrstigate the effect of LPC and PKC activator

treatment on neutrophil activity in LD muscle subjected to 3 h

of ischemia and 16 h of reperfusion ........................................................ 53

2.7 StatisticalAnalysis ..................... .. .................................................................. 55

3.0 Results ......................................................................................................... 56

3.1 Effect of PKC inhibitors on the infarct protective effect of IPC and

adenosine in pig LD muscle ................................ .. ............................................. 57

3.2 Effect of IPC. adenosine. PKC activator and PKC inhibitor on

muscle blood flow in LD muscle ....................................................................... 57

3.3 PKC isoforrns and the effect of IPC on PKC isoform distribution

in cytosol and membrane fractions of pig skeletal muscle

dwing sustained ischemia .................................................................................. 60

3.3.1 Fractionation of pig skeletal muscle ........................................................ 60

3.3.2 Identification of PKC isoforms in pig skeletal muscle ........................... 60

3.3.3 Effect of IPC on cytosol and membrane distribution of protein

kinase C ....................................... ,,.. ............................................................ 63

3.4 Effect of K, channel blockade on the anti-infarct effect of IPC

and PKC ac tivator* treatrnents. .................... .,. ................................................... 69

3.5 Effect of IPC and PKC activator treatment on neutropliil activity

in pig LD muscle flaps subjccted to 4 h of global ischemia and 16 h of

reperfusion .................................................................................. ..... .......... 7 1

Page

4.0 Discussion .................................................................................................. 73

4.1 Important New Findings in this Project ............................................................ 74

4.2 Central Role of PKC in IPC of SkeletaI Muscle against Infarction ................ 74

4.2.1 In vivo phmacologic studies ................................................................. 74

4.2.2 Protein kinase C translocation ............................................................... 78

4.2.3 Protein kinase C-KAp channel-linked pathway ..................................... 82

5.0 Summary and Perspectives .................................................................... 90

5.1 Summary ........................................................................................................... 91

5.2 Perspectives ....................................................................................................... 91

..................................................................................................... 6.0 References 93

7.0 Appendices ................................................................................................... 1 27

List of Tables

Table 1.

Page

Effect of IPC, adenosine, PKC activator, and PKC inhibitor on

muscle blood flow and systemic blood pressure .................................. 59

Table II. C h a n g in membrane fraction imrnunoblot band density during

ischernia in IPC muscle cornpared with time-matched paired

isc hemic control ..................................................................................... 65

List of Figures

Page

......... ................... Figure 1. Proposed pathophysiology of ischernic injury .... 6

- * . . Figure 2. Proposed pathophysiology of repertusion injury ................................... 9

Figure 3. The proposed role of protein kinase C in the mechanism of - . .

acute isc hemic precondi~oning.. .......................................................... 1 7

Figure 4. Schematic representation of the prim.uy structure of

conventional, novel. and atypical PKC isofoms ................................ 24

Figure 5. Local intraarteriai infusion technique for the denervated pig

island latissimus dorsi muscle flap .......................................................... 38

Figure 6. Effect of PKC inhibitors on the anti-infarction phenomenon

................................................ of IPC and adenosine .............................. 58

Figure 7. Confirmation of fractionation by western blot analysis for the

membrane protein Ca2+~TPase .............................................................. 6 1

Figure 8. Screening for nine PKC isoforms in membrane ( M ) and cytosol

(C)fractions of preconditionined pig skeletal muscle subjected to

........... 15 min of global ischemia .. ................................................. 67

Figure 9. The effect of direct PKC activation on the intracelluhr

......................................................... distribution of three PKC isoforrns 64

Page

Figure 10. PC-induced changes in ~ P K C E band density in membrane

and cytosol fractions during global ischemia

relative to paired control ......................................................................... 66

Figure 1 1 . Representative ~ P K C E immunoblots of membrane and cytosol

fractions from three separate PC-treated muscle flaps during global

ischemia ................................................................................................ 68

Figure 12. Effect of PKC activators on muscle infarction in the absence

or presence of a Km channel blocker ..................................................... 70

Figure 13. Neutrophilic rnyeloperoxidase (MPO) activity in pst-ischanic

....................................................................................... skeletal muscle 72

List of Appendices

Page

Appendix 1. Representative templates of ietrazolium-stained muscle sections

from a preconditioned and an ischemic control pig latissimus

dorsi muscle flap rxposrd to 4 hours of ischemia and 24 hours of

reperfusion ........ .... .................................................................... 1 28

Appendix U. Sarnple calculation of LD muscle infarct size using the paper

............ ............... template technique .. .. 129

Appendix III. Sample caIcuIation of change in band density in a preconditioned

.......................... muscle tlap relative to a paired ischemic control flap 130

Ab breviations

ANOVA

ATP

OC

Ca2+

cDNA

Che1

cm

Co

d

DAC

DPCPX

DMSO

G

E

x g

Glib

h

5-HD

IM

IP3

IPC

IV

analysis of variance

adenosine triphosphate

degree Celsius

calcium

cornplernentary

deoxyribonucleic acid

chelerythrine chloride

centimeter

CO bal t

days

diacylglycerol

8-cyclopentyl- 1.3-

dipropy lxanthine

dimethyl Sulfoxide

gauge

gr-

times gravitational force

glibenclarnide

hoiirs

sodium 5-hydroxy-

decanoate

in tramusculai-

inosi tol triphosphate

isc hemic preconditioning

intravenous

K.\-, channel ATP-sensitive potassium

KC

LD

mg

min

mL

MPO

mRNA

OAG

O.D.

PKC

PKM

PLC

PMA

SD

SEM

sec

wt

w /v

channel

ki logram

latissimus dorsi

rnilligram

minutes

millilitre

myeloproxidase

messenger ribonucleic

acid

1 -01eoyl-2-acetyl glycerol

optical density

protein kinase C

protein kinase M

phospholipse C

phorbol 12-myristate 1 3-

acetate

standard deviation

standard error of the mean

seconds

weight

weight per volume

1.0 Introduction

1.1 Overview of the Present Research Project

" Blood. ..appears to curry life to eveq part of the bu&, for whenever the whole or li pan

is deprived of fresh blood it v e y soon dies" - John Hunter. circa. 1780 '

Deprivation of blood, or ischemia, has long been recognized to have rapid and

serious consequences to living tissue; however the exact mechanism by which ischemia

exerrs its deleterious effects remains unknown. lnvestigating the mechanism of isc hemic

injwy in celis or a specific tissue or organ not only serves to increase Our understanding

of the underlying pathophysioiogy,

therapeutic agents for the prevention

the clinical situation.

but most likely will lead to the identification of

and/or ueatmen t of ischemic/reperfusion inj ury in

Ischemic preconditioning (IPC) is a phenomenon which was first demonstrated to

protect dog cardiac muscle against infarction resulting from ischemia '. but the

mechanism remains unclear. The anti-infarction effect of iPC has also been demonstrated

in pig skeletal muscle 3. in cardiac muscle. there is convincing evidence that adenosine.

mediated by the adenosine A, receptor. is the initiator of this phenomenon in a nurnber

of animal models '"7. with exception of the rat '. More recently. it has also brrn

demonstrated that adenosine A, receptors are also involved in [PC of pig skeletal muscle'.

However the post-adenosine receptor signal transduction pathway and the final effector

rnechanism of IPC remain controversial issues in the literature. This thesis is concerned

wi th elucidating the pst-receptor signal transduction pathway of isc hemic

preconditioning of

3

skeletal muscle against infarction with special emphasis on the role

and cellular rnechanism of the enzyme protein kinase C (PKC). Accordingly, this

introduction first describes the current clinical problem and pathogenesis of skeletal

muscle ischemic injury in surgery. In the context of the various rnethods described to

treat skeletai muscle ischemic injury, the potential role of ischemic preconditioning (WC)

in clinical practice will be discussed. as well as the possible involvement of PKC in the

proposed rnechanism of this phenornenon. The introduction will conclude with a

delineation of the objective. hypothesis, and specific a ims of this thesis.

1.2 The Clinical Problem of Skeletai Muscle Ischemic Injury in Surgery

In vascular and musculoskeletal reconstructive surgery, skeletal muscles are

frequently subjected to wann global ischernia. Cornrnon examples include the transfer of

autogenous muscle for wound coverage or restoration of function. prolonged tourniquet

or vascular clamp application. pst-uaumatic cornpartment syndrome, replantation of

amputated limbs. and pst-operative arterial thrombosis. Although human skeletai

muscles can tolerate warm global ischemia for up to 2.5 hours with minimal risk of

irreversible ischemic injury. prolonged ischemia, due ro unpredictable operative or post-

operative complications. can occasionally occur in these clinical situations. This

excessive ischemic insult can cause extensive muscle infarction. resulting in severe

rnorbidity, additional operations, and increased medical care costsl"-'".

In the specific context of transplantation of autogenous skeletal muscle flaps

requiring microvascular anastomosis (i.e. free flap surgery). the period of obligatory

4

warm intra-operative ischemia lasts frorn 1.5 to 3 hours depending on the complrxity of

the case 15. Quoted success rates for these procedures range from 8597% "-"-16-'7 . It must

be noted, however, that these reprted success rates largely arise €rom a relatively small

number of large acadernic centres around the world, employing the recognized "experts"

in the field: thus the reported high success rates in reconstnictive microsur_gery are

16 associated with advanced surgical experience. technical prowess. and facilities . In

addition, overall success rates can be misleading. Variables such as traumatic rtiology

of injury. choice of flap design, and presence of vein graft are associated with subgroups

at high risk of free flap failure 11*12. When it occurs. muscle flap failure can be

devastating. In a recent study, 22% of traumatically injured lower exuemities with a

failed free flap reconstruction went on to amputation. compared to 0% in the cases when

the flap was successful ". Muscle flap Mure not only increases patient morbidity. and

decreases the options available to treat the patients condition, it also represents an

average econornic Ioss to the health care system of at least $30.000 per case ".

1.3 Pathogenesis of S keletal Muscle Ischemic Injury

Skeletal muscle may be subjected to two different types of ischernic injury: disrd

isc*hrrnin places only a portion of the muscle at risk, such as during the raising of an

autogenous muscle flap that is too large for its inherent blood supply; whereas global

isc-kmia places the entire muscle at nsk. occumng ~econdary to extra- or intra-Iurnhal

obstruction of the artenal or venous supply of the muscle as a resuit of complications ".

Clinically, distal ischemic injury is observed infrequently due to an incseasrd

5

understanding of the underlying vascular anatomy of muscles. w hich lias allowed the

design of reliable skeletal muscle flaps with adequate distal b l d supply. Currently, what

is most relevant to the current and future practice of rrconstructive and vascular surgeons

is an understanding of the pathogenesis of global ischemic injury, which is the focus of

this thesis.

The exact pathogenesis of skeletal muscle infarction resulting from global

ischemia is unclear. The current theory is that muscle injury occurs both during the

penod of sustained ischemia. as well as during the subsequent repzrfusion of the

muscle1g. hence the overall term "ischernia-reperfusion injury". The pathophysiology of

these two phaszs of injury are believed to be different. although they may be inter-

related.

1.3.1 Ischemic injury

The term ischemia arises from the Greek tems "isch6" (to keep back), and

"hairna" (blood) "'. It represents an irnbalance between tissue supply of and demand for

oxygenated blood tlow, resulting in a conversion from aerobic to anaerobic cellular

metabolisrn as indicated in Figure 1 21. The less efficient means of enegy production in

aerobic metabolism resul ts in a depletion of cellular high energy phosphate stores, suc h

as adenosine-5'-triphosphate (ATP), leading to impaired synthetic and homeostatic

capabilites. The resulting oxygen debt and cellular energy depletion is compounded by

the cellular accumulation of metabolic by-products such as lactate and protons ".

Accumulation of these metabolites is believed to act as an osmotic load. causing cell

1 ISCHEMIA 1 ; Arterial 4 Venous

Inflow Outflow

7 402 f Hypoxanthine 4 ATP +Xanthine Oxidase &

f Additional Injury on Reperfusion (see fig.2)

v Homeostatic Capabilities

f Metabolic By-products

f Osmotic Load

4 Membrane lntegrity t Cell Swelling t Structural Damage f Edema

IRREVERSIBLE CELL INJURY

t Na+/Ca2+ & ? Na+/H+

Exchange

v

Figure 1. Proposed pathophysiology of ischemic injury. Ischemia results in an imbalance between tissue supply of and demand for oxvgenated blood tlow, causing both a deficiency in energy production, and an accumulation of toxic metabolic b y-products. Wi th prolonged ischemia, structural in tegritv of the ce11 is compromised, resulting in irreverçible ceIl injury (see text for dktails). ([H-1, : intracellular hydrogen ion concentration; [Ca'+Ii : intracellular calcium ion concentration

7

swelling and damage to the sarcolemma and cytoskeletal membrane. The increase in

intraceliular acidification is also believed to augment Na' / H' and consequently Na' /

Ca" exchange. resulting in CaL' overload, rnitochondrial membrane dysfunction, and

irreversible damage U-26. Secondary autolysis follows. characterized by lysosomal

swelling. endoplasmic reticulum dilatation and vesiculation, leakage of enzymes and

proteins. and loss of cellular compartmentalization. Membrane integrity cano t be

rnaintained. and the ce11 dies 17. Studies on dog and pig skeletal muscle ischemic models

are consistent with this current understanding of ischemic injury pathophysiology by

demonstrating ischemia-associated hiph-energy phosphate depletion, lactate and proton

accumulation. and increased osmotic load '? However. the exact cellular mec hanism by

which skeletal muscle sustains damage from an ischemic insult is not known.

1.3.2 Ischemia-induced reperfusion injury

Re-establishing blood flow to ischemic tissue is a prerequisite for stopping further

isc hemic injury; i t restores energy production. and removes accumulated toxic

metabolites. The paradox of ischemia-reperfusion injury is that this reperfusion of

oxygenated blood to previously ischemic muscle results in tissue damage in excess of that

sustained during ischemia. Parks and Granger 29 demonstrated in a cat intestine mode1

that 3 hours of ischernia and 1 hour of reperfusion resulted in more severe injury than 4

hours of ischrmia alone. This phenornenon of lethal injury to healthy viable cells in the

pen-reperfusion pe~iod must be distinguished from the accelerated necrosis of cells that

were already irreversibly injured at the onset of reperfusion by the preceding ischemic

8

insult. It has k e n observed that the extent of reperfusion injury is directiy related to die

duration of ischemia time i"-'2. as well as to the oxygen content of the blood during

reperfusion ")'"5 Reperfusion inj ury to the microvasculature and myoc ytes of skeletal

muscle has also been documented in rats "O. rabbits '", dogs30-4LJ3. and pigs 31.u-45.

however the exact pathognesis remains unclear.

The current consensus is that reperfusion injury is mediated by the production of

reactive oxygen intermediates (ROIS), including the superoxide anion radical (O;).

hydrogen peroxide (H,OJ. and the hydroxyl radical (OH') as illustrated in Figure 2.

Specifically. ischemia leads to an accumulation within endothelial cells of the end

product of ATP degradation. hypoxanthine, as well as a conversion of xanthine

dehydrogenase to the enzyme xanthine oxidase (XO). The reintroduction of oxygen

during reperfusion leads to the XO-catalyzed conversion of the accumulated

hypoxanthine to xanthine and the superoxide anion. This burst of superoxide production

starts a cascade of reactions which release the other ROIs into the endothelium. causing

cellular damage. Hydrogen peroxide is formed largely via a reaction catalyzed by the

enzyme superoxide dismutase. while the hydroxyl radical is derived from either the

Haber-Weiss or Fenton iron-catalyzed reactions

This early intracellular onslaught of ROIS in endothelial cells. in itself. does not

appear to be sufficient to account for the extent of injury during reperfusion. Instead. i t

is believed that the endothelial damage caused by the ROIs provides a chernotactic signal

which directs activated neutrophil migration to the site of injury ". The activated

neuthrophils adhere to the endothelium. and under the influence of cytokine and

ATP

AMP

Adenosine

lnosine

Xanthine Dehydrogenase

[Xanthine Oxidase] \\ u - Hypoxanthine i- Xanthine + 0;

+ pi [NADPH 7 \

Figure 2. Proposed pathophysiology of reperfusion injury. Ischemia leads to the breakdown of ATP stores and the accumulation within endothelial cells of hypoxanthine, as well as to the conversion of xanthine dehydrogenase to xanthine oxidase. The reintroduction of oxvgen upon reperfusion results in the generation of superoxide anion (O2-), as weli as the hydroxyl radical (OH-) and hydrogen peroxide (HZ02). The cellular damage caused by these reactive oxygen intermediates directs activated neutrophils into the reperfused tissue. The neutrophils adhere to the damaged endothelium and cause accentuated cellular darnage via NADPH oxidase-driven production of superoxide anion, as well as via myeloperoxidase (MPO) generation of hvpochlorous acid (HOCI). SOD: superoxide dismutase. (Adapted from Granger '9).

10

complement peptides. emigrate then degranulate proteolytic enzymes into the

perivascular space 15. Neutrophil adhesion creates a microenvironment which perrnits

high concentrations of neutrophil-generated injurious agents. such as elastase ". hydrogen peroxide, and hypochlorous acid, to increase microvascular permeability and tissue

edema ". Hypochlorous acid is also generated by the neutrophil enzyme myeloperoxidase

(MPO) ". The release of these cytotoxic substances and ROIS into the extravascular

space results in direct injury to the surrounding myocytes.

Recent experiments on pig and human skeletal muscle have supported the theory

that the endotheliai xanthine oxidase system likely does not account for the majority of

reperfusion injury, since xanthine oxidase amount 52. activity ". and mRNA tran~cripts'~ have been detected at only low levels in these tissues. Instead, the rnost widely held belief

is that the NADPH oxidase-dependent "respiratory burst" of neutrophil white blood cells,

migrating to the site of ischernic cet1 injury. is responsible for the superoxide radical-

mediated reperfusion injury in skeletal muscleY.

1.3.3 Potential rnethods of minimizing skeletal muscle ischernic injury

Ideaiiy, phmnacologic manipulation for the prevention and treatment of ischemia-

reperfusion injury should serve to prevent or minimize cell damage occumng during the

periods of both ischemia and reperfusion. The majonty of investigative work performed

to date has focussed on treating skeletal muscle reperfusion injury subsequent to ischemic

darnage using ROI scavengers ", xanthine oxidase inhibitors". iron chelators to prevent

hydroxyl radical formation neutrophil elastase inhibitorss". and monoclonal

I I

antibodies against neuuophil adhesion con~plexes ". Development of this a r a of

investigation is vital in developing a treatrnent for those clinical cases which present with

ischemic injury having already occurred. however. treatments which are only effective

during reperfusion are unable to address the cellular darnage which has already occurred

during ischemia, thus they c m only rninirnize reperfusion but not ischemic injury.

Another limitation of reperfusion treatments is that diey will need to continue to be

effective for at least 48 hours from the start of reperfusion. during which tirne tissue

injury has been observed to occur.

in those ciinicd situations when ischemia-reperfusion injury is either anticipated

or considered possible, the ideal treatment modality would be one which could safely and

efficiently increase skeletal muscle ischernic tolerance pior to the ischemic insult.

uicreased ischemic tolerance would not only prevent the cellular damage which occurs

during the period of ischemia it wouid also minirnize cellular inflammation, the stimulus

for white ce11 mediated reperfusion injury. Regional hypothermia is recognized as being

able to increase skeletal muscle ischemic tolerance likely through a reduction in

metabolism and preservation of ATP ''. Unfortunately. it is not clinically practical to

keep a muscle hypothermie rhroughout the entire inua-operative and 48 h pst-operative

pend during which it is at an increased risk of ischemic injury ". The ideal solution for

preventing ischemic injury therefore would be a safe. one-dose pre-operative

pharmacologie treatment which would provide infarct protection throughout the entire

pri-operative pxiod.

12

1.4 ischemic Preconditioni ng

In 1986. Murray et al. first descnbed the phenomenon of ischemic

preconditioning (IPC) of dog myocardiurn against infarction. They demonstrated that four

cycles of 5 min coronary artery occlusion and reperfusion pnor to 40 min of sustained

ischemia significantly decreased infarct size assessed after four days of reperfusion

compareci to non-preconditioned ischemic controls. This initial description of imrnediate

cardioprotection by IPC has been temed "acute isc hemic precondi tioning". More

recently, it has aiso been demonstrated that IPC cm ttiger a late phase of

cardioprotection that occurs 24 hours after the initial IPC stimulus. which has been

11 62-64 distinguished as " late preconditioning". or the "second window effect .

Since the original descriptions in the cardiac literature. the phenomenon of "acute

ischemic preconditioning" has been demonstrated to exist in a number of other species.

including rat 65. rabbit pig 68, and human 69*70, as well as being possible in a nurnber

of other organs. including brain 71.72 and skeletal muscle '. It has previously been

demonstrated that pig skeletal muscle requires three cycles of 10 min ischernia and

reperfusion to reduce infarct size when subjected to 4 h of ischemia and 48 h of

reperfusion '. Ischemic preconditioning could not only safely and efficiently protect

skeletal muscle against acute infarction intra-operatively. it could also provide a

prolonged incseasr in ischemic tolerance through the "second window effect". w hich

would protect skelttal muscle duing the first 24 to 48 pst-operative hours which has

been shown to be the highest risk penod for acute arterial occlusion following

microsurgical vascular anastomosis 13. The literature reviewed thus fkr indicmes tlmt the

13

most effective practiccil nietlmil of direct!\. rn in îm izing îscliemîc înjuq in skelr tu1 muscl r

îs ischernic precondirioning Since demonstrating that IPC of skeletal muscle was

possible. the present investigational efforts have been directed towards elucidating the

mediator and effector mechanisms of IPC in skeletal muscle with the eventuai goal of

developing pharmacological agents to safely and efficiently reproduce. or augment. the

IPC phenornenon to protect skeletal muscle against infarction in clinical situations.

1.4.1 Mecha~srn of ischernic preconditioning

The majority of reports on the possible mechanism of IPC concern in situ and

isolated heart rnodels. Examination of the findings from these reports may serve to

provide insight into the possible mechanism of IPC in skeletal muscle. Experimentai

evidence so far has excluded a number of potential major mechanisrns as being

responsible for the protective effect of acute iPC. These include mitochondrial ATPase

inhibitor protein activation 73-75. muscle stunning 76.'7. glycolytic flux an increase in

prostacyclin or nitrous oxide synthesis 65-'9"0. a neutrophil-related mechanism ''. increased

collateral blood flow L3.M.". synthesis of stress protein '2. and decreased oxy-radical

generation or anti-oxidant defences".". However. experimental evidence is accumulating

to indicate that adenosine is most likely the initiator of IPC in a number of animal

rnodelsJ-', with exception of the rat '.

The hypothesis for an adenosine receptor-linked pathway first came when Liu et

al. demonstrated that the non-selective adenosine receptor antagonists. 8-(p-

sulfophenyl) theophylline ( 8 - S R ) or N-[2-(dimethylamino)ethyl]N-methy1-4-(2.3.6.7-

14

tetrahydro-2.6-dioxo- 1.3-dipropyl- i -purin-8-yl) bznzosufonamide (PD 1 15.1 19). were

able to block the cardioprotective effect of IPC in the rabbit heart. They also showed that

an intracoronary infusion of adenosine, or the adenosine A, receptor agonist, N6-1-

(phenyl-2R-isopropy1)-adenosine (R-PM), could mùnic the cardioprotective effect of

IPC. Subsequent studies frorn this sarne laboratory "." demonstrated that intravenous

infusion of an A,-. but not an A,-, receptor agonist afforded infarct protection in the

rabbit heart, implicating a role for the adenosine A, receptor in the rnechanism of IPC in

rabbit cardiac muscle. More recent studies have suggested the possibility that the

adenosine A, receptor. may be also be involved as an initiator of WC in the rabbit

hea.~t''-~~. Specificall y. treatment of buffer-perfused rabbit hearts with the rabbit

adenosine A3-selective agonist, IB-MECA, mimicked the infarct protective effect of WC

when the h e m were exposed to 30 min regional ischernia and 120 min of reperfusiong9.

In addition. this protective effect of A, stimulation was unaffect& by the rabbit adenosine

A,-selective antagoonist. BWA1433 ''. The differential role of adenosine A, and A,

receptors in IPC has yet to be determined. Besides rabbit cardiac muscle, the infarct

protective cffect of adenosine treatment has also been demonstrated in dog and pig

heart rnodels. and the cardioprotective effect it elicits in these species has been shown

to occur dui-ing the isc hemic, rather then the reperfusion. pliase of injury "-". Finally.

adenosine also appears to mediate IPC in cultured human pediatric myocytes. although

the differential role of the adenosine A, and At receptors remains unclear %.

Adenosinr A, and A, receptor stimulation is known to be linked to a nurnber of

intracellular signalling pathways, including adenyl cyclase "7.'x. ATP-sensitive potassium

15

( K,) channels '". voltag-dependent Ca" channels ' . Na+-Ca'' çxchangersl"'.

acetylcholine-sensitive potassium channels lu'. and the phospholipase A? and C

s y ~ t e r n s ~ ~ ' - ~ ~ . One common theory of the rffector mechanism of LPC downstream of the

A, receptor involves activatior of the ATP-sensitive potassium (Km) channel. Gross et

al. observed that blockade of the Km channel with glibenclamide blocked the anti-infarct

effect of IPC in dog hem 'U5.1'>6. Since then. it has been demonstrated that die K,

channel antagonists glibenclamide 'O7 and sodium 5-hydroxydecanoate (5-HD) " blocked

the cardioprotective effect of WC. adenosine, and an adenosine A, receptor agonist (R-

PM) in pigs. and that glibenclamide blocked the cardioprotective effect of an allosteric

enhancer (PD 81.723) 92 and agonists (DCPCX '' and R-PIA lu') of the A, adenosine

receptor in dogs. As corroborating evidence. the K, channel openers. bimakalirn and

crornaketine. have been shown to mirnic the cardioprottctive effect of IPC in dogs '@'-'"'.

In small animal models such as the rabbit and rat, results of experiments investigating the

role of Km channels in PC are conflicting. and the matter remains unresolved " '-"' . One

potential rnechanism by which Km channel opening dunng sustained cardiac ischemia

might lead to protection is through a shortening of the action potential duration and

antagonization of membrane depolarization I L S . This would reduce the opening rime of

voltage-regulated (L-type) Ca" channels. consequently decreasing Ca2+ intlux. muscle

contractility and ATP catabolism Il6. This proposed mechanism of action of K,, channsl

opening has been supported by investigations in the guinea pig 1 1 7 - 1 1 X and dog I 19. IL() heait

models. However. it has been demonstrated that bimakalim. a K,, channel opzner.

administered to dog hearts at a low enough dose (0.1 pg/min) not to accrlerate the

16

shortening of the action potential duration is equally efficacious as higher doses in

reducing myocardial infarct size ''O. This would suggest that other cellular mechanisrns

besides action potential duration acceleration may also be involved in the

cardioprotective effect of Km opening.

Along with the K, channel theory, the most investigated proposed pathway

downsaeam of the adenosine A, receptor is the "Downey hypothesis" 12' as illustrated in

Figure 3. According to this hypothesis, adenosine. released from ATP hydrolysis during

the transient ischemic episodes of WC, stimulates the adenosine A, receptor which

activates phospholipase C (PLC) via a pertussis toxin-sensitive G-protein mechanism.

The activated PLC. in turn, cleaves phosphatidylinositol 4,5-bisphosphate (PIP,) to

produce two second messengers, inositol 1.4.5-triphosphate (IP,) and diacylglycerol

@AG). DAC then activates the serine-threonine kinase. PKC. which translocates from

the cytosolic pool to the membrane where it is activated. The spx f i c protein(s)

presumabiy phosphorylated by PKC on the membrane to elicit the infarct protective

effect of IPC has yet to be identified. Support for this hypothesis is largely from

observing bat the PKC inhibitors. staurosporine, chelerythrine, polymyxin B. can block

the cardioprotective effect of IPC and adenosine in rabbits. whereas the PKC activators.

4 P-pliorbol 12-myristate 13-acetate (PMA) and 1 -01eoyl-2-acetyl glycerol (OAG) c m

mimic the infarct protective effect of WC A recent study on human pediatric

myocytes has dso supported a link between endognous adenosine release and PKC

during preconditioning ? In other animal models. PKC has not been demonstrated to

play a role in IPC of pig hem '23."J. and evidence for its role in IPC of the dog heart

Adenosine t ATP

membrane

cytosol PKC

l nactive

Figure 3. The proposed role of protein kinase C in the mechanism of i schemic p r e c o n d i t i o n i n g . Adenosine, released du r ing ischemic preconditioning, preferentiallv activates Al-adenosine receptors, which results in phospholipase C (PLC) activ&on through a G-protein linked rnechanism. PLC, in turn, degrades phosphatidvlinositol-bis-phosphate (PIP,) to produce diacylglycerol (DAG). DAG cause; translocation of PKC from the cytosol to the membrane, where it is activa ted. Activated PKC presumably mediates the infarct- protective effector mechanism of IPC through phosphorylation of a n unidentified pro tective membrane protein (Adap ted from Downey 1'1).

remains equivocal 125,126

There have been reports in the literature suggesting that adenosine may not only

inititate the protective mechanism of IPC. but may also serve as an effector of this

phenornenon. Specifically. Kitakaze et al. observed an increase in venous adenosine

release during ischemia and reperfusion in preconditioned dog heart 12'. which they

atmbuted, based on pharmacologie studies. to a PKC- and Km channel-dependent

increase in ecto-9-nucleotidase activity 1"*128. This theory has been indirectly supported

by Akimitsu et al. '" in mouse cremasteric muscle, when they dernonstrated that the anti-

neutrophil adhesion effect of IPC could be rnimicked by topical adenosine. or fully

blocked by adenosine deaminase (ADA), an adenosine degradation enzyme, only when

they were applied b e f ~ r ~ and during both the ischemia and reperfusion periods. WC-

associateci postischemic a&mosine release could theoreticaily stimulate the adenosine AI-

receptors on activated neutrophils to decrease their endothelial adherence. thereby

contributing to the anti-infarct effect 13U.1'1. Altematively, the endothelial adenosine A,-

recep tors could be stimulated. conuibu ting to the anti-infarct effect through smooth

muscle relaxation and vasodilation l'L1". This speculation that adenosine may serve as

an effector in IPC is at variance w ith other published reports. w hich have demonstrated

that administration of a non-specific adenosine receptor antagonist during reperfusion did

not attenuate the anti-infarct eft'ect of WC in rabbit heart '), and that IPC of dog heart did

not result in an increasc in coronary venous adenosine concentration during 60 min of

regional ischemia and reperfusion ';'.

19

1.4.2 Windows of ischemic preconditioning

As mentioned previously, it has been demonstrated that the cardioprotection

observed after an P C stimulus appears to occur in two distinct "windows". The acute

preconditioning effect is believed to be transient. lasting less than three hours '. whereas the more recently descnbed "late" or "second window " of protection begins after several

hours and is still present 24 to 48 hours after the initial stimulus 1'5.i36. To date. most IPC

studies have examined the acute window, and the mechanism invoived in "late" as

opposed to "acute" preconditioning is not clea.. Some investigators have suggested that

a similar signalling pathway involving the adenosine A, receptor '" and PKC 1377.1'" i s

responsible. resulting in the synthesis of heat shock proteins u.

Protein denaturation as a result of ischernia or hyperthennia has been reported to

result in the onset of the heat shock response which increases synthesis of the so-called

heat shock proteins (HSPs) 13'. A direct correlation has been demonstrated between the

amount of one of these HSPs (HSP-70) and the degree of myocardial protection

following a hyperthermie treatment in rabbit '" and rat 139 hearts. The function of HSP-70

is not entirely known, although they rnay serve as intracellular molecular "chaperones"

w hich facilitate the transport of other protrins through the membranes of different

in tracellular compartrnents '4U.141. Their potential role in increasing ischemic tolerance

may therefore be to aid in the cellular localization of protective enzymes, or in the

removal denaîured proteins. Since rnessengr RNA for HSP-70 c m be detected within

five minutes of the onset of an ischemic stress. but the protein itself is only detectable in

significant quantities after severai hours la'. HSP-70 may be involved in late window IPC.

20

but is not likely to be responsible for acute IPC. The drtailed mechanism of HSPs is

beyond the scope of this thesis.

1.4.3 Ischemic preconditioning of skeletal muscle and the potential role of PKC

The phenornenon of iPC has also been demonstrated in skeletai muscle '. Evidence

has k e n accumulated indicating that the adenosine A, receptor and the K , channel are

involved in the infarct protective mechanism of IPC of skeletal muscle. Specifically. it

has b e n demonstrated that local intraarterial infusion of adenosine (0.5 mg/rnuscle flap)

over a penod of 10 min significantly reduced the infarct size of pig latissirnus dorsi

muscle flaps exposed to 4 hours of warm global ischernia and 48 hours of reperfusion '.

In addition. administration of 8-SPT. a non-selectivz adenosine receptor antagonist. and

DPCPX, a selective A, receptor antagonist, blocked the protective effect of P C and

adenosine treatrnents '. More recent investigations have demonstrated that pre-treatment

of muscle flaps with the Km channel antagonists. 5-HD or glibenclamide. blocked the

infarct protective effect of IPC and adenosine treatment, and that lemakalim. a Km

channel opener. imparted an infarct protective effect "". Finally. the infarct protective

effect of K. adenosine. and Km channel opening in skeletal muscle prior to sustained

global ischemia has been shown to be associated with a slower rate of high energy

phosphate depletion and lactate accumulation in the skeletal muscle during the sustained

period of global ischemia. and a lower neutrophilic myeloperoxidase activity during

reperfusion of the ischemic muscle compared with Ume-matched control muscle f lap~' . '~~.

21

To date, it lias not been detemiined whether PKC plays a role in the pst-receptor

transduction pathway of P C of skeletal muscle gainst infarction, and if it does, whether

it is linked to, or is distinct from, the adenosine A, receptor-Km channel-linked

mechanism. To formulate a hypothesis of the role of PKC in LPC of skeletal muscle, the

structure and activation of protein kinase C must first be discussed.

1.5 Protein Kinase C

1.5.1 Historical review of protein kinase C

In 1977, Nishizuka and colleagues descnbed a protein kinase partially purified

from bovine cerebellum which, unlike the previously identifed protein kinases A and G,

could phosphorylate histone and protamine independent of cyclic nucleotides 1449145. in

these initial reports, the enzyme was tentitively narned "protein kinase MW. and at that

time it was unknown whether it was "an artifact or a physiologically significant

enzyme"'? Subsequent work by Nishizuka's group showed that the previously ignored

proenzyme of protein kinase M was more likely to be the physiologically significant form

of the enzyme complex, king reversibly activated by a CaL+-dependent neutral protease,

hence the narne protein kinase "C" '?

PKC is now known to be not a single molecular entity, but rather a family of

closrly related serine- and threonine-specific protein kinase isoforms that are involved

in intracellular signal transduction '47-152. If is ubiquitously distributed and plays an

important role in the control of regdation of many different biological processes 153.1Y.

1.5.2 Protein kinase C isoforms

In 1986. Parker described the isolation of cDNA clones encoding PKC based on

peptide sequrnces from homogenous purified protein lJ7. From these results. it became

evident that PKC is not translated from a single mRNA, but that there exist multiple

mRNAs in the brain which are highly stnicturally related, and which potentially coded

for a family of enzymes with identical or similar propertiesL48~1J9~1s5~156 . In vi tro expression

of these cDNAs in rnamrnalian or insect expression systems demonstrated that they al1

encode proteins with PKC-specific properties. such as phorbol ester binding, and DAG-

or phorbol ester-ac tivated calcium- and phospholipid-dependent kinase activity 150.15&158

The first isolated fractions of purified PKC. identified in vivo by separation on a

hydroxyapatite chrornatography column. were referred to as types 1, II. and iIi '".

Molecular cloning experiments confumed that these fractions were cncoded by the earlier

described y. P. and a cDNA sequences respectively 15'. Additionally it was revealed that

PKC-P exists in two subfoms derived from alternative splicing of a single gene 1s0.1".161.

Subsequent molecular cloning identified several additionai PKC isoforms, such that the

mammalian PKC family at the present time consists of 12 known different polypeptides:

a. p,, Pi i , y . 6 . ~ . C. q.8.r . b n d y 162.

The PKC isoforms c m be classified into three groups according to their primary

structure and functional similarities '" as shown in Figure 4. The conventional or

classicai PKC isoforms (cPKCs a, Pl, PI,, and y ) fulfill the original definition of PKC

as a CaL+- and phospholipid- dependent protein kinase, whereas the novel isoforms

(nPKCs 6 . E . q. 8, and p) and atypical isoforms (aPKCs 5, T , and A) are Ca2'-

23

independent, and are also referred to as 'nonconventional' isoforms. The atypical PKCs

are differentiated from the novel PKCs by not being activated by phorbol esters since

they lack the zinc fmger-iike cysteine-rich phorbol ester binding motif (Figure 4)152-1a.165.

The tissue distribution of these isoforms varies. For example, some isoforms present in

h e m muscle, are not present in skeletal muscle 166.167

1.5.3 Translocation and activation of protein kinase C isoforms

Newly translated PKC isoforms Fust associate with the detergent-insoluble

fraction of cells '", where they undergoes triple phosphorylation to release the mature

catalpcally competent form into the cytosol "'). This cytosolic form is inactive since the

pseudosubstrate sequence of the enzyme occupies the substrate-binding cavity. DAG.

released from the hydrolysis of phosphatidylinositol- 4,5-bisphosphate by activated

P L C l 7 ' , binds to the enzyme and drarnaticdly increases its affinity for the membrane

fraction of the ceii. resulting in translocation 17'. A group of proteins collectively known

as RACKs, for Leceptors for gctivated C-&nase, have been identified which may bt:

involved in translocation of activated PKC isoforms '7L173 . It is hypothesized that RACKS

serve as "anchor proteins" which are located in rhe target membrane compartrnent of a

PKC isoform. A weak interaction that usually exists between a PKC isoform and its

anchor protein is accentuated by an activating signal, resulting in the PKC isoforni

translocating to its target membrane and binding to the anchor protein '". Membrane

phospholipid binding causes a conformational change in PKC that releases the

pseudosubstrate from the enzyme catalytic site. which dong with the substrate. and Ca:-

Regulatory Domain Catalytic Domain

4 b b

N dPsSuba PhEs ATP - Substrate C .

Figure 4. Schematic representation of the primary structure of conventional, novel, and atypical PKC isoforms. Al1 PKC isoforms consist of a regulatory and catalytic domain separated by a "hinge" region (arrow). The regulatory domain of the conventional PKC isoforms (cPKC) contains a pseudosubstrate (PsSub) domain, as well as phorbol ester (PhEs) and calcium/phosphatidyl serine (Ca'-/PS) binding sites. In contrast, the novel PKC isoforms (nPKC) contain a modified constant region which binds phosphatidvl serine but not calcium, and the atypical PKC isoforms (aPKC) contain neitherda calcium nor a phorbol ester binding site. The catalvtic domains of al1 isoforms contain both an ATP and a substrate binding site (kodified from Newton 155).

N - PsSub - ATP - Substrate C

25

for the conventional isoforms, maximaily activatzs the enzyme l6L175.i76

Following translocation and activation in vivo. PKC is believed to br

proteolytically cleaved at the V, hinge region (Figure 4) to produce two distinct

fragments. a protein comprising the regulatory domain. and a protein containing the

kinase domain 17'. It is still not clear whether this proteolytic degradation serves as a

means of preventing continuing kinase activity (so-called "downregulation"). or if the

kinase domain, released from the regulatory subunit and the membrane, can act as a

constitutive. activator-independent kinase 176.

PKC is therefore believed to be regulated by three means: (1) post-translational

maturation by phosphorylation or dephosphorylation 16). (2) distinct subcellular

distribution of both the enzyme and substrate '", which separates the two untii

translocation and activation of PKC has occurred, and (3) pst-translocation proteolysis.

1.5.4 Protein kinase C inhibitors and activators

The in vivo use of inhibitors and activators as pharmacologic probes is one of the

rnost direct methods for investigating the role of a particular enzyme in cellular

metabolism. Demonstrating that inhibitors of an enzyme block the end point of an

activated metabolic pathway. such as muscle infarction in WC. and that activators can

mimic the effect of the activated pathway. provides strong evidence that the particuiar

enzyme is involved in that metabolic mechanism. However, for this evidence to be valid.

both the dosage, systemic effects, selectivity and mechanism of action of the

pharmacologic probes must be detïned.

Protein kinase C inhibitors

Both the regulatory (phospholipid- and phorbol ester-binding sites) and catalytic

domains (peptide- and ATP-binding sites) on PKC provide targets for inhibitory design.

Due to the varying selectivity of currently available PKC inhibitors. it is important to

employ more than one inhibitor of different structure and mode of action to p rok PKC

activity 17'. Polyrnyxin B sulfate. is a cyclic polycationic peptide antibiotic that interacts

electrostatically with negatively charged phospholipids 179~ 'xU . Since PKC requires certain

phospholipids. such as phosphatidyl serine. as a c~factor"~. polymyxin B inhibits PKC

activity by disrupting PKC / phospholipid interaction. Its LC, for PKC inhibition is cl0

PM. however it is only seven fold more selective for PKC than it is for myosin light chain

kinase lu'"'. and it is possible that it can interact with the ATP-sensitive potassium ATP

channel lX4. An additional side-effect of polymyxin B that must be taken into

consideration is systernic and / or local vasodilation at higher concentrations 99.

By far the greatest progress has been made with the design of ATP-cornpetitive

inhibitors of PKC. One exarnple is the potent (IC',, = 660 nM) and selective inhibitor.

chelerythrine chlonde. Chelerythrine is a naturally occuning benzophenanthidine

alkaloid that acts on the catalytic domain of PKC as a cornpetitive inhibitor with respect

to the phosphate acceptor. and a non-cornpetitive inhibitor with respect to ATP. It is > 1 50

fold more selective for PKC than any other tested kinase 'w1.1n55. One concem with

chelerythrine is its limited aqueous solubility, requiring it to be first dissolved in dimethyl

sulfoxide (DMSO) prior to dilution. It has been documented that DMSO is a hydroxyl

37

radical scavenger, and therefore has an indepmdent anti-infarctive effect on ischernic

skeletaI muscle 186.187. In studies using DMSO as a vehicle for drugs, it must be ensured

that the concentration of DMSO is well below die therapeutic concentration of 25 mg /

100 mL blood (32 rnM)'?

Protein kinase C activators

As previously mentioned. there are four physiological activators of PKC '"1 (1)

DAG, (2). membrane lipids, (3) protein subsuate. and (4) Ca2' for the conventional

isoforms. The most commonly used pharmacobgic activators of PKC available today are

functional analogues of DAG. competing with "', and substituting for '" DAG in

activating conventional and novel PKCs both in vitro and in situ '"'-"' . The DAG binding

site on PKC has been localized to two conserved cysteine-rich zinc-finger motifs on the

regdatory domain of PKC 19L71w. one of which is not present on members of the atypical

PKC isofoms (5, r. and A) 15'. Pharmacologie PKC activators. therefore. do not activate

these three isofoms ' O 5 .

The two dnigs most comrnonly used as PKC activators are phorboi 12-myristate

13-acetate (PMA) and 1 -01eyl-2-acetyi-glycerol t OAG). Both are structural analogues of

DAG, however PMA is believed to be three orders more potent in its activation of PKC

than OAG 1 7 1 . Both PMA and OAG can cause coronary vasocons~iction and have a

negative cardiac inotropic effect at higher doses ? Dosage must therefore be tailored to

ensure that any observed effect is d w to PKC activation. and not to altered

hemodynamics. Like chzlerythrine. these two PKC activators also require DMSO as a

vzhicle in aqueous solution.

1.5.5 Measurernent and detection of protein h a s e C

The translocation of cPKCs and nPKCs from a soluble cytosol to a particulate

membrane form is often taken as being synonomous with activation Ig5. Direct

measurement of PKC activity in membrane and cytosol fractions is measured by the

phosphorylation of suitable mode1 susbstrates. Early PKC kinase assays used histone UIS

as a subsuate '*, which is suitable for conventional isoforms, but will not be

phosphorylated by the novel or atypical isoforms. The recently commercially available

oligopeptide E (modelled on the ~ P K C E pseudosubstrate site) c m measure both

conventional and novel isoform activity md similarily. use of an oligopeptide

sequence from the epidermal factor receptor has been reported 1860198-199 . Aiternatively,

binding of the phorbol ester [W] phorbol dibutyrate to c/nPKCs can also be rneas~red'~'.

The common shortcoming of al1 these methods is that they are unable to discriminate

unambiguously between PKC isoforms. In addition, these assays measure the rate of

phosphorylation by PKC under optimal artificial concentrations of calcium 1

phosphatidylserine / diacylglycerol "'. This measured maximal rate of phophorylation

therefore may not accurately represent the uue intraccllular PKC activity.

In order to discriminate the presence of PKC isoforms, i m r n u n o f l u o r e ~ c e n c e ~ ~

and western blot analysis 202203 using commercially available monoclonal or polyclonal

antibodies are now king employed to detzct PKC isoform translocation to the membrane

fraction. Immunofluorescence studies have the advantage of detecting subcellular

29

migration of PKCs while maintaining the native structure of the tissue. The disadvantage,

however. is that this technique is non-quantitative, and is more technically difficult in in

vivo tissue. as compared to cultured cells. In comparison, quantitative analysis of in vivo

samples is possible with western blot analysis. The fractionation procedure that is

required for this technique, however, may result in a high proportion of inactive PKC

isoforms in the membrane fraction, which can cause technical problems in detecting

translocation against a high background immunoreactivity lu.

The literature reviewed thus far therefore indicates that the mechanism of IPC of

skeletai muscle iikely involves the adenosine receptor which is linked downstream to the

KAT channel. The ptential role of PKC in this signal transduction pathway has not been

previously investigated. however one possiblity is that PKC is the "rnissing link" between

receptor stimulation and the end effector mechanism. PKC activators and inhibitors can

be used in vivo to investigate the potentiai role of PKC in IPC of skeletal muscle,

however, due the previously mentioned limitations of these pharmacologie probes, a

molecular technique such as western blot analysis is required to analyze the ptential

involvernent of individual isoforms of PKC at the cellular level. Only through increased

understanding of the mechanism of IPC can extremely selectivs phannacologic activators

be identified and used in clinical situations to protect skeletai muscle against infarction.

1.6 Objective

The objective of this project was to determine the role and cellular mechanism of PKC

in the pst-receptor signal transduction pathway of ischemic preconditioning of pig

skeletal muscle against infarction.

1.7 Hypothesis

The hypothesis was that PKC plays a cenual role in IPC of skeletal muscle against

infarction. and that the cellular mechanisrn of IPC involves an adenosine receptor-PKC-

K, channel-linkrd signal transduction pathway.

1.8 Specific Aims

Aim 1: To investigate the effect of PKC inhibitors on the infarct protective effect of IPC

and adenosine in pig latissirnus dorsi (LD) muscle subjected to 4 h of global ischemia and

24 h of reperfusion.

Aim II: To investigate the effect of IPC. adenosine, a PKC activator. and a PKC inhibitor

on muscle blood flow in pig LD muscle.

Aim III: To identify PKC isoforms in pig LD muscle. and to investigate the effect of IPC

on PKC distribution in cytosol and membrane fractions of pig LD muscle at various time-

points during sustnined ischemia.

Aim IV: To investigate the effect of KA, channel blockade on the anti-infarct effect of

IPC and a PKC activator.

Aim V: To investigate the effect of [PC and a PKC activator on neutrophil activity in pig

LD muscle subjected to 4 h of global ischemia and 16 h of reperfusion.

2.0 Materials and Methods

2.1 Expenmentai Animal

The domestic pig (Scrofa Domesticus) has been accepted in the plastic surgery

literature as a suitable animal mode1 for muscle and skin flap research due to the

sirnilarity in anatomy and vasculature to the those of the human 205306 . Yorkshire pigs

were used in these experiments due to their availability locally in large numbers. This

allowed selection of animals of the saine breed and sex, and of similar age and weight.

so as to rninimize inter-animal variability. The male pigs used in this study were casuated

by the supplier shortly after weaning to decrease aggression and facilitate animal a r c .

2.2 Animal Care

Al1 pigs were purchased from a specific pathogen free breeding herd in Ontario

(Albro Farms, Newcastle, Ontario). On arrivai. the anirnals were housed in a temperature-

(22 OC) and light- (0700-1900h) controlled environment in the Laboratory Animal

SeMces of the Hospital for Sick Children. AI1 pigs were provided a commercial pig dict

and tap water ad libitum. Food was withheld the evening prior to scheduled surgery.

Mean body weight at tirne of surgery was 19.0 kg t 1.4 (mean t SD). Animal care in this

project complied with "The Care and Lise of Laboratory Animals" (Canada Council on

Animal Care. 1980). and al1 expeiirnental pi-otocols were approved by the Animal Care

Cornmittee of The Hospital for Sick Childi-en.

2.3 Operative Procedure

2.3.1 Justification for the use of the pig LD muscle flap rnodel in this project

The human latissimus dorsi (LD) muscle flap is used rxtensively in clinical

reconstructive sugery 2u7.20'. The porcine counterpart is easil y dissec ted with minimal

blood loss. and the anatany is quite similar to that of the human . The pig LD

muscle flap model has been previously used by this laboratory 3.3 1.U.55 , and other

investigators m. to snidy skeletal muscle ischemia / reperfusion injury. The operative site

at the shoulder of the pig makes the flap less likely to be damaged inadvertently during

the pst-operative period. and as the muscle is bilateral. paired results c m be obtained

from each animal. As in the hurnan, these muscles are not essential for locomotion in the

pig, thus allowing observation of the long term effect (24 h) of ischemia / reperfusion

injury.

In the transfer of autogenous muscle for wound coverage or restoration of

function. such as in free flap surgery, a microvascular anastomosis is performed between

the vascular pedicle of the muscle flap and the recipient vessels. This surgical

manipulation is a vital cornponent of animal rnodels used to study pedicle thrombogenesis

in free muscle transfer. in these experiments, however. a microvascular anastomosis was

not performed. since the primary objective of the study was to examine muscle ischemic

tolerance. Not only would a rnicrovascular anastomosis significantly prolong the

operative time. it would also inuoduce confounding variables such as arterial or venous

thrornbosis.

35

After transection of rhe somatic thoracodorsal nerve in the LD muscle tlap model.

global ixhemia was achieved by occlusion of the muscle pedicle with a vascular clamp.

This method differs from a microvascular anastomosis in that the perivascular

sympathetic nerve fibers rernain intact. It is theoretically possible that sympathetic

innervation of the muscle could affect muscle metâbolism, and therefore ischemic

tolerance. However, it has k e n demonstrated that the sympathetic denervation provided

by severence of a skeletal muscle somatic nerve is not augmented by transecting the

muscle pedicle, since very few longitudinal sympathetic fibers travel dong the vessel

adventitia 210211. in addition. it has demonstrated that complete transection of the axillary

artery and nerve, with subsequent vascular a~astomosis. has no effect on muscle viablity

in ischemic control or WC-treated LD muscle flaps (W.He, unpublished data. 1996).

Previous studies examining the effect of sustained vessel clamping concluded that

greater than four to six hours of unintempted rnicrovascular clamping is required for

endothelid disruphon and mucosal necrosis 2''.21'. With this in mind, the positions of the

microvascular clamps were changed aiier two hours of ischemia without re-establishing

blood flow. As weU. to better distribute the occlusive force. double toothed clamps were

employed.

2.3.2 Anesthetic technique

Each animal was sedated with ketamine hydrochloridr 30 mgkg IM (Ketaleanw

100 rng/mL, MTC Phmaceuticals, Cambridge, ON). prior to shaving of the surgical area

and endotracheal intubation (interna1 diameter 6.5 mm). Normal Saline was infused

36

throughou t the operation via a 23-gauge angiocatheter inscned in the laterai auricular

vein at a constant rate of 2.5 Wmin. Surgical anesthesia was achieved with intravenous

sodium pentobarbitone at 10- 15 mgkg (Somnotol" 65 mg/rnL. MTC Pharmaceuticals.

Cambridge. ON). The pig was intubated and mechanically ventilated (Ventirnetera

Ventilator. Air Shields Inc., Hatboro, PA) with a mixture of 1 : I nitrous 0xide:oxygen

(tidai volume 15 mUkg). Intra-operatively, anesthesia was maintained with intravenous

pentobarbitone (2 mg/kg/h). The operating room temperature was regulated at 22 OC, and

rectal temperature was maintained between 38 to 39°C with a heating blanket set at 42°C

(Aquamatic K-Thermia Heating Blanket@. Mode1 RK-600. Amencan Hamilton Medical

Systems, Cincinnati. OH). Rectal temperature was monitored inuaoperatively (Thenno-

Fine$, Terumo. Japan). Post-operatively, anesthesia was withdrawn and the pig was

allowed to wake up and was returned to the animal holding room. Post-operative

analgesia was not administered, since upon waking, the animals did not appear to be in

discomfort. Presumably due to the denervation of the muscle flap, the only sensation

from the operation was from the skm incision. in consultation with the veterinary staff.

it was decided that catching the animals for administration of analgesia would be more

traumatic than any benctït resulting from analgesia.

2.3.3 Creation of the LD muscle flap

Hemostasis was achieved throughout the operation using grounded bipolar

cautery. Sterile operating technique was followed at al1 tunes. Bilateral island 8 by 12 cm

deneivated LD muscle flaps. based solely on the thoracodorsal pedicle and attached by

37

the humera1 tendon, was elevated from the intercostal musculature in each pig. The

humera1 tendon (-1 cm wide), used to support the vascular pedicle of the flap. was

isolated and ligated with 1-0 silk to obliterate any tendinous blood supply to the muscle

flap: the blood supply to the muscle flap was therefore denved entirely from the

thoracodorsal artery and drained by two thoracodorsal veins. The vascular pedicle was

cleaned of any fat or connective tissue. and the thoracodorsal nerve uansected to mimic

the clinical situation. The pedicled flap was then fixed in its original position and the skin

incision closed using 2-0 silk sutures. The animal was placed in the opposite lateral

decubitus position, and the procedure repeated to raise the contralateral muscle flap.

2.3.4 Global ischemia of the LD muscle flap

To render the muscle flap ischemic, a microvascular clamp (2 x 8 mm. Weck) was

applied to the vascular pedicle (Figure 5). Complete occlusion was verified by

intravenous injection of fluorescein dye (15 mgkg). Absence of yellow fluorecence under

ultraviolet light 10 min after dye injection indicated cornplete occlusion of the vascular

pedicle. During prolonged ischemia. the position of the microvascular clamp on the

pedicle was changed after two hours of ischemia without re-establishing blood flow.

According to the experimental protocols. the muscle flaps were subjected to 4h

of ischernia and 24 h of reperfusion. This duration of ischemia was used since it has been

previously demonstrated to result reliably in infarction of approximately 40% of the pig

latissimus dorsi muscle flap used in this study ". This allows detection of both a pro-

infarction as well as an anti-infarction treatment effect ". In the original study that

Axillary -, Artery

infusion Thoracodorsal site \

\

8 x I 2 c r n

-t- tatissimus dorsi muscle flap Brachial Artery

Thoracodorsal

Microclamp

Figure 5. Local intraarterial infusion technique for the pig denervated island latissimus dorsi muscle flap. The vascular supplv to the 8 by 12 cm island latissirnus dorsi muscle flap was entirely via the thoracodorsal arterv, and the muscle flap was drained by two accornpanying thoracodorsal ieins. The thoracodorsal nerve was cut to mimick the clinical situation. The ischemic preconditioning stimulus, as well a s the prolonged ischemia, was achieved by application of an approximator with double microvascular clamps to the flap pedicle. Drug and saline infusion to the flap was accomplished, following temporary occlusion (crosses) of the proximal brachial artery and the distal subscapular trunk, by cannulation of a small branch of the brachial artery (arrow). Oxygenated blood flow to the muscle flap was maintained during drug infusion via the axillary artery (see text for details).

39

examined the effect of different ischernia times on pig skeletal muscle, infarct analysis

was performed after 48 h of reperfusion, however since then. it has been demonstrated

that if the analysis is instead performed after 24 h reperfusion there is no difference in

infarct size.

2.3.5 Drug delivery

After raising the LD muscle flaps, the animal was secured in the supine position.

and the forelegs splayed to exposed the axillae. The skin incision used for raising the

muscle flap was extended dong the axillary crease. and the underlying soft tissue was

dissected to expose the axillary vasculature. A srnall0.5 to 1 mm branch (un-narned) of

the proximal brachial artery was consistently present in ail specimens. and was used as

an injection site for the muscle flap to prevent any damage which would have occurred

from direct injection into the thoracodorsal pedicle. To cnsure that the infusate flowed

retrograde through the brachial artery and down the axillary artery to the flap pedicle. the

brachial artery just distal to the injection vessel. and the subscapular trunk just distal to

the thoracodorsal artery. were isolated and temporarily ligated with 1-0 silk just prior to

injection of a drug (Figure 5). Oxygenated blood flow to the flap via the axillary artery

was therefore maintained during the infusion period. AI1 drugs were dissolved in 20 mL

of normal saline and infused via a 23-gauge anggiocatheter over a 10 minute pcriod.

Following the infusion, the temporary ties on the brachial arteiy and subscapular uunk

were released, and the angiocatheter was removed from the small brachial branch, which

was ligated for hernostasis. After h g infusion, a 10 min stabilization period was allowed

40

before preconditioning or starting sustained ischemia. When DMSO was used as a

vehicle. the maximum final concentration was 0.7 mM. well below the previously

rnentioned therapeutic concentration of 32 mM la6.

2.3.6 Muscle sampling

Tirned intra-operative full thickness muscle biopsies were sequentially harvested

1 cm frorn the dorsal edge of the LD flap in a cephalad direction starting -8 cm from the

vascular pedicle (see Appendix 1. page 128). It has been previously documented that

muscle infarction during prolonged ischemia. as well as the protective effect of IPC.

occur consistently in this biopsied region of the LD muscle flap 3 . 3 1 . With this technique.

removal of each biopsy should n ~ t interfere with the blood supply to the remaining

muscle. A preliminary study using this technique demonstrated no significant difference

in infarct size between a biopsied muscle flap and the un-biopsied contralateral muscle3.

Each biopsy was irnrnediately trimmed of visible connective tissue and fat to a weight of

0.5 to 0.6 g. rinsed in normal saline. and imrnersed in liquid nitrogen. The biopsies were

stored at -85' C and fractionated within 24 h of harvesting.

2.4 Muscle Anal ysis

2.4.1 Assessment of muscle viability

Immediately after the pig was sacrificed with an overdose of intracardiac

pentobarbitone ( 100 mgkg Euthanyl". MTC Phmaceuticals. Cambridge, ON), the LD

muscle flaps were excised. and twelve 1-cm thick transverse sections were harvested

from each muscle flap, with the most cephalad section starting at the natural junction of

the long head of the triceps and the LD muscle. The harvested sections were then

completely irnmersed in a 1 mg/mL solution of nitroblue tetrazoliurn (NBT) dye 2'4 in

0.28 Tris buffer pH 7.4 for 30 minutes at 22°C. Tissue dehydrogenase present in viable

muscle reduces tetrazolium salts to produce a dense, non crystalline pigment called

"fonnazan" which stains the muscle dark blue. Non-viable muscle remains unstained due

to the absence of dehydrogenase activity "'. The advantage of this technique is that

unlike histologie examination, it can discriminate between reversible and irreversible

ischemic lesions within minutes. instead of days, of the injury '15. This technique of

nitroblue tetrazolium dye test has been vaiidated in dog gracilis muscle using electron

microscopy and '"technetium pyrophosphate uptake to confirm that the resolution of the

staining between live and dead skeletal muscle it accurate to within less than 1 mm 2'".

NBT staining has also been used previously in pig latissimus dorsi myocutaneous flaps

7 7 1.44.C.YN -.-

The pirentage infarct in the flap was calculated using the previously desctibed

papa template technique 31*217 (see Appendix II, page 129). Areas of viable and nonviable

42

muscie on the caudal surface of each muscle section were traced on a uansparcnt acetate

sheet. and paper templates of these areas were cut and weighed. The extent of muscle

necrosis was calculated as follows:

weight of paper templates of infarct areas

weight of paper templates of entire area

of the muscle flap

It has been previously demonstrated that the extent of pig LD muscle infarction

measured by tracing the caudal transverse surface of muscle segments is highl y correlatrd

(mean coefficient of variation less than 4 percent) with that obszrved by tracing the

cranial transverse surface, or by weighing viable and nonviable muscle dissectrd from

the flap according to NBT stain on both transverse cut surfaces ".

2.4.2 Radioactive microsphere technique for assessrnent of muscle b l d fiow

nCo-labelled microspheres (New England Nuclear, Boston. MA) of 1 5.5 1 0.1 p m

diameter (mean I SD) were injected (- 150,000 microsphereskg) into the left venuicle

of pigs with bilateral LD muscle flaps for measurement of muscle blood tlow. using the

reference blood sampling technique . This method has been pl-eviously uscd for

assessrnent of muscle tlap blood tlow in the dog and pig, and the exact technique and

validity have been discussed -'-'*."'.

43

Bilateral LD muscle flaps were raised on pigs. The left carotid artery was

catheterizsd with a cutdown radiopaque catheter ( 1 J G ) , which was gently descendrd to

the level of the left ventricle for injection of microspheres. Both femoral artenes were

catheterized with polyethylene tubes (PE 240). The right femoral artery catheter was used

for monitoring mean arterial blood pressure ( Hewlett Packhard Model 78534A Monitor.

Andover, MA), whiie the left femoral anery catheter was connected to the syringe of a

withdrawl pump (Harvard Model 940. South Natick, MA) for collection of reference

arterial blood samples. The microspheres were suspended in 10 rnL of 0.99 saline

containing 5 8 sucrose and 0.05% Tween 80. sonicated for 5 min, then vortexed

vigoroudy for 1 min immediately prior to injection. The withdrawl pump was turned on

for 10 sec before and 60 sec after rnicrosphei-e injection at a rate of 4.12 mL 1 min. The

reference blood sample in the collection syringe was uansfemed to a counting vial. The

catheter and syringe were flushed to dislodge any adherent rnicrospheres, which were

then transferred to counting vials as reference blood sarnpie. The LD muscle flaps were

excised, cut uansversely into twelve 1-cm thick sections, and weighed. The

radioactivities of the muscle sections and the reference blood samples were measured on

a gamma counter (1282 Cornpugamma CS". Pharmacia Wallac, Turky, Finland). The pig

was sacrificed with an intracardiac overdose of pentobarbitone.

The following equation was prograrnmed on a microcornputer to calculate thé

bIood flow ratio:

radioactivity (cpm) in muscle sections

Muscle blood flow = -- x Withdrawlrate

(mL/min ) radioactivity (cpm) in reference blood (mumin)

sample

2.4.3 Myeloperoxidase (MPO) assay

MPO activity has been shown to be directly proportional to the number of

7 7 ?

activated neu~ophils in injured tissue -, and was used in this study as a measure of p s t -

ischemia neutrophil activity. Neutrophilic myeloperoxidase (MPO) activities were

determined by a modified method of Krawisz ? Al1 steps were performed at 4 "C or on

ice. Muscle samples (-300 mg) were homogenized (Polytron Kinematica GmbH. speed

setting 4.5) in 3 mL of ice-cold sodium chloride (0.9%) solution. and cenuifuged at

20,000 x g for 15 min. The supernatant was decanted, and the pellet suspended in ice-

cold 50 mM potassium phosphate buffer containing 0.5% hexadecyl trimethyl ammonium

bromide at pH 6.0 to release MPO from the neutrophil primary granules. This suspension

was homogenized, sonicatrd in an ice bath for 10 sec, and then ccnuifugrd at 20.000 x

g for 15 min. The supernatant was assayed for MPO activity by msasuring the H201-

dependent oxidation of 3.3'.5,5'-teuamethylbenzidine (TMB). A spectrophotorneter

(Beckman DU-50, Fullriton. CA) was used to measure the generation of oxidized TMB

at 655 nrn. The seaction mixture contained 10 pL of supernatant. 1.6 mM TMB, 0.3 mM

HP, 80 rnM sodium phosphate buffer (pH 5.4), 8% Np N-dimçthyl formamide, and 409

Dulbecco's phosphate-buffer saline in a total volume of 550 PL. MPO activity was

45

txpressed as initial velocity of absorbance increase at 655 nm per minute per gram wet

tissue. One unit of enzyme activity was defined as the amount of MPO that produced an

absorbance change of 1 .O O.D. min" - g wet wt-' at 37" C. This analysis was performrd

with the assistance of Dr. Huai Xu in our laboratory.

2.4.4 Protein kinase C translocation detection

Muscle ce11 fractionation

Crude membrane and cytosol fractions were prepared by a modifieci methcd of

Farese 223"24. Al1 steps were perform