Associate editor: C.L. Wainwright

Pharmacological modulation, preclinical studies, and new clinical features

of myocardial ischemic preconditioning

Claudio Napolia,b,*, Aldo Pintoc, Giuseppe Cirinod

aDepartment of Medicine, Federico II University of Naples, P.O. Box, Naples 80131, ItalybDepartment of Medicine-0682, University of California, San Diego, CA 92093, USA

cDepartment of Pharmacological Sciences, University of Salerno, Fisciano-Salerno 84100, ItalydDepartment of Experimental Pharmacology, Federico II University of Naples, Via D. Montesano 49, Naples 80131, Italy

Abstract

The term `̀ ischemic preconditioning (PC)'' was first applied to canine myocardium subjected to brief episodes of ischemia and

reperfusion that tolerated a more prolonged episode of ischemia better than myocardium not previously exposed to ischemia. Protective effect

of myocardial ischemic PC was demonstrated in several animal species, resulting in the strongest endogenous form of protection against

myocardial injury, jeopardized myocardium, infarct size, and arrhythmias other than early reperfusion. New onset angina before acute

myocardial infarction, episodes of myocardial ischemia during coronary angioplasty or bypass surgery, and the `̀ warm-up'' phenomenon

may represent clinical counterparts of the PC phenomenon in humans. Here, we have attempted to summarize pharmacological modulation,

preclinical studies, and new clinical features of ischemic PC. To date, the pathophysiological basis of the `̀ chemical PC'' is still not well

established, and `̀ putting PC in a bottle'' for clinical applications still remains a new pharmacological venture. D 2001 Elsevier Science Inc.

All rights reserved.

Keywords: Ischemic preconditioning; Pharmacology; Myocardial ischemia; Oxygen radicals; Adenosine; KATP channels

Abbreviations: AMI, acute myocardial infarction; CGRP, calcitonin gene-related peptide; DOG, 1,2-dioctanoyl-sn-glycerol; 5-HD, 5-hydroxydecanoate; hsp,

heat shock protein; IAA-94, indanyloxyacetic acid 94; IB-MECA, N6-(3-iodobenzyl)-adenosine-50-N-methyluronamide; JNK, Jun NH2-terminal kinase; KATP,

K+ channel sensitive to adenosine triphosphate; KIR, inward-rectifier K+; 125I-ABA, [125I]N6-4-amino-3-iodobenzyladenosine; MAPK, mitogen-activated

protein kinase; MIDCAB, minimally invasive direct coronary artery bypass; MLA, monophosphoryl lipid A; MOR-14, N-methyl-1-deoxynoirimycin; NO,

nitric oxide; iNOS, inducible nitric oxide synthase; PC, preconditioning; PIA, N6-(2-phenylisopropyl)adenosine; PKC, protein kinase C; PL, phospholipase; PP,

protein phosphatase; TK, tyrosine kinase; TNF, tumor necrosis factor.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

2. Pharmacological modulation of ischemic preconditioning . . . . . . . . . . . . . . . . . . . . 312

2.1. Triggers of myocardial ischemic preconditioning . . . . . . . . . . . . . . . . . . . . . 313

2.1.1. Adenosine receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

2.1.2. Opioid receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

2.1.3. Bradykinin and B2 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

2.2. Transducers of myocardial ischemic preconditioning . . . . . . . . . . . . . . . . . . . 316

2.2.1. Kinase cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

2.2.2. Phospholipase D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

2.3. End-effectors of myocardial ischemic preconditioning . . . . . . . . . . . . . . . . . . 317

2.3.1. Sarcolemmal and mitochondrial KATP channels . . . . . . . . . . . . . . . . . 317

2.4. Glycemic control of myocardial ischemic preconditioning? . . . . . . . . . . . . . . . . 318

* Corresponding author. Department of Medicine, Federico II University of Naples, P.O. Box, Naples 80131, Italy. Tel./fax: +39-81-5603990.

E-mail addresses: claunap@tin.it or cnapoli@ucsd.edu (C. Napoli).

Pharmacology & Therapeutics 88 (2000) 311 ± 331

0163-7258/01/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved.

PII: S0 1 6 3 - 7 2 5 8 ( 0 0 ) 0 0 0 93 - 0

2.5. A possible role in preconditioning for monophosphoryl lipid A and RC-552 . . . . . . . 318

2.6. Other possible mechanisms involved in myocardial ischemic preconditioning . . . . . . 319

3. From preclinical studies to new clinical features in myocardial ischemic preconditioning . . . . 320

3.1. Preinfarction angina and `̀ new-onset'' angina as models of myocardial ischemic

preconditioning in humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

3.2. Is the development of myocardial tolerance to ischemia in humans due

to ischemic preconditioning or to collateral recruitment? . . . . . . . . . . . . . . . . . 321

3.3. Myocardial stunning and ischemic preconditioning . . . . . . . . . . . . . . . . . . . . 322

3.4. Preconditioning and coronary artery bypass surgery. . . . . . . . . . . . . . . . . . . . 323

3.5. Arrhythmias and myocardial ischemic preconditioning . . . . . . . . . . . . . . . . . . 323

3.6. A loss of preconditioning in the aging heart? . . . . . . . . . . . . . . . . . . . . . . . 324

4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

1. Introduction

In 1926, Einstein told Heisenberg that it was nonsense to

found a theory on the basis of observable facts alone: `̀ In

reality the very opposite happens. It is theory which decides

what we can observe.'' Restating this comment in a some-

what less contentious fashion, we may add that a paradigm,

albeit a powerful influence upon what is observed, is

basically a complex framework that accumulates new data

with increasing time. Data are obtained that represent an

anomaly, which does not fit the accepted paradigm. Subse-

quently, this theory may be replaced by one that becomes

the new paradigm; the cycle then continues until a period of

anomaly arises again. Pursuing the idea of Einstein, we need

to comprehend that solid evidence in some experimental

models is only a good beginning of the story. Although a lot

of data provides renewed optimism for the inclusive under-

standing of `̀ ischemic preconditioning (PC),'' to date, we

continue to look for the anomaly(ies) that enables us to

resolve the puzzle in clinical conditions.

The results of the outstanding study of Murry et al.

(1986) undeniably represent a significant advance in our

broad understanding of the pathophysiology of tissue

ischemia. More recently, ischemic PC has been defined

as a powerful endogenous protective mechanism against

prolonged ischemic injury that has been shown to occur in

a variety of organ systems, including the heart, brain,

spinal cord, retina, liver, lung, and skeletal muscle. In

particular, myocardial ischemic PC is the phenomenon by

which a brief episode(s) of myocardial ischemia increases

the ability of the heart to tolerate a subsequent prolonged

period of ischemic±reperfusion injury; PC has both imme-

diate (early phase or first window of protection) and

delayed (late phase or second window of protection)

protective effects, the importance of which varies between

species and organ systems (reviewed in Li et al., 1990;

Downey, 1992; Walker & Yellon, 1992; Kloner & Yellon,

1994; Yellon & Baxter, 1995; Bolli, 1996; Connaughton &

Hearse, 1996; Kloner et al., 1998a). The distinction in

mediators and pathways activated in delayed PC (second

window) are summarized to the latest acquisition in Table

1. Several mediators involved in the classical early

response are also implicated in the second window of

protection. While the exact mechanisms of both protective

components are still unclear, ischemic PC could be defined

as a multifactorial pathophysiological process, requiring

the interaction of numerous cellular signals, second mes-

sengers, and end-effector mechanisms. Myocardial protec-

tion induced by PC develops approximately 7 days after

birth, and the inability of neonatal hearts to precondition

appears not to be due to insufficient stimulus or extended

ischemia (Awad et al., 1998). Stimuli other than ischemia,

such as hypoxic perfusion, tachycardia, and pharmacolo-

gical agents, may have PC-like effects (reviewed in Kloner

et al., 1998a).

We have examined here two aspects of research primarily

in the classical early phase of myocardial ischemic PC. First,

we have sought to examine pharmacological studies on the

modulation of PC by agonists and antagonists/inhibitors of

possible biological pathways involved in this phenomenon.

Second, we have focused the remaining part of the review

on the new clinical features, in the effort to elucidate which

aspects of experimental evidence and pharmacological

approach are involved in the human response.

2. Pharmacological modulation of

ischemic preconditioning

The hallmark of ischemic PC, documented in virtually

all species and experimental models evaluated to date in

countless laboratories worldwide, is the profound reduction

in infarct size and jeopardized myocardium seen in pre-

conditioned groups versus time-matched controls (Przyk-

lenk & Kloner, 1998). Of course, the main objective in

recent years has been to identify the possible triggers,

transducers, and end-effectors involved in the classical

early phase of ischemic PC to allow mimicking of the

physiological response by chemical agents. For example, it

has been observed that following mild chemical inhibition

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331312

of oxidative phosphorylation, it is possible to mimic PC

(Riepe & Ludolph, 1997). Efforts to identify the clinical

setting of possible therapeutic applications are all topics of

intensive on-going investigation, and this research field is

known as `̀ chemical PC'' (Wainwright, 1992; Riepe &

Ludolph, 1997).

2.1. Triggers of myocardial ischemic preconditioning

2.1.1. Adenosine receptors

Adenosine receptor involvement has been widely stu-

died, and there is a growing body of evidence for adenosine

receptor involvement in PC (for a review, see Miura &

Tsuchida, 1999). From the scientific rationale provided from

preclinical studies, several clinical approaches are now

undertaken using adenosine-mediated PC (see below and

Section 3).

In anesthetized dogs subjected to 1 hr of coronary artery

occlusion, followed by 4 hr of reperfusion, ischemic PC

was elicited by 10 min of coronary occlusion, followed by 1

hr of reperfusion before the 1-hr occlusion period (Yao et

al., 1997). PC resulted in a marked reduction in infarct size,

whereas administration of adenosine or bimakalim, fol-

lowed by a 1-hr drug-free period, had no effect. However,

the simultaneous administration of adenosine and bimaka-

lim resulted in a marked decrease in infarct size. One hour

after ischemic PC, administration of glibenclamide blocked

the protective effect of ischemic PC, whereas a selective

A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine

or a nonselective adenosine receptor antagonist PD-

115199 did not affect it. These data suggested that adeno-

sine and the K + channel sensitive to adenosine triphosphate

(KATP) may have a synergistic interaction that is important

for the memory phase of PC (Yao et al., 1997). A possible

role for the A1 receptor has been further addressed by using

PD 81,723, which acts allosterically to increase agonist

binding to A1 receptors and to enhance functional A1

receptor-mediated responses in the heart and other tissues

(Mizumura et al., 1996). In anesthetized dogs subjected to

ischemic PC, PD 81,723 was infused intracoronarily for

17.5 min before the 60-min occlusion period in nonprecon-

ditioned dogs or in dogs preconditioned with 2.5 min of

ischemia. Infarct size was not significantly affected by 2.5

min of PC alone or by PD 81,723 alone, but was decreased

by PD 81,723 plus PC or by a longer period (5 min) of PC

alone. Administration of the selective antagonist of A1

receptors such as 8-cyclopentyl-1,3-dipropylxanthine or

the KATP blocker glibenclamide for 15 min before PD

81,723 plus PC blocked the protection. Radioligand-bind-

ing studies were conducted using membranes derived from

COS-7 cells expressing recombinant canine receptors and

agonist radioligands. PD 81,723 enhanced the binding of

[125I]N6-4-amino-3-iodobenzyladenosine (125I-ABA) to A1

receptors by increasing the t1/2 for dissociation by 2.18-fold,

but PD 81,723 had no effect on the dissociation kinetics of125I-ABA from A3 receptors or [125I]-[2-(4-amino-3-iodo-

Table 1

Summary of the mechanisms involved in late preconditioning or second window of protection

Mechanism proposed Experimental model Reference

Nuclear factor kB Activation by NO Conscious rabbits Xuan et al., 1998

NO NO hypothesis of late PC Bolli et al., 1998

NO iNOS activation Rabbits Imagawa et al., 1999

iNOS activation Conscious rabbits Takano et al., 1998

PKC Activation by NO Conscious rabbits Ping et al., 1999a

PKC Translocation of PKCe from cytosolic

to particulate fraction

Conscious rabbits Qiu et al., 1998

PP1A, PP2A Undetermined Ca2 + -tolerant rabbit

cardiomyocytes

Armstrong et al., 1998

KATP channels Inhibition of MLA-induced late PC by

glibenclamide or 5-HD

Anesthetized rabbits Baxter et al., 1997

MLA Additive effect with adenosine Chick ventricular myocytes Stambaugh et al., 1999

d1 opioid KATP channel activation Rat Fryer et al., 1999

ND Rat Tsuchida et al., 1998

KATP channels Glibenclamide and 5-HD blockade Rabbits Bernardo et al., 1999

A1 receptor activation Protection against myocardial infarction

by repeated A1 stimulation

Rabbit Dana et al., 1998a

Not involved Conscious rabbit Maldonado et al., 1997

Hsp72 Not involved Rats Qian et al., 1999

TK Blockade by genisteine of late PC Rabbit Imagawa et al., 1997

Diacylglycerol Activation of PKC isoenzymes Anesthetized rabbits Mei et al., 1996

Free radicals Reactive oxygen species protect

coronary endothelium

Anesthetized rats Kaeffer et al., 1997

Blockade of late PC by

antioxidant therapy

Conscious pigs Sun et al., 1996

Lack of evidence of increased levels

of myocardial antioxidants

Conscious pigs Tang et al., 1997

ND, not determined.

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331 313

phenyl)ethylamino] adenosine from A2 receptors. Gliben-

clamide, at concentrations up to 10 mmol/L, had no effect on

the binding of radioligands to recombinant canine A1, A2,

or A3 receptors. These data suggested an important role for

the A1 receptor, and suggested that KATP receptor blockade

prevented the protection afforded by A1 receptor activation

by a mechanism not involving adenosine receptor blockade

(Mizumura et al., 1996). Moreover, the A1 receptor agonist

N6-(2-phenylisopropyl)adenosine (PIA) treatment reduced

myocardial infarction in the rabbit (Hashimi et al., 1998).

The anti-infarction effect of ischemic PC and adenosine was

significantly blocked by the A1 receptor antagonist 8-

cyclopentyl-1,3-dipropylxanthine and the KATP-channel

blockers Na + 5-hydroxydecanoate (5-HD) and glibencla-

mide. These observations indicated that adenosine, through

A1 receptors, initiates the mechanism of ischemic PC with

postreceptor involvement of KATP channels in the heart

(Hashimi et al., 1998).

The cardioprotective effect of a selective A1 receptor

agonist GR79236 also has been tested in a porcine model

of myocardial ischemia±reperfusion injury with controver-

sial results (Louttit et al., 1999; Smits et al., 1998).

GR79236 appeared not to reduce infarct size in pigs,

which suggests that under these experimental conditions,

stimulation of adenosine A2 receptors was important for

the cardioprotective effect of the A1/A2 receptor agonist

AMP 579 (see below). The adenosine-regulating agent

acadesine also failed to reduce infarct size (Smits et al.,

1998). However, when pigs were subjected to a 50-min

coronary artery occlusion, followed by a 3-hr reperfusion,

GR79236 significantly reduced infarct size whether given

10 min before the onset of ischemia or reperfusion. This

effect was independent of the bradycardia induced by

GR79236, as it was also observed in animals in which

heart rate was maintained by electrical pacing (Louttit et

al., 1999). However, GR79236 administered 10 min after

reperfusion did not reduce infarct size, and it had no effect

on the incidence or outcome of ventricular arrhythmias.

Also, in this case, the selective adenosine A1 receptor

antagonist 8-cyclopentyl-1,3-dipropylxanthine abolished

the hemodynamic and cardioprotective effects of

GR79236 (Louttit et al., 1999). More recently, the effect

of different body core temperatures was examined on

GR79236- or PC-induced cardioprotection when adminis-

tered prior to ischemia and on cardioprotection induced by

GR79236 administered 10 min prior to the onset of

reperfusion (Sheldrick et al., 1999). When rabbits were

subjected to a 30-min occlusion of the left coronary artery,

followed by a 2-hr reperfusion, GR79236 or PC, adminis-

tered or applied 10 min prior to the occlusion, significantly

limited the development of infarction. The cardioprotective

effect was significantly greater than that seen after admin-

istration of GR79236. Pretreatment with the selective

adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipro-

pylxanthine prevented the cardioprotective effect of

GR79236, but not that of PC. Maintaining body core

temperature at 38.5°C rather than at 37.0°C did not

influence infarct size in controls, but reduced the cardio-

protective effect of GR79236 when administered 10 min

prior to occlusion or 10 min prior to the onset of reperfu-

sion. The effect of PC was not temperature-dependent.

Thus, protection conferred by GR79236 in anaesthetized

rabbits is mediated via adenosine A1 receptors (Sheldrick

et al., 1999). Myocardial protection was conferred when

GR79236 was administered before the onset of ischemia or

reperfusion, and it was reduced when body core tempera-

ture was maintained at 38.5°C rather than at 37.0°C. In

contrast, protection by PC was not reduced by adenosine

A1 receptor blockade or by maintaining body core tem-

perature at 38.5°C rather than at 37.0°C. These findings

highlight the need for careful control of body core tem-

perature when investigating PC (Sheldrick et al., 1999).

The ability of mixed adenosine inhibitors to afford

cardioprotection has also been tested in pigs. The mixed

agonist AMP 579 was also tested in a model of myocardial

infarction in anesthetized pigs induced by a 40-min occlu-

sion of the left coronary artery, followed by 3 hr of

reperfusion (Smits et al., 1998). Administration of AMP

579 30 min before ischemia resulted in marked cardiopro-

tection, with a 98% reduction in infarct size. The cardio-

protective effect of AMP 579 was a consequence of

adenosine receptor stimulation, since it was completely

inhibited by pretreatment with the specific adenosine recep-

tor antagonist CGS 15943. Cardioprotection was shown not

to be dependent on changes in afterload or myocardial

oxygen demand, and was a consequence of adenosine

receptor stimulation.

The involvement of A3 receptors in PC has been studied

by using a Langendorff model of myocardial ischemia±

reperfusion injury (Tracey et al., 1997). Rabbit hearts were

exposed to 30 min of regional ischemia and 120 min of

reperfusion, and PC by 5 min of global ischemia and 10

min of reperfusion was able to reduce the infarct size.

Replacing global ischemia with a 5-min perfusion of the

rabbit A1-selective agonist N6-(3-iodobenzyl)-adenosine-50-N-methyluronamide (IB-MECA) elicited a concentration-

dependent reduction in infarct size. An A1-selective agonist

(R-PIA; 25 nM) in this same study reduced infarct size to a

similar extent. However, while the cardioprotective effect

of R-PIA (A1 selective) was significantly inhibited by the

rabbit A1-selective antagonist BWA1433, the IB-MECA-

dependent cardioprotection was unaffected. However, a

nonselective higher dose of BWA1433 significantly atte-

nuated the IB-MECA (A3-selective)-dependent cardiopro-

tection (Tracey et al., 1997). In a model of simulated

`̀ ischemia'' and `̀ reperfusion'' in quiescent human ventri-

cular cardiomyocytes, cellular injury and metabolic para-

meters were assessed after various interventions: cells were

preconditioned with anoxia, hypoxia, anoxic supernatant,

or hypoxic supernatant, with or without an adenosine

receptor antagonist or adenosine deaminase (Cohen et al.,

1998). Adenosine was applied before, during, or after

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331314

ischemia or continuously, with and without the adenosine

receptor antagonist. Cells were treated with the protein

kinase C (PKC) agonist phorbol myristate acetate (PMA)

and preconditioned cells were incubated with the PKC

antagonist calphostin-C. PC with anoxia was most protec-

tive. Protection was transferable via anoxic supernatant,

which produced the highest concentrations of adenosine,

and was lost when adenosine receptor antagonist or ade-

nosine deaminase was used. Exogenous adenosine was

most protective when administered before ischemia,

whereas if it was administered during ischemia, it was only

partially protective. An adenosine receptor antagonist abol-

ished the protective effects of adenosine. Adenosine repro-

duced the protective effects of PC, preserved ATP, and

increased lactate production, perhaps by stimulating glyco-

lysis (Tracey et al., 1997). In a rabbit Langendorff model of

myocardial ischemia±reperfusion injury (Hill et al., 1998),

adenosine was shown to be 19-fold selective for inhibition

of 125I-ABA binding to recombinant rabbit A1 when

compared with rabbit A3 receptors. Ischemic PC and

adenosine-mediated cardioprotection were completely

blocked in the presence of the rabbit A1-selective antago-

nist BWA1433 (Hill et al., 1998).

Although many agents that were thought to attenuate

reperfusion injury have been unsuccessful in clinical inves-

tigation, adenosine resulted in a significant reduction in

infarct size. These data have indicated that adenosine should

be investigated in large multicenter clinical outcome trials.

2.1.2. Opioid receptors

The role of opioid receptors in PC has also been widely

studied, and there are several lines of evidence for involve-

ment of the d opioid receptor type. The first evidence for a

role for opioids in PC was proposed by Schultz et al. (1995,

1996). By using the rat heart, they demonstrated that

naloxone pretreatment reduced the protection afforded by

PC (Schultz et al., 1995) and that morphine mimicked the

cardioprotective effect (Schultz et al., 1996). Similarly,

naloxone blocked myocardial ischemic PC in open-chest

rabbits (Chien & Van Winkle, 1996). Following this initial

evidence, opioid receptor involvement has been studied.

Naltrindole, a selective d opioid receptor antagonist, com-

pletely abolished the cardioprotective effect induced by PC

and morphine in open-chest rats (Schultz et al., 1997). d1

receptors have been shown to be involved in the cardio-

protective effect of ischemic PC in rats; however, the

mechanism by which d opioid receptor-induced cardiopro-

tection is mediated remains unknown. Several of the known

mediators of ischemic PC, such as the KATP channel and Gi/

o proteins, may be involved in the cardioprotective effect

produced by d1 opioid receptor activation. Involvement of

the d1 receptor has been shown by using a selective d1

agonist. Infusion of TAN-67, a new selective d1 agonist, for

15 min before the long occlusion and reperfusion periods

significantly reduced the infarct size, as compared with

control (Schultz et al., 1998b). The cardioprotective effect

of TAN-67 was completely abolished by 7-benzylidenenal-

trexone, a selective d1 antagonist; glibenclamide, a KATP-

channel antagonist; and pertussis toxin, an inhibitor of Gi/o

proteins. These results suggested that stimulating the d1

opioid receptor elicits a cardioprotective effect that is

mediated via Gi/o proteins and KATP channels in the intact

rat heart (Schultz et al., 1998b).

The possible involvement of a m or k opioid receptor has

also been studied in rats (Schultz et al., 1998a). Anesthe-

tized, open-chest rats were subjected to 30 min of occlusion

and 2 hr of reperfusion. Ischemic PC markedly reduced

infarct size, but naltriben, a selective d2 opioid receptor

antagonist; b-funaltrexamine, an irreversible m opioid recep-

tor antagonist; and the k opioid receptor antagonist nor-

binaltorphimine were unable to block the cardioprotective

effect of ischemic PC. Similarly the m opioid receptor

agonist D-Ala,2N-Me-Phe,4glycerol5-enkephalin had no

effect on infarct size. These results also indicated that d1

opioid receptors play an important role in the cardioprotec-

tive effect of ischemic PC in the rat heart. There is also some

evidence that k opioid receptors could play a role in PC in

isolated rat ventricular myocytes by using a selective kopioid receptor antagonist nor-binaltorphimine, or a k opioid

receptor agonist (Wu et al., 1999).

2.1.3. Bradykinin and B2 receptors

It was shown in 1996 by Miki et al. that captopril

potentiates PC without increasing kinin levels, and that

the effect of captopril can be reversed by HOE140, a

specific bradykinin receptor antagonist. This finding has

been further extended using B2 kinin receptor knockout

mice, as well as kininogen-deficient rats, demonstrating a

loss of the protective effect in these strains. The results

obtained in these experiments suggest that activation of

prekallikrein may contribute to the effect of PC and that an

intact kallikrein±kinin system is necessary for the cardio-

protective effect of PC (Yang, X. P. et al., 1997). Similarly,

in the human heart, captopril and lisinopril have been shown

to increase ischemic PC via B2 receptor activation (Morris

& Yellon, 1997). However, in one study, B2 receptor

involvement has been ruled out by using isolated rat hearts

(Bouchard et al., 1998). A possible cooperation between

adenosine receptors (A1/A3) and the B2 receptor has been

proposed (Giannella et al., 1997; Kaszala et al., 1997).

Using 1,1-diphenyl-2-picryl-hydrazyl to trigger free

radical injury in guinea-pig heart and PC to protect the

myocardium, it was demonstrated that bradykinin perfu-

sion protected the heart against radical injury (Jin & Chen,

1998). Pretreatment with PC and bradykinin resulted in

cardiac protection against free radical injury through the

activation of B2 receptors, suggesting that endogenous

generation of bradykinin may mediate PC in the guinea-

pig heart. Finally, bradykinin appears to be essential during

periods of PC ischemia of shorter duration (i.e., 3 min);

adenosine is more important during PC of longer duration

(i.e., 10 min) (Schulz et al., 1998).

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331 315

2.2. Transducers of myocardial ischemic preconditioning

2.2.1. Kinase cascade

The role of PKC in ischemic PC remains controversial

(for a review, see Simkhovich et al., 1998). However, there

are several studies that have addressed the role of PKC in

PC. It has been suggested that kinase activity itself is not

required to precondition the rabbit heart, but that some

processes upstream of PKC activation are responsible for

the triggering and memory of PC in rabbits (Yang X. M. et

al., 1997). PKC isoforms, namely a, d, and e, could be

implicated in PC (Yoshida et al., 1997). Using rabbit heart, it

has been shown that PC can be induced by b-adrenoreceptor

stimulation (Yabe et al., 1998). In rat hearts subjected to 40

min of ischemia and 30 min of reperfusion, isoproterenol

pretreatment for 2 min followed by 10 min of normoxic

perfusion enhanced the recovery of the rate-pressure product

of the ischemic/reperfused heart and attenuated the release

of creatine kinase during 30 min of reperfusion, similar to a

PC stimulus of 5 min of ischemia and 5 min of reperfusion.

Cardioprotection and ischemic PC was removed by treat-

ment with polymyxin B. In the same study, similar changes

were observed in the subcellular distribution of PKC fol-

lowing b-adrenergic stimulation and PC, suggesting trans-

location of PKC d, a marker of PKC activation, from the

cytosol to the membrane fraction. These results imply that

the cardioprotection induced by b-adrenoreceptor stimula-

tion, like ischemic PC, is mediated by PKC activation. The

role of PKC in PC has also been studied by using diacyl-

glycerol, an endogenous activator of PKC. Using the

diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG),

Galinanes et al. (1998) have evaluated the possible protec-

tive effect against injury during ischemia and reperfusion

and if the effect is mediated via PKC activation in rat heart.

A cardioprotective effect was achieved with DOG. How-

ever, it was less cardioprotective than ischemic PC, and the

achieved protection did not appear to necessitate PKC

activation prior to ischemia. Recently, a new 1,4-benzothia-

zepine derivative, JTV519, was investigated on ischemia/

reperfusion injury in isolated rat hearts (Inagaki et al.,

2000). The protective effect of JTV519 was completely

blocked by pretreatment of the heart with GF109203X, a

specific PKC inhibitor. In contrast, the effect of JTV519 was

not affected by a1-, A1-, and B2-receptor blockers that

couple with PKC in the cardiomyocyte. Immunofluores-

cence and immunoblots demonstrated that this agent

induced concentration-dependent translocation of the d iso-

form, but not the other isoforms of PKC, to the plasma

membrane. Thus, JTV519 may provide a novel pharmaco-

logical approach via a subcellular mechanism for mimicking

ischemic PC (Inagaki et al., 2000).

Tyrosine kinase (TK) inhibitors have also been shown to

attenuate ischemic PC. However, it is unclear whether TK is

involved in the initiation of and/or the maintenance of this

phenomenon. This hypothesis has been tested by using

genistein, a nonspecific TK inhibitor; daidzein, an inactive

structural analog of genistein; and lavendustin A, a more

specific TK inhibitor (Fryer et al., 1998). In the rat heart,

PC-induced cardioprotection was attenuated by genistein

pretreatment. However, genistein, administered during the

first or third occlusion period of PC, did not significantly

attenuate cardioprotection. Lavendustin A pretreatment also

attenuated PC, whereas daidzein had no effect, suggesting

that activation of a TK was involved in the initiation, but not

the maintenance, of PC. TK also appears to be involved in

ischemic PC in the rabbit heart (Baines et al., 1998) and in

pigs (Valhaus et al., 1998).

Recently, it has been shown in conscious rabbits by using

the MEK1 inhibitor PD-98059 that selective overexpression

of PKC induced activation of both p44 and p42 mitogen-

activated protein kinases (MAPKs), accompanied by trans-

location from the cytosol to the nucleus, and reduced lactate

dehydrogenase release during ischemia reperfusion (Ping et

al., 1999b). Another recent interesting observation in the

rabbit heart suggested the involvement of 12-lipoxygenase

metabolism in the cardioprotection induced by PC (Chen et

al., 1999), since WEB 2086 administration or PC reduced

the incidence of arrhythmias during reperfusion.

In another experimental setting, isolated perfused rat

hearts were adapted to ischemic stress by repeated ischemia

and reperfusion, and hearts were pretreated with genistein

to block TK, whereas SB-203580 was used to inhibit p38

MAPK (Maulik et al., 1998). Western blot analysis demon-

strated that p38 MAPK is phosphorylated during ischemic

stress adaptation. Phosphorylation of p38 MAPK was

blocked by genistein, suggesting that activation of p38

MAPK during ischemic adaptation was mediated by a TK

signaling pathway. Immunofluorescence microscopy with

an anti-p38 antibody revealed that p38 MAPK was loca-

lized primarily in perinuclear regions, and moved to the

nucleus after ischemic stress adaptation (Maulik et al.,

1998). Corroborating these results, myocardial adaptation

to ischemia improved left ventricular function and reduced

myocardial infarction, both of which were reversed by

blocking either TK or p38 MAPK. These results indicate

that myocardial adaptation to ischemia triggers a TK-

regulated signaling pathway, leading to the translocation

and activation of p38 MAPK, and implicating a role for

MAPK-Activated Protein Kinase-2 (MAPKAPK-2), a

kinase immediately downstream from p38 MAPK. Interest-

ingly, small heat shock proteins (hsps) have been implicated

in mediation of classic PC in the rabbit; hsp27 is a terminal

substrate of the p38 MAPK cascade (Armstrong et al.,

1999). p38 MAPK and hsp27 phosphorylation levels,

respectively, were also determined during in vitro ischemia

in control, calyculin A-treated [protein phosphatase (PP)

inhibitor], SB203580-treated, and preconditioned isolated

adult rabbit cardiomyocytes (Armstrong et al., 1999). The

dual phosphorylation of p38 MAPK was increased by early

ischemia (30±60 min), after which there was a loss of total

cytosolic p38 MAPK. The ischemic increase of p38 MAPK

dual phosphorylation was enhanced by PC. Calyculin A

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331316

strongly activated dual phosphorylation of p38 MAPK in

oxygenated cells, and this was maintained into early ische-

mia. SB203580 inhibited the dual phosphorylation of p38

MAPK and attenuated the loss of total cytosolic p38

MAPK. In each protocol, ischemia translocated hsp27 from

the cytosolic fraction to the cytoskeletal fraction at similar

rates and extents. After 90 min of ischemia, cytoskeletal

hsp27 was markedly dephosphorylated. During ischemic

incubation, calyculin A blocked ischemic dephosphoryla-

tion, SB203580 accelerated ischemic hsp27 dephosphoryla-

tion and injury, PC insignificantly decreased the initial rate

of ischemic dephosphorylation of hsp27, but not the extent

of dephosphorylation in later ischemia. Thus, the PC effect

was not correlated with a significant increase in cytosolic or

cytoskeletal hsp27 phosphorylation levels during prolonged

( > 60±90 min) ischemia (Armstrong et al., 1999). Finally,

in a very recent study, the regulation of MAPKAPK-2 and

the activity of c-Jun NH2-terminal kinase (JNK) were

examined in isolated, preconditioned rabbit hearts (Nakano

et al., 2000). Ventricular biopsies before treatment and after

20 min of ischemia were homogenized, and the activities of

MAPKAPK-2 and JNK were evaluated. For the MAP-

KAPK-2 experiments, seven groups were studied as fol-

lows: control hearts; preconditioned hearts; hearts treated

with R-PIA, an A(1)-adenosine receptor agonist; precondi-

tioned hearts pretreated with 8-( p-sulfophenyl) theophyl-

line, an adenosine receptor antagonist; preconditioned

hearts treated with SB 203580; hearts treated with aniso-

mycin (a p38 MAPK/JNK activator); and hearts treated

with both anisomycin and the TK inhibitor genistein. There

was a 3.8-fold increase in MAPKAPK-2 activity during

ischemia in preconditioned hearts. Activation of MAP-

KAPK-2 in preconditioned hearts was blocked by both 8-

( p-sulfophenyl) theophylline and SB 203580. MAPKAPK-

2 activity during ischemia increased 3.5- and 3.3-fold in

hearts pretreated with PIA or anisomycin, respectively.

MAPKAPK-2 activation during ischemia in hearts pre-

treated with anisomycin was blocked by genistein. In

separate hearts, anisomycin mimicked the anti-infarct effect

of PC, and that protection was abolished by genistein.

There was a comparable, modest decline in JNK activity

during 30 min of global ischemia in both groups. As a

positive control, a third group of hearts was treated with

anisomycin before global ischemia, and in these, JNK

activity increased by 290% above baseline. These data

confirm that the p38 MAPK/MAPKAPK-2 pathway is

activated during ischemia, only if the heart is in a pre-

conditioned state (Nakano et al., 2000).

2.2.2. Phospholipase D

Phospholipase (PL)D might play a role in myocardial

ischemic PC. There is a receptor-dependent PLD present in

the myocardium (Eskidilsen-Helmond et al., 1996). Activa-

tion of PKC via stimulation of PLC degrades membrane

phospholipids to diacylglycerol, an important PKC cofactor.

Propranolol, which blocks diacylglycerol production from

metabolites produced by PLD catalysis, completely abol-

ished the protective effects of PC in isolated rabbit hearts

(Cohen et al., 1996). These results were also confirmed in

the isolated rat heart (Tosaki et al., 1997). PLD also acts in a

synergistic activation by G-protein and PKC in the rat atria

(Lindmar & Loffelholz, 1998), suggesting a similar effect

during PC in vivo.

2.3. End-effectors of myocardial ischemic preconditioning

2.3.1. Sarcolemmal and mitochondrial KATP channels

KATP channels have been shown to play a critical role in

ischemic PC, and this hypothesis is supported by several

pieces of experimental evidence (Gross & Fryer, 1999;

Kevelaitis et al., 1999). Nicorandil, a KATP-channel opener,

has been shown to lower the threshold for the infarct-

reducing PC effect in dogs by activation of myocardial

KATP channels (Mizumura et al., 1997). The effects of

nicorandil and pinacidil have also been evaluated in isolated

and ischemic rabbit cardiomyocytes using the whole-cell

recording technique (Critz et al., 1997). Pinacidil increased

KATP current � 4-fold in isolated cardiomyocytes, and this

increase was reversed rapidly after treatment with the KATP-

channel blocker glibenclamide. Pinacidil protected cardio-

myocytes from simulated ischemia, but the protection

achieved from pinacidil was completely eliminated by

pretreatment with glibenclamide. In contrast, nicorandil,

which opens KATP channels in some tissues, caused no

detectable effect on the KATP current. Similarly, nicorandil

did not produce cardioprotection in this model of PC. These

results indicated that pinacidil and nicorandil have very

different effects on rabbit cardiomyocyte KATP channels.

Furthermore, because protection correlated with the ability

of the agent to open the channel, this supported a role for

KATP channels in PC.

Mitochondrial KATP channels have also been proposed to

be involved in PC (Gross & Fryer, 1999). Diazoxide, a

mitochondrial KATP-channel opener, was administered

before ischemia was protective, and protection was lost

when diazoxide was given after the onset of ischemia.

Anisomycin, a p38/Jun kinase activator, also reduced infarct

size, but protection from both diazoxide and anisomycin was

abolished by 5-HD, a selective inhibitor of mitochondrial

KATP channels (Baines et al., 1999). Moreover, it is possible

to simulate ischemia of isolated adult rabbit cardiomyocytes

in vitro by centrifuging the cells into an oxygen-free pellet

for 3 hr and to induce PC by prior pelleting for 10 min,

followed by resuspension for 15 min. Under these experi-

mental conditions, PC delayed the progressive increase in

osmotic fragility seen in nonpreconditioned cells. Incubation

with diazoxide or pinacidil was as protective as PC. Aniso-

mycin reduced osmotic fragility, and this was reversed by 5-

HD. Interestingly, protection by PC, diazoxide, and pinacidil

could be abolished by disruption of the cytoskeleton by

cytochalasin D. Nicorandil also exerts a direct cardioprotec-

tive effect on heart muscle cells, and this effect is believed to

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331 317

be mediated by selective activation of mitochondrial KATP

channels (Sato et al., 2000).

More recently, an interaction between KATP channels and

Na + /K + -ATPase, through sarcolemmal ATP, has been

shown to modulate the infarct size reducing effect of

ischemic PC (Haruna et al., 1998). To further complicate

this issue, d opioid receptors appeared to improve recovery

of cold-stored hearts to a similar extent as ischemic PC,

most likely through an opening of KATP channels (Kevelaitis

et al., 1999).

2.4. Glycemic control of myocardial

ischemic preconditioning?

By using isolated human right atrial trabeculae, it has

been shown that human myocardium from patients without

long-term exposure to oral hypoglycemic agents is function-

ally protected by PC, whereas long-term oral hypoglycemic

intake blocks the protection by PC (Cleveland et al., 1997).

Recent results in rats have shown that the sulfonylurea

receptor couples to several types of inward-rectifier K +

(KIR) channels, suggesting that glibenclamide blockade of

PC may not be the result of this compound selectively

blocking the KATP channel (Schultz et al., 1998c). Terikalant,

a KIR-channel blocker, did not abolish the cardioprotective

effect of ischemic PC at any dose tested, while glibencla-

mide completely abolished the cardioprotective effect, sug-

gesting that endogenous myocardial KIR channel does not

mediate ischemic PC in the rat heart, as opposed to the KATP

channel. Another new sulfonylurea recently studied is gli-

mepiride, a drug that is supposed to impact less on extra-

pancreatic KATP channels than the conventional drug glib-

enclamide. Glimepiride did not reduce myocardial PC, while

glibenclamide was able to prevent it (Klepzig et al., 1999).

N-methyl-1-deoxynoirimycin (MOR-14), an a-glucosi-

dase inhibitor, reduces the glycogenolytic rate by inhibiting

the a-1,6-glucosidase of the glycogen-debranching enzyme

in the liver, in addition to possessing an antihyperglycemic

action by blocking a-1,4-glucosidase in the intestine (Arai

et al., 1998). MOR-14 dose-dependently decreased the a-

1,6-glucosidase activity in rabbit heart extract, and also

dose-dependently reduced the infarct size without altering

the blood pressure or the heart rate (Arai et al., 1998). In

addition, MOR-14 decreased a-1,6-glucosidase activity to

� 20% in vivo, reduced glycogen breakdown, and attenu-

ated lactate accumulation during ischemia. Pre-ischemic

treatment with MOR-14 preserved glycogen, attenuated

the accumulation of lactate, and reduced infarct size by

69%, indicating that this effect could be associated with a-

1,6-glucosidase inhibition.

It has been suggested that increased mortality after acute

myocardial infarction (AMI) is correlated to alteration of

protection afforded by ischemic PC. This hypothesis was

tested in dogs subjected to a prolonged (60 min) coronary

artery occlusion and 3 hr of reperfusion, where glycemic

levels were simulated using an intravenous infusion of 15%

dextrose in water (Kersten et al., 1998). Modest degrees of

hyperglycemia (300 mg/dL) had no effect on infarct size,

but abolished the protective effect of ischemic PC. In

contrast, profound hyperglycemia (600 mg/dL) increased

infarct size without altering hemodynamic and coronary

collateral blood flow, indicating that acute hyperglycemia

adversely modulated myocardial injury in response to

ischemia in vivo.

2.5. A possible role in preconditioning for monophosphoryl

lipid A and RC-552

Monophosphoryl lipid A (MLA) represents a novel agent

capable of enhancing myocardial tolerance to ischemia/

reperfusion injury. This cardioprotective activity of MLA

manifests itself as a reduction in infarct size, myocardial

stunning, and dysrhythmias in multiple animal species (for

reviews, see Elliott, 1998; Zhao & Elliott, 1999).

The drug was effective in dogs and rabbits at doses of

10±35 mg/kg, with larger doses required in the rat. In the

rabbit infarct model, protection appeared 6 hr following

drug administration, and lasted for 36 hr. Although multi-

factorial mechanisms of ischemic tolerance may be induced

by MLA, current evidence suggests that the cardioprotective

effects of MLA involve myocardial inducible nitric oxide

synthase(s) [iNOS(s)] enzyme activation [for the role of

nitric oxide [NO] in the cardiovascular system, see Ignarro

et al., (1999)] with NO-coupled activation of myocardial

KATP channels upon ischemic challenge (Elliott, 1998;

Tosaki et al., 1998; Zhao & Elliott, 1999). MLA presently

is being evaluated in Phase 2 clinical trials in patients

undergoing cardiopulmonary bypass associated with coron-

ary artery bypass engraftment or aortic valve replacement or

reconstruction. Severity of lethal and reversible myocardial

injury and dysrhythmia are study endpoints. Although

further clinical testing will establish the utility of MLA as

a cardioprotectant against ischemia±reperfusion injury in

the human, presently this agent is proving very useful in

expanding our understanding of mechanisms responsible for

delayed cardiac PC against ischemia±reperfusion injury.

The role of opening of KATP channels in MLA-induced

myocardial protection after ischemia reperfusion has been

evaluated in rabbits using 5-HD to block MLA-stimulated

cardiac protection (Janin et al., 1998). Pretreatment with

MLA reduced infarct size in rabbits, whereas infarct size

increased with 5-HD in MLA-treated rabbits, suggesting

that MLA also exerted its protective effect through activa-

tion of KATP channels.

More recently, it was determined whether RC-552, a

novel synthetic glycolipid related in chemical structure to

MLA, could afford similar protection (Xi et al., 1999;

Zhao & Elliott, 1999). Adult mice were pretreated with

RC-552 24 hr before global ischemia and reperfusion in a

Langendorff isolated, perfused heart model (Xi et al.,

1999). A group of RC-552-treated mice received S-methy-

lisothiourea, a selective inhibitor of iNOS, 30 min before

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331318

heart perfusion. Myocardial infarct size was significantly

reduced in the RC-552-treated group, and treatment with

S-methylisothiourea abolished the RC-552-induced reduc-

tion in infarct size. More importantly, RC-552 failed to

reduce infarct size in isolated hearts from iNOS knockout

mice compared with that in hearts from control knockout

mice without drug treatment. Thus, RC-552 induces

delayed cardioprotection via an iNOS-dependent pathway.

2.6. Other possible mechanisms involved in myocardial

ischemic preconditioning

There are several other mediators whose involvement in

PC has been proposed. Here we will comment on some of

the most recent acquisitions.

It has been shown that endogenous calcitonin gene-

related peptide (CGRP) may play an important role in the

mediation of ischemic PC (Zhou et al., 1999). Whether

nitroglycerin provided a PC stimulus and whether the

cardioprotective effects of nitroglycerin-induced PC

involved endogenous CGRP has also been examined (Hu

et al., 1999). Pretreatment with nitroglycerin for 5 min

before ischemia improved cardiac function, decreased crea-

tine kinase release, and increased the content of CGRP-like

immunoreactivity in the coronary effluent.

Isoflurane may have cardioprotective effects that mimic

PC. Since KATP channels and adenosine receptors are

implicated in ischemic PC, it has been proposed that

isoflurane-induced PC and ischemia-induced PC share simi-

lar mechanisms, which include activation of KATP channels

and adenosine receptors (Ismaeil et al., 1999). Recent data

support a cardioprotective effect of isoflurane and, more

generally, demonstrate the feasibility of pharmacologically

PC the human heart during cardiac bypass surgery (Bel-

homme et al., 1999).

Cl ÿ channel involvement in the myocardial protection

afforded by ischemic PC has been shown in isolated rabbit

ventricular myocytes using two inhibitors [indanyloxyacetic

acid 94 (IAA-94) and 5-nitro-2-(3-phenylpropylamino)ben-

zoic acid] that abolished the protection afforded by ischemic

PC (Diaz et al., 1999).

Among the signal transduction pathways involved in the

mechanism of PC, mention has to be made of the role of

cyclic nucleotide phosphodiesterase, as well as of pH,

vacuolar proton ATPase, apoptosis, and diacylglycerol.

Fluctuations in cyclic nucleotides cyclic GMP and cyclic

AMP during PC has been associated with concomitant

changes in phosphodiesterase activity (Lochner et al.,

1998). Moreover, blockade of vacuolar proton ATPase

prevented the effect of PC, suggesting that acidification,

even in the absence of Na + /H + exchange, may lead to cell

death. This implies that a possible target of PKC in mediat-

ing PC could be the activation of vacuolar proton ATPase

(Gottlieb et al., 1996).

The role of PPs has also been investigated. Fostriecin, a

potent inhibitor of PP2A, at a concentration selective for

the inhibition of PP2A, mimicked PC in both rabbit and

pig cardiomyocytes (Armstrong et al., 1997). The role of

PPs during ischemic PC in the rabbit heart was further

examined. Fostriecin was administered to rabbit hearts, and

the effect of fostriecin pretreatment was assessed by

measuring changes in cell osmotic fragility during simu-

lated ischemia. PP1 and PP2A activities of control and

ischemically preconditioned cells were also measured

(Weinbrenner et al., 1998). In hearts pretreated with fos-

triecin, only 8% of the ischemic zone infarcted, signifi-

cantly less than that in untreated control hearts, but

comparable with that in ischemic preconditioned hearts.

In isolated myocytes, fostriecin also provided protection

comparable with that produced by metabolic PC. PC had

no apparent effect on the activity of either PP1 or PP2A in

isolated ventricular myocytes. Thus, fostriecin protected the

rabbit heart from infarction, even when administered after

the onset of ischemia.

b-adrenoreceptors have been proposed to play a role in

PC, and several studies indicate that ischemia-induced

activation of the b-adrenergic signaling pathway during

PC should also be considered a trigger in eliciting PC

(Lochner et al., 1999). A brief period of stimulation of

cardiac b-adrenoreceptors with isoproterenol or norepi-

nephrine, but not phenylephrine, caused a PC mimetic

effect against postischemic contractile dysfunction in per-

fused rat heart (Nasa et al., 1997). This effect seems to be

mediated in part by activation of PKC (Yabe et al., 1998).

Moreover, overexpression of the rat-inducible hsp70 in

transgenic mouse increased the resistance of the heart to

ischemic injury (Marber et al., 1995).

The role of inositol 1,4,5-trisphosphate in PC initially

has been proposed by using neomycin as a pharmacological

tool (Bauer et al., 1999). Ischemic PC in the rabbit heart

causes an increase in inositol 1,4,5-trisphosphate, which is

prevented by neomycin treatment. However, infarct size

was similar in both neomycin and control preconditioned

hearts. In contrast, it was determined whether an agonist

and an antagonist of the second messenger inositol 1,4,5-

trisphosphate signaling, D-myo-inositol-1,4,5-trisphosphate

hexasodium salt and 2-aminoethoxydiphenyl borate, respec-

tively, given that they mimic this biphasic profile, would

mimic infarct size reduction with PC in isolated rabbit hearts

(Gysembergh et al., 1999). Infarct size was reduced with PC

and in all D-myo-inositol 1,4,5-trisphosphate hexasodium

salt- and 2-aminoethoxydiphenyl borate-treated groups ver-

sus control. Thus, pharmacological manipulation of the

second messenger inositol 1,4,5-trisphosphate signaling

appeared to mimic the cardioprotective effects of PC in

isolated rabbit heart (Gysembergh et al., 1999).

Furthermore, it is noteworthy that the hypothesis has

been forwarded that tumor necrosis factor (TNF)-a also has

a role in ischemic PC. Using perfused rat heart, it has been

shown that ischemia reperfusion induced increases in TNF-

a in the rat heart and impaired myocardial function, while

sequestration of myocardial TNF-a improved postischemic

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331 319

myocardial function (Meldrum et al., 1998). TNF-a also

establishes a complex network with the protease-activating

receptor-2 (Cirino et al., 2000) during myocardial ischemic

injury in the rat (Napoli et al., 2000). Finally, some studies

have suggested that free radicals (oxygen radicals) can

contribute to PC by directly stimulating PLs and/or tran-

scription signaling (Baines et al., 1997; Tritto et al., 1997),

and that hypercholesterolemia did not appear to limit the PC

effect (Kremastinos et al., 2000) in the rabbit heart.

3. From preclinical studies to new clinical features in

myocardial ischemic preconditioning

Several clinical conditions may mimic ischemic PC

(reviewed in Dana et al., 1998b; Schwarz et al., 1999;

Yellon & Baxter, 2000). Repeated coronary artery occlu-

sions during coronary angioplasty might simulate PC

(Deutsch et al., 1990; Cribier et al., 1992). In this condition,

adenosine can mimic the protective effect (Leesar et al.,

1997) and, accordingly, the adenosine antagonists block it

(Claeys et al., 1996). Preliminary data in humans also

suggest that naloxone may reduce the effects of PC during

angioplasty (Tomai et al., 1999b). Pharmacological PC has

also been shown to be induced by nicorandil in patients

undergoing coronary angioplasty (Matsubara et al., 2000).

Finally, in a recent study (Leesar et al., 1999), bradykinin

appears to precondition human myocardium against ische-

mia in vivo in the absence of systemic hemodynamic

changes. Pretreatment with bradykinin appeared to be just

as effective as ischemic PC, and could be used prophylacti-

cally to attenuate ischemia in patients undergoing coronary

angioplasty. Thus, several compounds with PC-like effects

could reduce myocardial ischemia during angioplasty.

In the Acute Myocardial Infarction Study of Adenosine

trial, the hypothesis was tested in humans that adenosine

given as an adjunct to thrombolysis would reduce myocar-

dial infarct size (Mahaffey et al., 1999). The Acute Myo-

cardial Infarction Study of Adenosine trial was a

prospective, open-label trial of thrombolysis, with randomi-

zation to adenosine or placebo in 236 patients within 6 hr of

infarction onset. The primary endpoint was infarct size, as

determined by 99Tc-Sestamibi single-photon emission-com-

puted tomography imaging. Secondary endpoints were

myocardial salvage index and in-hospital clinical outcome

(death, reinfarction, shock, congestive heart failure, or

stroke). There was a significant reduction in infarct size

with adenosine (33%; P < 0.05) associated with a better

clinical outcome, indicating a possible application of ade-

nosine-mediated PC in larger multicenter clinical studies.

Interestingly, nucleoside transport inhibitors, that is, dipyr-

idamole, have been shown to enhance infarct size limitation

by PC in the rabbit heart, most likely as a consequence of

elevated interstitial adenosine levels (Suzuki et al., 1998).

Since dipyridamole is widely used clinically, a possible role

for modulating adenosine levels in PC of humans has been

debated (Seiler & Billinger, 1998). Previous studies showed

that intracoronary application of dipyridamole in patients

undergoing coronary angioplasty significantly improved

myocardial function during the angioplasty procedure

(Strauer et al., 1996). Similarly, intravenous low-dose dipyr-

idamole increased peak adenosine plasma levels and

mimicked PC with respect to electrocardiographic and

echocardiographic findings (Pasini et al., 1996). Low-dose

dipyridamole infusion increases exercise tolerance in

patients with chronic stable angina, possibly by endogenous

adenosine accumulation acting on high-affinity A1 myocar-

dial receptors involved in PC or positively modulating

coronary flow through collaterals (Tommasi et al., 2000).

Finally, in the double-blind randomized Clinical European

Studies in Angina and Revascularization-2 investigation, the

addition of nicorandil to anti-anginal treatment was shown

to reduce transient myocardial ischemia and arrhythmias in

patients with unstable angina, an effect that can be attributed

to chemical PC (Patel et al., 1999).

PC is also observed in preinfarction angina and during

coronary artery bypass surgery (see Section 3.4). The

`̀ warm-up'' phenomenon may represent another clinical

counterpart of myocardial ischemic PC (Okazaki et al.,

1993; Maybaum et al., 1996; Baxter, 1997; Cohen &

Downey, 1999). In fact, patients with coronary heart disease

(usually with stable angina) are able to exercise longer

before developing chest pain, and may develop less angina

and signs of myocardial ischemia during a second exercise

test compared with the first exercise when exercise tests are

divided by a rest period. The pathophysiology and the

mechanisms of the warm-up phenomenon are not well

established (Cohen & Downey, 1999). In patients with

exertional angina, the size of the warm-up response is

related to the maximum intensity rather than the duration

of first exercise (Kay et al., 2000). Patients with coronary

heart disease were randomized to receive glibenclamide or

placebo, and then stimulated to perform serial exercise tests

(Tomai et al., 1999a). After placebo administration, rate-

pressure product at 1.5 mm ST-segment depression signifi-

cantly increased during the second exercise test compared

with the first, but it did not change after glibenclamide.

Thus, glibenclamide, at a dose previously shown to abolish

ischemic PC during coronary angioplasty, prevented the

increase of ischemic threshold observed during the second

of two sequential exercise tests.

Mortality in diabetic patients in the Diabetes and Insu-

lin-Glucose Infusion in Acute Myocardial Infarction study

with AMI is predicted by age, previous heart failure, and

severity of the glycometabolic state at admission, but not

by conventional risk factors or sex (Malmberg et al., 1999).

Intensive insulin treatment reduced long-term mortality,

despite high blood glucose at admission and glycosylated

hemoglobin. Patients receiving oral hypoglycemic agents

for diabetes mellitus have been indicated to be at an

increased relative risk of cardiovascular mortality after

direct angioplasty for myocardial infarction (Garratt et al.,

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331320

1999). Since oral hypoglycemic agents are inhibitors of the

KATP channel, it has been suggested that myocardium from

patients taking long-term oral hypoglycemic agents would

be resistant to protection by ischemic PC.

Although the research is very active in understanding

the mechanisms of PC and several large studies are actually

in progress, we sought to analyze the `̀ state of the art''

clinical evidence for ischemic PC. In particular, we attempt

to describe the newly developed hypothesis that has arisen

from preclinical studies and, therefore, new possible cli-

nical challenges.

3.1. Preinfarction angina and `̀ new-onset'' angina as

models of myocardial ischemic preconditioning in humans

Several studies (Iwasaka et al., 1994; Kloner et al., 1995;

Nakagawa et al., 1995; Ottani et al., 1995; Napoli et al.,

1998b) support the idea that preinfarction angina may act as

a common PC stimulus. In this condition, two key points

should be kept in mind. First, the analysis of the mechan-

ism(s) by which previous episodes of angina exert a

beneficial clinical effect on myocardial function after myo-

cardial infarction. Second, the exact temporal definition of

the term `̀ preinfarction angina.''

For instance, patients with angina prior to AMI have

been shown to have better preserved left ventricular perfor-

mance (Matsuda et al., 1984; Hirai et al., 1992; Iwasaka et

al., 1994), and the Thrombolysis and Angioplasty in Myo-

cardial Infarction Study Group (Muller et al., 1990) reported

less short-term complications and fewer episodes of re-

occlusion after thrombolysis in patients with preinfarction

angina within 1 week. Probably, the difference in residual

contractile function was due in part to recruitment of

coronary collateral flow. In fact, Cortina et al. (1985)

reported better preservation of ventricular function in

patients with preinfarction angina (ranging from 1 month

to 8 years), but it was shown angiographically that visible

collaterals played a significant protective role. More

recently, Nakagawa et al. (1995) also showed the protective

effect of previous angina in patients with reperfused anterior

wall myocardial infarction. However, we have to consider

that large studies showed that those with angina did worse in

terms of prognosis (Barbash et al., 1992; Behar et al., 1992).

Indeed, patients with antecedent stable angina often have

multivessel coronary heart disease, and are prescribed sev-

eral anti-ischemic drugs that are taken for several months or

years. These considerations can additionally affect the

clinical outcome following myocardial infarction. In order

to minimize the impact of several variables, we analyzed the

recovery of regional myocardial contractile function in

patients after thrombolysis, in whom myocardial infarction

occurred unheralded, in comparison with patients who had

new-onset angina within 48 hr before their first myocardial

infarction (Napoli et al., 1998b). We showed for the first

time that recovery of myocardial contractile function after

thrombolysis was more prompt and durable in patients in

whom myocardial infarction was preceded by new-onset

angina, as compared with patients in whom infarction

occurred unheralded. Moreover, TIMI-9B prospectively

determined the importance of the time of onset of preinfarc-

tion angina in relation to 30-day outcomes (Kloner et al.,

1998c). Of the 3002 patients entered into the study, 425

reported angina before their myocardial infarction, and,

more important, patients with angina onset within 24 hr of

infarction had a lower 30-day cardiac event rate (4%) in

terms of mortality, recurrent myocardial infarction, heart

failure, or shock than those with onset of angina >24 hr

(17%). Therefore, these temporal observations (Kloner et

al., 1998c; Napoli et al., 1998b) suggest that human PC is

induced by new-onset preinfarction angina. Interestingly,

although a complete myocardial reperfusion is mandatory

for PC (Ovize et al., 1992b), it has been shown that the

presence of a critical coronary artery stenosis does not

abolish the protective effect of PC (Kapadia et al., 1997).

This phenomenon may be similar to the clinical scenario in

which brief ischemic episodes and reperfusion, superim-

posed on a critical coronary stenosis, precede a prolonged

occlusion determining AMI. Thus, new-onset angina may

be a representative clinical condition in which the presence

of a critical stenosis does not abolish the beneficial effect of

ischemic PC. Moreover, further studies are necessary in

order to better understand pathophysiological mechanisms

underlying atherogenesis in humans (Napoli et al., 1999a;

Ross, 1999), coronary vasomotion (Maseri et al., 1999), and

myocardial microcirculation (Tritto & Ambrosio, 1999).

Finally, it is well known that early administration of

thrombolysis after AMI is crucial in both limiting infarct

size and preserving left ventricular function (Lincoff &

Topol, 1993). Andreotti et al. (1996) proposed that the

benefit of preinfarction angina with respect to infarct size

may partially depend on faster coronary thrombolysis,

besides ischemic PC. New-onset angina may be associated

with newly formed thrombi that are thrombolyzed faster

than isolated and persistent growth thrombi, and a trend

toward shorter reperfusion times was also seen in other

studies (Ottani et al., 1995; Napoli et al., 1998b).

3.2. Is the development of myocardial tolerance to

ischemia in humans due to ischemic preconditioning or

to collateral recruitment?

The time needed for developing new collaterals after

acute coronary occlusion in humans is still unclear

(Ambrose & Fuster, 1983; Sasayama & Fujita, 1992).

Schwartz et al. (1984) showed that � 2 weeks is required

to develop new visible collateral vessels after myocardial

infarction. Better preservation of ventricular function in

patients with preinfarction angina may depend upon collat-

erals (Cortina et al., 1985). However, the retrospective

analysis of patients enrolled in the Thrombolysis in Myo-

cardial Infarction 4 Trial has shown that preinfarction angina

resulted in a lower incidence of in-hospital death, severe

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331 321

heart failure, or shock, and a smaller infarct size, and this

protection was not dependent on angiographically visible

epicardial coronary collateral blood vessels (Kloner et al.,

1995). Similarly, the absence of visible collaterals during the

beneficial effects of PC was seen in other studies (Ottani et

al., 1995; Napoli et al., 1998b).

In a recent study, the contribution of ischemic, as well as

adenosine-induced, PC and of collateral recruitment to the

development of tolerance against repetitive myocardial

ischemia has been elegantly investigated in patients with

quantitatively determined, poorly developed coronary col-

laterals (Billinger et al., 1999). Myocardial adaptation to

ischemia was measured using intracoronary electrocardio-

graphic ST segment elevation changes during three subse-

quent 2-min balloon occlusions in 30 patients undergoing

coronary angioplasty. Simultaneously, an intracoronary

pressure-derived collateral flow index was determined as

the ratio between distal occlusive minus central venous

pressure, divided by the mean aortic minus central venous

pressure. Results showed that collateral flow index at the

first occlusion was not different between the groups, and it

increased significantly during the third occlusion. Thus,

even in patients with few coronary collaterals, the myocar-

dial adaptation to repetitive ischemic episodes appeared to

be closely related to collateral recruitment. When adenosine

was used before coronary angioplasty, the phenomenon did

not occur. Therefore, the variable responses of ECG indi-

cators of ischemic adaptation to collateral opening suggest

that ischemic PC is a relevant factor in the development of

ischemic tolerance.

3.3. Myocardial stunning and ischemic preconditioning

The postischemic myocardial dysfunction termed `̀ myo-

cardial stunning'' should not be considered as a single entity,

but rather, as a `̀ syndrome'' observed in several experi-

mental settings, which include the following: (1) stunning

after a single, completely reversible episode of regional

ischemia in vivo; (2) stunning after multiple and reversible

episodes of regional ischemia in vivo; (3) stunning after a

partly reversible episode of regional ischemia in vivo (sub-

endocardial infarction); (4) stunning after global ischemia in

vitro and in vivo; and (5) stunning after exercise-induced

ischemia (high-flow ischemia) (Bolli & Marban, 1999).

Although the pathogenesis of myocardial stunning has not

been definitively established, the two major hypotheses are

that it is caused by the generation of oxygen radicals (radical

hypothesis) and/or by a transient Ca2 + overload (Ca2 +

hypothesis) during reperfusion (Bolli & Marban, 1999).

However, these hypotheses are not mutually exclusive and

are likely to represent different aspects of the same patho-

physiological scenario. In fact, increased oxygen radical

formation could also cause cellular Ca2 + overload, which

would damage myocardial contractile function, and this

could also directly alter filaments of myocytes in a manner

that renders them less responsive to Ca2 +.

Murry et al. (1991) firstly studied the relationship

between ischemic PC and stunning in an experimental

model. If the reperfusion phase between the brief PC

ischemia and the 40-min occlusion was extended to 120

min, the myocardium remained severely stunned after the

brief ischemia, but the myocardial infarct size plotted against

collateral flow returned toward nonpreconditioned values.

Matsuda et al. (1993) showed that dobutamine could be used

to reverse stunning induced by four 5-min coronary occlu-

sions in the dog model, but that reversing stunning did not

prevent PC. These studies suggested that although brief

periods of ischemia induce both stunning and PC, the two

phenomena could be dissociated and PC may be not due

only to reduced contractile function of stunned myocardium.

In other studies, the efficacy of ischemic PC in reducing

stunning has not been as consistent as its ability to reduce

necrosis. It is unclear, therefore, whether the protection

afforded by PC against stunning is mediated by the same

mechanism that mediates its protection against lethal cell

injury (Asimakis et al., 1992; Ovize et al., 1992a). More

recently, experimental studies have shown that ischemic PC

significantly increased the maximal inotropic response and

diminished the contractile dysfunction of early stunning

(Mosca et al., 1998), and PC also reduced myocardial

stunning, preserving high-energy phosphates after cardiac

transplantation (Landymore et al., 1998).

However, it is important to note that contractile dysfunc-

tion may persist for hours, or even for as long as several

days, before function recovers (Bolli & Marban, 1999). In

contrast, prolonged contractile impairment may stem from

development of reversible myocyte damage caused by

ischemic episodes, whereas evidence of contractile dysfunc-

tion in the presence of abnormalities of myocardial flow

manifests persisting or recurrent ischemia (Bolli & Marban,

1999). Thus, the effects of late PC (second window of

protection) may affect prolonged contractile impairment. In

this respect, we showed that patients with new-onset angina

before infarction had an endogenous protection of left

ventricular ejection fraction and wall motion until 3 months

of follow-up (Napoli et al., 1998b). Moreover, ischemic PC

also improved cardiac function at 30 min and 12 hr after

reperfusion in valve-replacement patients (Li et al., 1999).

The possible protective mechanism was that ischemic PC

decreased the production of oxygen radicals. Taken

together, these studies (Napoli et al., 1998b; Li et al.,

1999) strongly suggested that late PC may exert beneficial

effects on postischemic contractile dysfunction, that is,

stunning, in humans. Accordingly, it was also demonstrated

recently in a single-center prospective study that the bene-

ficial effect of preinfarction angina on left ventricular wall

motion is independent of collateral flows (Noda et al.,

1999). The greater protective effect of a longer time interval

between angina pectoris and AMI also suggested that the

protection was due to a delayed PC effect.

MLA was shown to attenuate myocardial stunning in

dogs, and a recent study investigated this drug in stunning

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331322

and PC (Elliott et al., 1998). To induce stunning, anesthe-

tized dogs were subjected to five cycles of 5 min of

coronary occlusion with 10 min of reperfusion, and finally,

followed by 2 hr of reperfusion. Single intravenous doses of

MLA in the range of 10±35 mg/kg given 24 hr before

ischemia resulted in an improvement in the number of

hypokinetic segments over a 2-hr reperfusion period. Car-

dioprotection against stunning with MLA appeared to

require activation of KATP channels during ischemia,

because glibenclamide completely blocked the protective

effect of MLA. Thus, MLA improved stunning by a KATP-

sensitive channel-dependent process during late ischemic

PC. These studies suggest that MLA also may exert positive

effects on stunning and PC in humans.

3.4. Preconditioning and coronary artery bypass heart

surgery

Mentzer et al. (1997) were the first to apply adenosine-

mediated PC to patients undergoing coronary artery bypass

surgery. More recently, minimally invasive direct coronary

artery bypass (MIDCAB), which utilizes alternative inci-

sions and `̀ port-access'' technology, provides many anes-

thetic challenges, including intense monitoring, managing of

myocardial ischemia, and pain control (Chitwood et al.,

1999). Recent advances in videoscopic visualization and

evolving mechanisms of myocardial protection may justify

the expanding application of MIDCAB. A recent study has

evaluated the monitoring requirements and the potential

benefits of PC and intrathecal morphine sulfate in MIDCAB

patients (Heres et al., 1998). Transesophageal echocardio-

graphy was used, and a pulmonary artery catheter was used

in 43% of the patients. PC did not prevent increases in

systemic or pulmonary artery pressures during coronary

occlusion. MIDCAB may reduce the length of hospital stay

for patients with single-vessel coronary artery lesions, when

compared with classical median sternotomy. Early PC

induced by a single 5-min test coronary occlusion did not

protect against subsequent regional ischemic dysfunction in

the subset of patients with normal baseline function. The

effects of early ischemic PC during MIDCAB were also

investigated in another recent study (Malkowski et al.,

1998). Left ventricular wall motion score increased signifi-

cantly from baseline to coronary occlusions 1 and occlusion

2, whereas no difference in wall motion was noted between

coronary occlusions 1 and 2. Pulmonary artery systolic

pressure increased significantly from baseline to coronary

occlusion 1 and occlusion 2. Pulmonary artery diastolic

pressure also increased significantly from baseline to cor-

onary occlusion 1 and occlusion 2, whereas no significant

differences in pulmonary artery pressures were noted

between coronary occlusions 1 and 2. Thus, ischemic dys-

function was precipitated by the early ischemic PC induced

by a 5-min coronary occlusion, as shown by the increase in

left ventricular wall motion score and pulmonary artery

pressure. This study indicated that early ischemic PC

induced by a 5-min coronary occlusion and the resulting

ischemia did not alter regional left ventricular systolic

function during subsequent ischemia in humans.

3.5. Arrhythmias and myocardial ischemic preconditioning

The efficacy of early ischemic PC in reducing arrhythmias

in experimental models has not been as consistent as its

ability to reduce necrosis (Sariahmetoglu et al., 1998; Przy-

klenk & Kloner, 1995; Hagar et al., 1991; Shiki & Hearse,

1987). More recently, it was demonstrated that KATP chan-

nels and opioid receptors may be partly involved in the

suppression of reperfusion arrhythmias, although their roles

may be compensated for by other antiarrhythmic mechan-

isms in repetitive PC (Kita et al., 1998). In contrast with the

effects of early PC against infarction, PKC is unlikely to play

a major role in protection afforded by PC against reperfusion

arrhythmias in the rat (Kita et al., 1998). Interestingly, in a

very recent study, it was shown that in rats neither maturation

nor gender influence the antiarrhythmic effect of early

ischemic PC; however, female rats exhibit a lower level of

arrhythmic activity during sustained coronary artery occlu-

sion than male rats, both in vivo and in vitro (Humphreys et

al., 1999). There are also effects of PC that are species-related

(reviewed in Wainwright, 1992; Sun et al., 1996;). For

example, ischemic PC increases both the arrhythmic index

and the incidence of ventricular fibrillation during the early

phase of a subsequent ischemic period in the pig (Grund et

al., 1997). The progressive electrocardiographic deteriora-

tion and increasing incidence of ventricular arrhythmias

during repetitive 15-min occlusions in pigs suggested

increasing metabolic derangement. However, the progres-

sively faster normalization of the ST segment and the reduced

incidence of ventricular arrhythmias during reperfusion sug-

gested an increasingly faster restoration of the metabolic and

ionic balance (Figueras et al., 1996).

An important question on the possible mechanism

exerted by PC in protecting the myocardium from arrhyth-

mias in humans was postulated by Lawson and Hearse

(1994). It is important to understand whether this beneficial

effect is due to an antiarrhythmic action per se or to a

general anti-ischemic phenomenon. At present, the exact

nature of the protective mechanism(s) exerted by PC on

arrhythmias is still not well established.

It is well known that QT interval dispersion reflects

regional variations in ventricular repolarization and cardiac

electrical instability. The effect of ischemic PC on the

manner of ventricular repolarization was investigated by

assessing the change in QT dispersion during coronary

angioplasty (Okishige et al., 1996). The gradual decrease

in QT dispersion provoked by coronary artery occlusion and

reperfusion during coronary angioplasty may be associated

with the electrophysiologic effects of ischemic PC on

human myocardium. Moreover, animals treated with cardiac

pacing (Parratt et al., 1996) and patients with severe angina

within 24 hr of onset of their first myocardial infarction, but

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331 323

not those without preceding angina (Tamura et al., 1997),

had reduced occurrence of life-threatening ventricular

tachyarrhythmias and late potentials mainly associated with

reperfusion. Early PC also induced a significant protection

against ischemia-induced complex ventricular arrhythmias

(more than five premature ventricular beats per minute or

repetitive ventricular arrhythmias) in patients with variant

angina (Pasceri et al., 1996). This beneficial effect was not

related to a reduction in either severity or duration of

ischemia, suggesting that arrhythmic protection was a direct

consequence of PC rather than an epiphenomenon of

ischemic protection.

3.6. A loss of preconditioning in the aging heart?

The geriatric population is considerably increased around

the world. Thus, more elderly patients have coronary heart

disease, which is often the cause of sudden death (Gersh,

1986; Toefler et al., 1988).

Abete et al. (1996) first showed that ischemic tolerance

induced by PC was reduced in the rat senescent heart. This

finding appeared to be species-related. In fact, in the ovine

senescent heart, the cellular pathways involved with the PC

response were well preserved (Burns et al., 1996). Later,

Tani et al. (1997) confirmed that hearts became more

vulnerable to ischemia with age, and that the beneficial

effects of PC were reversed in middle-aged rat hearts. More

recently, it was demonstrated that in the senescent rabbit

myocardium, the cellular pathways involved ischemic PC,

such as postischemic dysfunction, and that functional reco-

very was worse compared with that of the adult myocardium

(Uematsu & Okada, 1998). Moreover, using adenosine-

enhanced ischemic PC, it was shown that this treatment

provided similar protection to Mg2 + -supplemented K +

cardioplegia, significantly enhancing postischemic func-

tional recovery and decreasing infarct size in the rabbit

senescent myocardium (McCully et al., 1998). PC also

failed to lessen the increased [Na + ]i or to protect the aging

hearts, probably due to the pre-existence of increased

glycogen level (Tani et al., 1999). However, preliminary

data indicate that exercise training may partially restore

ischemic PC in the senescent rat heart (Abete et al., 2000).

It is well known that there is a shorter survival time after

AMI in elderly patients compared with younger patients

Fig. 1. Some of the inhibitors, agonists, and antagonists used in experimental procedures of PC to determine involvement of different triggers, transducers, and

end-effectors, and possible signaling pathways activated. The continuous arrow indicates stimulation while the dotted arrow indicates inhibition. Substances

quoted in the figure are reported with the corresponding reference in parentheses: RO31-8220, inhibitor of PKC (Tosaki et al., 1997); 5-HD, inhibitor of KATP

mitochondrial channels (Baines et al., 1999); R-PIA, A1 receptor agonist (Smits et al., 1998); [D-Ala(2),D-Leu(5)]enkephalin (DADLE), d1 opioid receptor

agonist (Kevelaitis et al., 1999); DOG, inhibitor of PKC (Yabe et al., 1998); icatibant, inhibitor of B2 receptor (Bouchard et al., 1998); TAN67, d1 opioid

receptor agonist (Schultz et al., 1998b); PMA, PKC stimulator (Fryer et al., 1998); Lavendustin A, tyrosine kinase inhibitor (Galinanes et al., 1998);

isoproterenol, b-adrenoreceptor agonist (Yoshida et al., 1997); fostriecin, PP inhibitor (Armstrong et al., 1997; Weinbrenner et al., 1998); captopril, ACE

inhibitor (Armstrong et al., 1998; Yang, X. P., et al., 1997); sodium oleate, PLD agonist (Eskidilsen-Helmond et al., 1996); MLA, KATP channel and iNOS

stimulator (Elliott, 1998; Kersten et al., 1998; Tosaki et al., 1998); 7-benzylidenenaltrexone (BNTX, d1 opioid receptor agonist (Schultz et al., 1997); IB-

MECA, A3 adenosine receptor agonist (Tracey et al., 1997); naloxone, opioid receptor antagonist (Schultz et al., 1995); cromakalim, KATP channel opener

(Haruna et al., 1998); BWA1443, A1 receptor agonist (Cohen et al., 1998); IAA-94, chloride channel inhibitor (Diaz et al., 1999); hsp70 (Marber et al., 1995);

nicorandil and pinacidil, KATP opener (Critz et al., 1997; Kita et al., 1998); MAPK and JNK (Ping et al., 1999b); TK (Fryer et al., 1998; Ping et al., 1999b;

Valhaus et al., 1998); genistein, TK inhibitor (Fryer et al., 1998); glibenclamide, KATP inhibitor (Gross & Fryer 1999; Tomai et al., 1999b). aPKC, activated

PKC; MEK, MAPK kinase, MEK2 resembles MEK1 in its substrate specificity; NFkB, nuclear factor kB.

C. Napoli et al. / Pharmacology & Therapeutics 88 (2000) 311±331324

(Gersh, 1986; Toefler et al., 1988). Retrospective studies

strongly suggest that ischemic PC (Abete et al., 1997) and

the warm-up phenomenon (Napoli et al., 1998a, 1999b) are

insufficient to protect the senescent, but not the adult,

myocardium in humans. Another retrospective study, how-

ever, suggested that preinfarct angina was still beneficial in

older patients (Kloner et al., 1998b). Since multiple

mechanisms may be responsible for age-related effects of

myocardial ischemic PC (Napoli & Ambrosio, 1998), the

hypotheses presented here need to be tested in large

prospective clinical studies. In particular, these studies will

have to also evaluate the exact contribution of collaterals to

myocardial function in elderly people with previous angina.

Nevertheless, in a very recent study, it was confirmed that

beneficial effects of prodromal angina are lost in elderly

patients with AMI (Ishihara et al., 2000). Nine hundred

ninety patients (722 < 70 years and 268 >70 years) who

underwent coronary angiography within 12 hr after the

onset of AMI were studied. Prodromal angina in the 24 hr

before infarction was found in 190 of 722 nonelderly

patients and in 66 of 268 elderly patients (26% vs. 25%,

P =0.61). In nonelderly patients, prodromal angina was

associated with lower peak creatine kinase levels, lower

in-hospital mortality rates (3.7% vs. 8.8%, P =0.02), and

better 5-year survival rates ( P =0.007). On the contrary, in

elderly patients, there was no significant difference in peak

creatine kinase levels ( P =0.51), in-hospital mortality rate

(21.2% vs. 17.4%, P =0.49), and 5-year survival rates

( P =0.47). A multivariate analysis showed that prodromal

angina in the 24 hr before infarction was associated with a

5-year survival rate in nonelderly patients (odds ratio 0.49,

P =0.009), but not in elderly patients (odds ratio l.12,

P =0.65). Thus, in nonelderly patients, but not in elderly

patients, prodromal angina in the 24 hr before infarction

was associated with a smaller infarct size and better short-

and long-term survival, suggesting a relationship to

ischemic PC.

4. Concluding remarks

It is very difficult to understand which is the leading

mechanism(s) in the PC phenomenon. Even though several

triggers, transducers, end-effectors, agonists, antagonists,

and inhibitors have been tested in experimental settings,

the question whether it is possible to chemically simulate PC

remains open. In an effort to give to the reader an overview

of what has been discussed in this review, some of the drugs

used in experimental models and some of the most acknow-

ledged mechanisms are reported in Fig. 1. Obviously, this

figure does not pretend to summarize all that is known, but

attempts to give an outline on the complexity of this

phenomenon. Delayed effects of PC (second window of

protection) and their possible mediators are reported in

Table 1. The same criteria used to create Fig. 1 have also

been used for Table 1. However, at the present, the possi-

bility to develop a safe drug that will mimic PC still seems

to be far away, except for a preliminary clinical study with

adenosine (Mahaffey et al., 1999).

Obviously, results from clinical trials are more compli-

cated than those obtained from models used in experimental

studies. PC is more likely to occur in patients with `̀ new-

onset'' angina before myocardial infarction, as well as

during MIDCAB or coronary angioplasty. The role of

ischemic PC in preventing arrhythmias, stunning, and with

increasing age is still poorly understood. Large prospective

multicenter trial studies are needed to better understand the

possible pathophysiological role of the endogenous protec-

tion induced by PC in these clinical conditions.

Acknowledgments

C. Napoli would like to dedicate this paper to Dr. G.

Ambrosio (Perugia, Italy), his mentor in the pathophysiol-

ogy of myocardial ischemia and oxygen radicals. The

authors also gratefully acknowledge Drs. P. Abete, A.

Liguori, F. Cacciatore, M. Chiariello, and L. Sorrentino

(Naples, Italy) and Drs. V. Anania, F. Franconi, and M.P.

DeMontis (Sassari, Italy) for valuable discussions in this

field, and Dr. C.L. Wainwright (Glasgow, UK) for insight-

ful suggestions regarding the organization of the paper.

This work was supported by grants from Ministero della

Universita' e Ricerca Scientifica e Tecnologica (MURST

97/60%, 96/40%) to the Federico II University of Naples

(G. Cirino and C. Napoli), by grant ISNIH.99.56980 (C.

Napoli), and by grant MURST 98/60% to the University of

Salerno (A. Pinto).

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