Cardiovascular Research 1992;26:641-655
64 1
Masterclass
The oxygen free radical system: from equations
through membrane-protein interactions to
cardiovascular injury and protection
Rakesh C Kukreja and Michael L Hess
Highly toxic oxygen radicals and their metabolites have been implicated in the pathogenesis of ischaemid
reperfusion injury. These reactive oxygen species include the superoxide anion, hydrogen peroxide, hydroxyl
radical, and singlet oxygen. The central theme of this review is first to discuss the basic mechanisms of free
radical generation from various potential sources, to point out and emphasise the growing importance of the role
of singlet oxygen, and then to discuss in depth membrane-protein interactions that ultimately lead to myocardial
damage and dysfunction. With this background, we highlight several novel therapeutic strategies aimed at
interrupting the oxygen free radical mediated component of ischaemidreperfusion injury. It is hoped that this
thesis will then serve as a future impetus to challenge these hypotheses and further build on this truly unique
system that has assumed such an important role in the pathophysiology of myocardial ischaemidreperfusion and
inflammation.
“To those of us who are humbly exploring the mysteries of science, we must project our findings and model
our systems. For if correct, we have made a small contribution and, if wrong, we have forced others to eventually
think.” A V Hill, on the occasion of the 50th anniversary of E H Starling’s Linacre lecture (University College,
London, 1968).
A
major concern during cardiopulmonary bypass,
coronary reperfusion and thrombolytic therapy, and
percutaneous transluminal angioplasty procedures is
minimising ischaemic damage to the myocardium, thereby
avoiding depressed myocardial performance in the
postreperfusion period. Prolonged ischaemia, such as that
following myocardial infarction or occurring during long
term coronary bypass procedures, can cause serious damage
to the myocardium. Early reperfusion is an absolute
prerequisite for the survival of ischaemic tissue. However,
reperfusion has been referred to as the “double edged
sword”’ because abundant evidence suggests that reperfusing
ischaemic myocardium carries with it a component of injury
known as “reperfusion injury”. Two popular theories of how
reperfusion injury might occur are (1) the calcium hypothesis
and (2) the free radical hypothesis.’ The former suggests that
ischaemia induces a defect in the ability of the cell to
regulate calcium such that upon reperfusion the cell
accumulates toxic levels of calcium. The free radical
hypothesis is based on the premise that partially reduced
forms of molecular oxygen including the superoxide anion
(.Oi), hydrogen peroxide (HzOz), and hydroxyl radical (-OH)
are produced at the time of reperfusion. Many investigators,
but not all3 have found that free radical scavengers such as
superoxide dismutase (SOD), with or without catalase, can
reduce the injury in the isolated heart model and argue in
favour .of a free radical mechanism. In fact, free radicals
might be inducing the membrane defects which promote
calcium entry, thus unifying both these theories.
Biochemistry of free radicals
Superoxide serves a key role in this scheme. It is thought to
be cytotoxic and is a requirement for production of the .OH
radical. The hydroxyl radical can be formed by the HaberWeiss reaction when an .02 anion and an HzOz molecule
spontaneously combine to form molecular oxygen and two
.OH radicals. A much more efficient means of producing
.OH radical is via the Fenton reaction where H202 accepts
an electron from a reduced metal ion such as Fez+.
Superoxide serves a critical role here as well since it is the
primary reducing agent to replenish the reduced metal ion.
Recently, a second pathway4 for the generation of .OH
radical has been described, in which .OH is generated from
an iron independent reaction involving the interaction of .OI
and nitric oxide (.NO), the latter probably identical to the
~
Division of Cardiopulmonary Laboratories and Research, Department of Medicine, Medical College of Virginia, Virginia
Commonwealth University, MCV Station, Box 281, Richmond, VA 23298, USA: R C Kukreja, M L Hess. Correspondence
to Professor Hess.
642
Kukreju, Hess
endothelial cell derived relaxing factor. The proposed
chemistry is:
.NO + .O? --* ONOO- (peroxynitrite anion)
ONOO- + H’ t+ ONOOH
ONOOH tf .OH + .NO? (nitrite)
(1)
(2)
(3)
s
NO,
This reaction process ultimately produces nitrites and
nitrates. The high reactivity of the .OH radical causes it to
react at diffusion limited rates, and it reacts with the first
molecule it comes into contact with (usually within 14 A
following a period of 10“ s). In acid media such as the
vacuole of the phagocyte or the microenvironment of cell
membranes (the cell surface, containing polyanions, attracts
H’ ions) .OI is protonated into the perhydroxy radical HOz.
(fig 1). The HOT radical is a stronger oxidant than .OI and
is cytotoxic. Singlet oxygen ( ‘ 0 2 ) is formed if one of the
unpaired electrons of molecular oxygen is transferred via
energy absorption to a higher energy orbitals and its spin is
inverted. Singlet oxygen exists in two states, ie, as delta (A)
‘0: in which the newly paired electrons occupy the same
orbital, and sigma (Z) ‘ 0 2 in which the electrons of op osite
spins are in different orbital. The life time of ‘c 0
2 is
sufficiently short that all ‘ 0 2 chemistry in solution involves
A ‘ 0 2 state. The surplus energy of ‘ 0 2 may be spent through
thermal decomposition, light emission, or chemical reaction
(as indicated by chemiluminescence).‘ ti The importance of
various pathways of free radical production in this condition
and the hierarchial roles of different active oxygen
intermediates in the damage are currently in dispute.
P
Mechanisms of oxygen radical generation
Several mechanisms have been described for the production
of free radicals in biological tissues. Some of the widely
accepted are: xanthine oxida~e,’-~activated neutrophils, (’
direct donation of electrons from the reduced mitochondria1
electron transport chain (NADH dehydrogenase, ubiquinonecytochrome b regions) to molecular oxygen,” catecholamine
oxidation,” and c clo oxy enase and lipoxygenase enzymes
(prostaglandins).1.7 The
antineoplastic drug, doxorubicin,
known to induce free radicals, is an example of an
exogenous source of free radicals which induces myocardial
damage.
Xunthine oxidase
The breakdown of homeostatic mechanisms during hypoxid
ischaemia can result in increased cytosolic calcium,
Excitation
1
A ’02
Delta
singlet oxygen
/-
1’02
Sigma
singlet oxygen
_--
Tetravalent reduction
D
HO;
Perhydroxyl
radical
Figure I Forniation of reactive oxygen intermediates during
reduction and excitation of molecular ox.vgen.
activating a calcium dependent (and maybe calmodulin
regulated) cytsolic protease that covalently modifies xanthine
dehydrogenase, converting it to xanthine 0xida~e.I~
This
conversion can also take place by simple degradation in the
absence of enzyme. This enzyme catalyses the univalent
oxidation of purine substrates with the concomitant
formation of .O? radical, H 2 0 I 5~ Ih and perhaps I0:.17
Accumulation of these substrates during ischaemia has been
established by Jennings and Reimer,18 and by Chambers et
ul,’ who reported that conversion of xanthine dehydrogenase
to xanthine oxidase increased during the course of canine
myocardial ischaemia. Other studies have shown oxygen free
radical mediated damage in isolated organ systems by
providing exogenous xanthine oxidase along with
appropriate purine substrate.” 2o Allopurinol or oxypurinol,
which are xanthine oxidase inhibitors, protected against
oxidative dama e in ischaemidreperfusion injury in the cat,”
7 F
rat,” and dog.-. Feeding animals a low manganese, tungsten
supplemented diet results in synthesis of inactive xanthine
dehydrogenaseloxidase, and has been shown to have a
protective effect in reperfused cat intestine.” However,
studies in other vertebrates, most notably man24and rabbit,”
suggest a lack of cardiac xanthine oxidase through failure of
xanthine oxidase inhibitors to protect against ischaemid
reperfusion injury. Additionally, Eddy et al” have shown
that there is no measurable xanthine dehydrogenase or
xanthine oxidase activity in human myocardium. Recently,
Yokoyama et a t 7 postulated that large amounts of xanthine
oxidase can be released from the liver following ischaemia.
and initiate systemic production of free radicals.
Polymorphonucleur leucocytes
The NADPH dependent oxidase system on the membrane
surface of neutrophils is a highly efficient source of the . 0 5
radical.” This enzyme is normally dormant, but when
activated, eg, by bacteria, mitogens, or cytokines, it catalyses
the sudden reduction of oxygen to .O; and HzOz. The
phenomenon, termed the “respiratory burst”, accounts for
more than 90% of the oxygen consumption by stimulated
neutrophils.” Hydrogen peroxide in the presence of
myeloperoxidase and chloride ion forms hypochlorous acid
(HOCI). Myeloperoxidase is secreted by the azurophilic
granules of the neutrophil into the intracellular, lysosomal,
and extracellular compartments, resulting in production of
HOCl both inside and outside the cell. Most of the highly
reactive HOCl combines non-enzymatically with nitrogenous
compounds to generate monochloramines, a group of stable
non-radical oxidants of varying reactivity and ability to cross
membranes.
Romson and coworkers3”first proposed the neutrophil as
a source of oxygen radical production during the course of
myocardial ischaemia and reperfusion. They observed that
neutrophil depletion reduced infarct size to a degree
comparable to the reductions seen using the oxygen radical
scavengers SOD and catalase. In primary myocardial
ischaemia, there is a documented decrease in intracellular pH
(approaching pH 6.4), an increase in reducing equivalents,
and a decrease in ATP levels with concomitant increase in
hypoxanthine and xanthine; all of these reactions favour the
generation of .OI and the more toxic .OH radicals during
reperfusion. Superoxide anion released from metabolically
active neutrophils also activates a latent chemoattractant and
thereby amplifies the inflammatory process.” This process
has been reported by Mullane et 4.’’
who showed migration
of neutrophils to a developing infarct in dogs. Arrival of
neutrophils and their subsequent activation in the
643
Oxygen free radicals and myocardial injury
myocardium results in further reduction of 0 2 to .Oi,
contributing to the pool of toxic metabolites. One of the
products, monochloramine (NHzCl), is a lipophilic oxidant
that is both highly permeable and reactive with membranes.
It has been shown to react with thiol, thiol ethers, aromatic
compounds, and peptide bonds.33 It is quite possible,
therefore, that NHzCl produced during ischaemidreperfusion
by activated neutrophils could enter the myocyte and attack
the contractile proteins, sarcoplasmic reticulum, and
sarcolemma. Kukreja et a t 4 have shown that physiologically
obtainable concentrations of HOCl and NHzCl inhibit Ca2'ATPase and calcium uptake by cardiac sarcoplasmic
reticulum, whereas much higher non-physiological concentrations of HzOz were needed to obtain similar inhibition.
The myeloperoxidase/HzOdhalide system from human
(equations 4 and 5):
neutrophils can also result in
H20z
HzOz
+ H' + C1+ HOCl
+
HzO + HOCl
+ H' + C1- + ' 0 2
Myelopcmridaw
+
H20
(4)
(5 1
There is a great deal of evidence to support a physiological
role for the neutrophil in oxygen radical production.
Leucocyte depletion decreases damage due to ischaemia and
reperfusion as measured by infarct size36 and acute
myocardial fail~re.'~
38
Free radicals generated by
neutrophils can inhibit Na' + K' ATPase activity, destroy
ouabain binding sites,39 and increase capillary permeabilit~.~'
41 Indirect support comes from the use of antiinflammatory agents. SOD has been shown to have an antiinflammatory effec?' and certain anti-inflammatory drugs
can reduce infarct size." Semb et a142have recently shown
that neutrophils activated by phorbol myristate acetate
depressed the function of isolated rat heart by an oxygen free
radical mechanism. In addition to free radical scavengers,
lipoxygenase inhibitors also protect, implicating the role of
leukotrienes in neutrophil mediated cardiovascular injury.4'
Mitochondria1 respiration
Mitochondria are perhaps the largest intracellular source of
.01 and Hz0z4 via the electron transport system. These, of
course, are vital for the oxidation of NADH and FADHz, p
oxidation of fatty acids, and other metabolic pathways.
"Leakage" of electron caniers out of the chain reduces the
oxygen in the mitochondria and forms *O;.45There are two
pathways by which this can occur: (1) breakdown of
ubisemiquinone,46 and (2) NADH dehydr~genation.~~
It is
not known if free radicals generated from these sources
contribute significantly to ischaemidreperfusion damage.
Catecholamines
The autoxidation of catecholamines (adrenaline, noradrenaline, and isoprenaline) results in the formation of
0-quinone, 2 H', and 2 e-. These electrons can then be
captured by molecular oxygen to produce .O;, propagating
the further chain reactions leading to .OH formation and then
membrane lipid peroxidation. This the02 was advanced and
reinforced by the studies of Singal et al. These investigators
have shown that rats fed the antioxidant vitamin E were
resistant to lipid peroxidation (as shown by estimating the
change in concentration of the product malondialdehyde) and
theorised that the source of the free radicals was from
catecholamine autoxidation. The histology of catecholamine
induced myocardial injury is similar to that seen in
reperfused postischaemic hearts. SO;produced as a byproduct
of oxidative metabolism of catecholamine has been proposed
in catecholamine induced cardiomyopathy.'2 Noradrenaline
release has been shown in the coronary sinus of hearts
reperfused following a period of ischaemia, and oxidation of
these catecholamines could provide an additional mechanism
for free radical generation at the time of postischaemic
reperfusion. The extent of participation of this source of free
radicals in myocardial ischaemidreperfusion injury is
unclear and merits further investigation.
Prostaglandin
Reperfusion is a potent stimulus for the synthesis of
pro~taglandin.~~
Prostaglandin H2 synthase (PGH2 synthase)
contains two distinct enzymatic activities: cyclo-oxygenase
catalyses the oxygenation of arachidonic acid to PGGz and
hydropetoxidase converts this to PGH.. Kontos et al" have
shown that a number of free radical species are generated in
this process. Purified PGHz synthase produces
when
NADH or NADPH is a ~ a i l a b l e 'in
~ the presence of the
substrates, arachidonic acid, linoleic acid, or PGGz.
Superoxide radical production was not blocked by cyclooxygenase inhibitors when PGGz was used as a substrate,
indicating that .O; was produced by the action of hydroperoxidase during the conversion of PGGz to PGHr.
Peroxidases can cause ' 0 2 generation during the horseradish
peroxidase catalysed oxidation of mal~ndialdehyde.~'
Prostaglandin hydroperoxidase activity of PGH synthase also
forms ' 0 2 , through a mechanism involving an oxoferil
complex intermediate [(FeO)3']:
a
PGG2 + Fe3'
PGHz + (Fe0)j'
PGG2 + (FeO)3' + PGH2 + Fe3'
+ '02
0
5
(6)
(7)
Experiments using the antioxidant n a f a ~ a t r o mhave
~ ~ further
implicated an arachidonic acid metabolism source of free
radicals. Feuerstein et als3have shown that nafazatrom also
blocks cyclo-oxygenase activity and this ma ex lain why
this drug reduces myocardial infarct size.54 Y, However, it
also blocks li oxy enase activity, a potential source of
singlet oxygen.R
Doxorubicin
Doxorubicin and other anthracyclines are highly effective
anticancer agents. Unfortunately, their use is limited by dose
related cardiot~xicity.~~
Controversy remains regarding the
specific agent responsible for this toxicity, but substantial
evidence points to oxygen radical mediated lipid
per~xidation.'~"~
Anthracyclines such as doxorubicin contain
a quinone moiety which can be reduced to a semiquinone (ie,
a free radical) by single electron donors such as NADPH or
cytochrome P-450 reductase, and NADH or NADPH
dehydrogenase. Once formed, the anthracycline semiquinone
transfers an electron to molecular oxygen forming
leadin to li id eroxidation and eventually, cell injury and
p p
death.
Systolic and diastolic dysfunctions occur after acute and
chronic exposure to doxorubicin. Decreased cardiac output
and left ventricular peak systolic pressure were reported
following doxorubicin injection in dogs," " as was
decreased diastolic fungion in humans .63 Tachycardia,
hypotension and arrhythmias have also been reported when
doxorubicin has been given acutely to cancer
Llesuy et a t 9 document ultrastructural damage to myofibril
and sarcoplasmic vacuolation following doxorubicin
treatment in rabbits. As such, administration of doxorubicin
at doses exceeding 400 mg.m-2 body surface area is limited
due to a high incidence of cardiotoxicity and resultant
myocardial failure. Lee et a t 7 reported that exposure of
papillary muscle to doxorubicin reduced developed force and
increased lipid peroxidation by 200%. Catalase and mannitol
a
B
0
3
644
Kukreja, Hess
showed significant protection, demonstrating HzOz and .OH
radical in doxorubicin induced cardiac muscle dysfunction.
The mechanisms of doxorubicin cardiotoxicity were
investigated by Ondrias et alhXby incorporating sarcoplasmic
reticulum vesicles into planar lipid bilayers to evaluate how
doxorubicin affected calcium permeable channels. They
found a biphasic effect: doxorubicin initially activated the
channel, releasing large amounts of calcium; then, after
approximately eight minutes, the channel became
irreversibly inhibited, stopping calcium release. The above
experiments can explain the acute (tachycardia, arrhythmias)
and the long term (cardiomyopathy) effects of doxorubicin
cardiotoxicity: ie. acute increases in intracellular free
calcium, and in the long term, generation of cell destructive
oxygen free radicals.
Calcium overload
Several investigators have suggested that an influx of
calcium initiates free radical productionhy7" which is
independent of "classic" calcium channels and probably
represents direct sarcolemmal injury. Calcium influx was
shown to initiate oxidation in isolated perfused rat hearts
following low oxygen condition^.^' Steenberger et a17' used
nuclear magnetic resonance measurement of intracellular
calcium concentrations to show that an increase in calcium
precedes lethal myocardial ischaemic injury and accelerates
the depletion of cellular ATP. Calcium overload damages
cardiac m i t o ~ h o n d r i a ~affecting
~
their ability to reduce
oxygen tetravalently to water. This in turn probably diverts
oxygen to univalent pathways and thus to the formation of
free radicals. In addition, perfusion of isolated hearts in the
absence of calcium does not immediately damage
membranes: however, reperfusion with calcium results in
severe immediate tissue damage (the calcium paradox). This
phenomenon is associated with an increase in phospholipase
A1 activity," 74 liberating arachidonic acid from the cell
membrane" to be metabolised to PGG?. This suggests that
a large disturbance of calcium homeostasis may cause
production of free radicals via arachidonic acid
metabolism.
Recent evidence from electron spin resonance studies also
suggests that the endothelial cell is a potential cellular source
of free radical^.^' It is apparent from various studies that the
source( s)/mechanism(s) of free radical production are highly
dependent on the injury model employed. For example, (a)
it is unlikely that free radicals produced by the isolated
perfused postischaemic heart originate from l e u c ~ c y t e s ~
(b)
~;
in the neonatal myocyte, free radical production is probably
17
an intracellular source : and (c) in isolated vascular
endothelial cell cultures, the source of free radical production
is from the xanthine oxidase and prostaglandin system^.^' All
of these sources are able to generate .OS, H.02, and .OH
radical. The highly reactive .OH radical and ' 0 2 initiate lipid
peroxidation (LOOH) of cell membrane components and
cause the release of proinflammatory mediators that activate,
attract, and promote the adherence of neutrophils to the
vascular end~thelium.~'
The neutrophils release long acting
oxidants and elastase which further damage cells (fig 2).
bigure 2 Schrnzatic representation of rhe relationship of superoxide anion. neutrophils, and endothelial cells in ischaemic reperfision injrr y
of the myocyte: superoxide anion i s generated from the endothelial cells via one or more pathways (xanthine o.widase, cyclo-oxygenuse,
catecholamine oxidation, or mitochondria). The superoxide derived oxidants such as H?O?or .OH radical (fimned either bv reaction of .01.
HzO?mid iron, or interaction qf' .OI and nitric oxide) interact with membrane coniponents of endothelial cells to ,form potent granulocyte
cheniotrttractarits. During ischaetniu and reperfusion. neutrophils adhere to the vessel wall at sites of injammation and release a variety
of' toxic products such as .Oi,HzOz, .OH rudical. hypochlorous acid. monochloramine. and elastase. The tqenemtion of the cvtotoxic
metabolites within the microerivironnimt .formed between the adherent activated neutrophils arid altered endothelial cells leads to an increase
in \wscular permeability and mvncyte damuge.
Oxygen free radicals and myocardial injury
Possible sources of singlet oxygen
Woodward and Z a k a ~ i aand
~ ~Manning et af8' have proposed
that oxygen derived free radicals generated during the early
moments of reperfusion may initiate membrane injury,
leading to the development of severe ventricular
arrhythmias. It has been suggested that singlet oxygen and
.OH radicals are the direct initiators of lipid peroxidation by
concerted addition-abstraction reactions with the diene bonds
of unsaturated lipid hydroperoxides." During myocardial
ischaemia, especially after reoxygenation of the heart, there
is an apparent accumulation of lipid peroxides in the tissue.82
Since lipid peroxides are extremely unstable, they tend to
break down rapidly to form lipid peroxy radicals. Singlet
oxygen is generated as a result of the self reaction of lipid
peroxy radicals in the termination step of lipid
peroxidation.s3 Any agent interfering with this reaction
sequence during the course of ischaemidreperfusion would
offer protection. Therefore, exogenously administered
scavengers of .O; and H202, as well as scavengers of .OH
radical, have all been shown to offer protection. We recently
reported that histidine, which has the fastest reaction rate
with ' 0 2 (k = 1 X 10' m o l d ) and is an excellent quencher
of ' 0 2 , significantly reduced infarct size following ischaemid
reperfusion in v ~ v o . ' ~Histidine appears to protect by
scavenging 'Or, which is the end product of free radical
chain reactions and directly inactivates proteins. Singlet
oxygen can interact with other molecules in essentially two
ways: it can react with them chemically, or it can transfer its
energy to them (quenching). When histidine reacts with ' 0 2
it forms endoperoxide which then decomposes to a complex
mixture of non-reactive products.
Direct evidence of ' 0 2 generation is currently lacking.
Organ chemiluminescence is an accepted assay to determine
the production rate of ' 0 2 and the steady state peroxyl
radicals under physiological condition^.^^ Using this assay,
Ferreira et alah showed a significantly higher
chemiluminescence in reperfusion biopsies as compared to
control (preischaemic) biopsies (table). In the case of human
heart, simultaneous determinations of mitochondria1 damage
by electron microscopy showed that in post-reperfusion
samples, the percentage of severely damaged mitochondria
was higher than in control samples. Kumar et a t 7 also
reported luminol enhanced production of oxygen radicals
during ischaemidreperfusion in the isolated rat heart, which
probably includes ' 0 2 . The main sources of
chemiluminescence detected appear to be the dimol emission
of ' 0 2 [equation (8)lS8and the photon emission from excited
carbonyl groups [equation (9)Ig9as indicated by spectral
analysis of the chemiluminescence of liver homogenate
supplemented with organic hydroperoxide."
2 '02
2 0 2 + hv (634-703 nm)
(8)
RO* + RO + hv (380-460 nm)
(9)
where RO* = excited carbonyls.
There are several known sources of ' 0 2 , eg, the
rnyeloperoxidasehI202halide system from human neutro+
Chemiluminescence in repe@sion biopsies compared to control
biopsies. From ref 86. Values are mean (SEM)
Experimental situation of treatment
Rario post stress/control
Ischaemidreperfusion
Ischaemia-reperfusion+ mannitol
Ischaemidreperfusion + desferrioxamine
Ischaemidreperfusion + vitamin E
2.1(0.4)
1.2(0.2)
l.l(O.3)
0.9(0.2)
645
phil? [equation (4) and ( 5 ) ] . Steinbeck et a19' recently
showed that ' 0 2 is produced during the process of
phagocytosis. Interaction of .OI with H202 via the nonenzymatic dismutation reaction results in ' 0 2 formation
[equations (10) and (1 l)].
.O? + .OI + 2H'
HrO? + 0
HrO2 + .Oj .OH + OH- +
+
+
(10)
(11)
2
' 0 2
It is believed that the Haber-Weiss reaction does not occur
in biological tissue under normal conditions, but its
occurrence during pathological states (such as ischaemia,
which is accompanied by significant reduction of
intracellular pH) is possible. Beauchamp and Fridovichg2
suggested that the concentration of .Oi is reduced at lower
pH and the concentration of the perhydroxyl radical (HO?.)
would be correspondingly increased. Furthermore, the
spontaneous dismutation of these radicals to form HrOz
would be markedly accelerated at the low pH. Spontaneous
disproportionation of HzOz was indicated as generating ' 0 2
by monitoring the formation of 5 a-hydroperoxide of
cholesterol, a specific trap of
[equation (12)]. These
conditions would also favour the formation of .OH radical,
which can initiate events leading to formation of ' 0 2
(equation 12).
HzO? + H202
4
2H20
+'02
(12)
Singlet oxygen generation is also reported during the
horseradish peroxidase catalysed oxidation of malondialdeh~de.~'
Prostaglandin hydroperoxidase activity of PGH
synthase also forms ' 0 2 through the following mechanism
involving an 0x0-ferry1 complex intermediate [(FeO)3']
[equation (1 3a,b)]:
PGG2 + Fe3' + PGH2 + (FeO)3'
PGH2 + Fe3'
PGG2 + (FeO)3'
-+
+ '02
(13a)
(13b)
Studies on the photoemission associated with lipid
peroxidation show that the excited species formed are related
to the interaction of lipid derived radicals formed in the
propagation steps of lipid peroxidation.8' These studies
confirmed spectroscopically the generation of ' 0 2 in the
termination step of microsomal lipid peroxidation. As a
consequence, the self reaction of lipid peroxyl radicals (lipid00.) with the formation of a cyclic intermediate can
decompose to carbonyl corn ound in the triplet state or to
oxygen in a singlet state.94 92
Generation of ' 0 2 by enzymatic reactions such as xanthine
oxidase and lipoxygenase has also been rep~rted.'~
96 Most
of these sources have been suggested to be the cause of free
radical mediated injury in various injury
Targets of free radical injury
Alterations in membrane proteins by free radicals are among
the important factors in the evolution of myocardial
ischaemidreperfusion damage. Membranes are composed
mostly of phospholipids and proteins. Oxygen free radicals
can attack subcellular structures resulting in metabolic and
structural changes leading ultimately to cell death and
necrosis (see fig 3). One well documented process by which
oxygen radicals cause cell injury is microsomal lipid
peroxidation,
with
"oxidative
deterioration
of
polyunsaturated lipids".97 Cell membranes contain large
amounts of polyunsaturated fatty acid complexed to
phospholipid which when peroxidised results in loss of the
cell integrity and function. Mechanistically, .OH attacks
646
Kukreju, Hess
2.5
1
high reactivity, ' 0 2 can damage lipids"" and constituents of
biological membranes, and can lead to inactivation of many
enzymes."' DNA damaFe'"' and oxidation of mitochondria1
components"' due to 0 2 have also been demonstrated.
Although
and .OH have been shown to be formed in the
ischaemic/reperfused myocardium, our recent work
established that .Oi is relatively inactive in causing damage
to the cardiac sarcoplasmic reticulum and sarcolemma.39It is
either the .OH radical or the toxic long acting oxidants such
as HOCl and monochloramine, which are generated by
myeloperoxidase catalysed oxidation of CI- by Hz02 from
activated human neutrophils, that disrupt yrdiac sarcoplasmic reticular function by inhibiting the Ca-'-ATPase and
calcium uptake.'"" These oxidants also inhibit the sarcolemma1 Na'K'-ATPase activity and ouabain binding sites.
The role of ' 0 2 in myocardial cell damage was first
investigated by Donck et d."' This was achieved by
exploiting the photodynamic properties of rose bengal to
generate reactive oxygen species. They observed a rapid
rounding up and ultrastructural injury in isolated myocytes
when exposed to ' 0 2 . Subsequently, Hearse ef
showed
that low concentrations of rose bengal induced extremely
rapid electrocardiographic changes ( 4 s) that rapidly
deteriorated to arrhythmias (<30 s), indicative of severe
membrane dysfunction. Photosensitisation of rose bengal
also resulted in pro ressive impairment of coronary flow.
%I3
Tarr and Valenzeno reported that photosensitised rose
bengal induced a prolongation followed by a reduction of
action potential duration in atrial myocytes. Vandeplassche et
u1lm showed that oxidant stress may modify the activity of
a number of ion translocation proteins in the sarcoplasmic
reticulum resulting in intracellular calcium overload,
oscillatory release of calcium from sarcoplasmic reticulum,
and potential oscillation in membrane potential. Stuart and
Abramson"" have reported that the photoactivation of rose
bengal can induce calcium release from isolated sarcoplasmic reticular vesicles.
a
RB* = rose bengal illuminated with light
Figure 3 Effect of singlet oxygen generated from irradiated rose
hrngal on Ca2'-ATPase activit.v of sarcoplasmic reticulum: note
that histidine (10 mM) had .significantprotective effect as compared
to superoxide dismutase (SOD) or catalase which were without any
protective effect.
*p<0.05 v control
unsaturated fatty acids, abstracting a hydrogen atom to
generate a carbon centred radical [equation (14)].
Lipid-H + .OH
H20
+ lipid.
(14)
Molecular rearrangement results in formation of a
conjugated diene, with uptake of an 0 2 at its centre, to yield
oxygen centred lipid peroxyl radical (lipid-OO.)[equation
(131:
Lipid. + 0
2
--t
-+
(15)
lipid-00.
This peroxyl radical (lipid-00.) is capable of propagating a
chain reaction by extracting a hydrogen atom from another
fatty acid molecule [equation (16)].
Lipid-00.
+ lipid-H
--t
lipid-00H
+ lipid.
(16)
Lipid peroxides (lipid-00H) can break down into either
alkoxy (lipid 0.) or peroxyl (lipid 00.) radicals in the
presence of transition metals such as iron and copper.
Kellogg" and MeadWhave both shown through in vitro
studies of purified membrane preparations that structural and
functional alterations can be attributed to free radical
mediated lipid peroxidation. Polymerisation, breakage of
polypeptide chains, and changes in amino acid structure may
occur in proteins and enzymes exposed to lipid peroxides in
aqueous solution. Proteins containing amino acids
(tryptophan, tyrosine, phenylalinine, histidine, methionine,
and cysteine) are mostly sensitive to modification of their
structure." Proteins are also constituents of membrane, so
their damage might also contribute to the membrane
damaging effects of free radicals. Loss of specific activity of
enzymes may also have several consequences.Iw One
specific functional alteration is disruption of membrane
permeability and viscosity which can affect membrane
bound enzymes, receptors, and ion channels. Fragmentation
of the membrane and an increased permeability for Ca2' and
other ions leads to irreversible cell destruction.
Singlet oxygen in myocardial injury
Singlet molecular oxygen is not a radical but rather is an
electronically excited state of oxygen which results from the
promotion of an electron to higher energy orbital. Due to its
0
3
Free radicals in sarcoplasmic reticulum protein damage
We first hypothesised that the first primary target organelles
attacked by the ischaemic process is that portion of the
excitation-contraction coupling system that regulates calci um
delivery (the sarcolemma and sarcoplasmic reticulum) and
not the contractile proteins per se.lll-'lh Sarcoplasmic
reticulum is an intracellular organelle which rapidly
sequesters and releases calcium, thereby regulating muscle
relaxation and contraction. Sequestration of the released
calcium into the sarcoplasmic reticular lumen is mediated by
magnesium dependent Ca"-ATPase enzyme. Impaired
calcium handling may affect cardiac contractile function. In
support of our hypothesis, we first observed a decrease in
calcium uptake in isolated sarcoplasmic reticulum after
exposure to xanthine/xanthine oxidase, an enz matic system
capable of generating oxygen free radicalsII Y or activated
human neutrophils."' Ito er u1"* showed a marked reduction
of Ca'*-ATPase activity following ischaemidreperfusion,
which was accompanied by a fivefold increase in free radical
generation. Longer periods of ischaemic/reperfusion insults
have shown significant degradation of major ATPase
proteins,'" together with appearance of contraction bands.
These findings suggested that reperfusion injury occurs as a
result of degradation of intracellular calcium regulation due
to production of free radicals. It has been shown that
recovery of contractility following reperfusion is greater in
hearts treated with oxygen radical scavengers. This finding
Oxygen free radicals and myocardial i n j q
Figure 4 Denaturing polvacrylamide electrophoresis gels of
surcoplasmic reticulum ( S R ) exposed to singlet oxygen (rose bengal
and light) and protection bv histidine: SR was incubated with rose
bengal (SO nM) plus light for varying lengths of time: lane 2 (0
min). lurie 3 ( I min), lane 4 ( 2 min). lane 5 (4 rnin). lane 6 (6 rnin).
lane 7 ( 8 niin). lane 8 (10 min), lane 9 (12 niin), lane 10 (14 min),
lane I I (SR irose bengal + light i10 mM histidine, 14 rnin), lane
12 (SR control), lane 13 ( S R + rose bengal without exposing ,to
light): lane I (stnndurds). Note that the monomeric band of C d + ATPase disappears by increasing the duration of exposure to
irradiated rose bengal.
has been observed in hearts subjected to severe ischaemic
injury and in non-irreversibly injured “stunned” hearts,’”””
supporting the hypothesis that reperfusion is associated with
the development of a specific form of myocardial damage.
Kim and Akera’I5 reported a significant inhibition of
sarcolemmal Na’K’-ATPase activity during ischaemid
reperfusion. Scavengers of free radicals and ‘Or (histidine)
significantly protected sarcolemmal function. Indirect
evidence of free radical mediated effects in the rat and dog
has been obtained by numerous groups that have treated their
models with free radical scavengers.’”-”’
Exposure of isolated cardiac sarcoplasmic reticulum to
irr!diated rose bengal resulted in significant inhibition of
Ca-’-ATPase activity which was inhibitable by histidine.’”
SOD or catalase were without any protective effect, thus
confirming that the damage was due to ‘ 0 2 (fig 3). SDSpolyacrylamide gel electrophoresis of ‘ 0 2 exposed
sarcoplasmic reticulum demonstrated almost complete loss
of the Ca”-ATPase monomer band (fig 4), and histidine
preserved this protein (lane 1 I). Rose bengal derived oxygen
intermediates caused progressive loss of ‘H-ryanodine
binding and the degradation of heavy molecular weight
proteins such as calcium release channel.”” There was no
significant effect of .OI on Ca”-ATPase protein (lane 2
control versus lane 4 for xanthine/xanthine oxidase) or .OH
radical (lane 5, xanthine/xanthine oxidase + Fe3’-EDTA) (fig
5). There was no significant effect of HzO? on Ca”-ATPase
in sarcoplasmic reticulum (fig 6). Thus it Fppears that the
damage of cardiac sarcoplasmic reticular Ca-’-ATPase is due
to ‘ 0 2 and not .O;, HzO2 or -OH radicals. Higher
concentrations of HzOr inhibited Ca”-ATPase activity,los
which was probably related to the oxidation of sulphydryl
groups (unpublished data) similar to that of other oxidants
such as hypochlorous acid’2y and not to ‘02induced
fragmentation or aggregation of the enzyme.
The process of aggregation of Ca2’-ATPase enzyme by ‘02
is not clear. However, it has been proposed that .OH radical
647
Figure 5 SDS-PAGE of sarcoplasmic reticulum (SR) exposed to
.Oj anion, H202, and .OH radical: isolated SR vesicles were
exposed to .Oj anion and H 2 0 2 (jiim xanthine ixanthine oxidase)
or .OH radical by including Fe-’-EDTA in the enzyme reaction
mixture or Fe’+-EDTA with 0.5 mM H202. Lane I (standard),lane
2 (xanthine 100 M),lane 3 (xanthine rixidase 0.05 U d ) ,lane
4 (xanthine plus xanthine oxidase). lane S (xanthine plus xanthine
oxiduseplus 50 p M Fe.”-EDTA). lane 6 (0.5 m M H202). lane 7 (SO
Fe-’-EDTA), lane 8 ( H 2 0 2 plus Fe2’-EDTA). Note that .02.
H202 or .OH radical do not have signiJicunt effects on 97 kdalton
band of Ca2t-ATPase.
causes protein aggregation due to intermolecular bityrosine
formation. Essentially any amino acid radical formed within
a peptide chain could cross link with an amino acid radical
in another
Protein fragmentation was explained
by Garrison et
as a result of 0 2 addition to a-carbon
radicals induced by .OH radical. The resulting peroxyl
radical would further react to produce a peroxide, the
decomposition of which can cause chain scission to produce
a carboxyl and an amide. The peptide chain would remain
intact during such reactions. A modified version of the
scheme of Garrison et all.”’ was recently proposed by
Schuessler and Schilling.”‘ In the Schussler and Schilling
model, bovine serum albumin is cleaved by .OH + .05 + Or
by oxidative destruction of proline residues. Wolff e? af’”
have further explored the question of proline attack and have
suggested that peptide bond hydrolysis occurs. Whether such
a mechanism is possible by ‘ 0 2 in Ca-’-ATPase needs further
investigation.
Free radicals and sarcolemmal damage
Na’K’-ATPase is an important sarcolemmal enzyme, which
regulates intracellular sodium concentration and therefore
regulates membrane potential.”’ Transsarcolemmal Ca2’
transport is crucial to the regulation of myocardial contractility.’” The sodium-calcium exchange system is specifically located on sarcolemma and is electrogenic, with three
or more N.3’ ions being exchanged for each calcium ion.’40
The inhibition of Na’K’-ATPase activity would result in the
648
Kukreja, Hess
suggested that sarcoplasmic reticulum is more vulnerable to
neutrophil derived oxidants than sarcolemma.
Singlet oxygen also caused significant inhibition of
sarcolemmal Na'K'-ATPase activity and histidine (25- I00
mM) protected the enzyme in a dose dependent fashion,
whereas neither SOD, catalase, nor mannitol (.OH radical
scavenger) had any effect.'" These considerations call for
further studies on detection and quantification of 'Or in
ischaemidreperfusion, along with investigation of '02
toxicity at the organic, cellular, subcellular, and biochemical
levels. Thus it appears that the exchanger protein as well as
sarcolemmal receptors, including adrenergic and muscarinic,
may be subject to attack by oxygen radicals, ' 0 2 , and
oxidants generated during the course of ischaemid
reperfusion and myocardial infarction, thus contributing to
cell death.
Can histidine protect against ischemidreperfusion
injury?
Figure 6 Dose dependent effect qf H 2 0 2 on CCI"ATPU.Wenzyme
of snrcoplasmic reticulum (SR). Lane I (srandurd.s),lane 2 (conrrol
SR without HrOz), lane 3 ( S R plus 0.25 niM HrOr), lane 4 (SR plus
0.5 mM H 2 0 2 ) , lane 5 (SR plus I mM HzO?), lane 6 (SR plus 2 m M
H 2 0 2 ) , lane 7 (SR plus 5 m M HrO?). lane 8 ( S R plus 10 niM H 2 0 2 ) ,
lane 9 ( S R control).
occurrence of intracellular Na' overl~ad.'" This increase in
intracellular Na' will, increase Ca-' influx through the
participation of Na'-Ca-' exchange. Reeves et a/'" reported
a 10-fold increase in Na'-Ca'' activity by reduced oxygen
species in bovine cardiac sarcolemmal vesicles. The
stimulation of activity required the presence of reducing
agents (such as .O?) as well as oxidising agents (H?Oz) and
probably resulted due to their influence on the conformation
of the Na-Ca exchange carrier by promoting thiol-disulphide
interchange in the carrier protein. Kim and Akera1I6reported
decreased Na'K'-ATPase
activity, specific ['H]-ouabain
binding, and lowered sodium pump activity of the
sarcolemma in ventricular muscle homogenate. Their studies
revealed that scavengers of all the species of activated
oxygen (.02, HrOr, .OH, ' 0 2 ) had protective effects to
various degrees. Studies in isolated cardiac sarcolemma
showed that .O? had no direct effect on the Na' + K'-ATPase
activity." The GOvalue for Hr02 was 1 mM which is
extremely high and is not in the range of physiological levels
generated from free radical producing systems in vivo.
Therefore it may not be directly responsible for the
inactivation of the enzyme in vivo. The effect of HrO2
differed greatly from sarcoplasmic reticular Ca"-ATPase,
where its concentration up to 1 mM had no significant
effect."'" In contrast, phorbol myristate acetate stimulated
human neutrophils severely depressed the Na'K'-ATPase
activity, which could be partially inhibited by SOD and
completely inhibited by catalase and SOD plus catalase. This
strongly incriminated the involvement of either .OH radical
or non-radical oxidants such as HOCl and NHKI, or perhaps
I
0 2 , in damaging Na'K'-ATPase
enzyme. This is proved by
lower ICSUvalues for HOCl and N H K I as compared to HrO2.
The susceptibility of Na'K'-ATPase to these oxidants
differed greatly from that of Ca"-ATPase of sarcoplasmic
reticulum. The ICWof Ca"-ATPase was 8 pM and 6 pM for
HOCl and NHrCI respectively."'6 These observations
These studies suggested that beneficial effects of histidine in
I
Or mediated sarcolemmal and sarcoplasmic reticulum
damage. We therefore hypothesised that histidine would also
protect against ischaemidreperfusion induced myocardial
injury (Kukreja RC et al, unpublished data). Figure 7 shows
typical changes in ventricular pressure and electrophysiological tracings from hearts subjected to normal reperfusion
(A-no ischaemia), 30 min ischaemia followed by 20 min
reperfusion (B), and ischaemidreperfusion with 25 mM
histidine (C). The contractile force markedly declined
immediately after the cessation of flow and reached almost
zero in 6-8 min. Reperfusion after ischaemia did not produce
recovery until 16-17 min (fig 7B) whereas histidine perfused
hearts showed immediate return of contractile function
(within 2-3 min) (fig 7C). The protective effect of histidine
was dose dependent and was comparable to classical free
radical scavengers such as soD/catalase/mannitol. Histidine
itself was without effect on the normal function of the heart.
Electron microscopy of the tissue revealed that histidine
significantly reduced ultrastructural damage caused by
ischaemidreperfusion in a dose dependent fashion, as
evidenced by (a) reduction in mitochondria1 swelling, (b)
preservation of myofibrillar architecture, and (c) preservation
of sarcolemmal integrity. The classic free radical scavengers,
SOD, catalase, and mannitol, also reduced the damage but not
as impressively as hi~tidine.'~
I W These results suggest that
I
0 2 may be one of the damaging species (in addition to .OH
radical) during reperfusion induced myocardial injury. Other
compounds which scavenge ' 0 2 include DABCO, diphenylisobenzofuran, sodium azide, tryptophan, and p carotene.
Unfortunately, most of these (except tryptophan) are either
toxic or have limited solubility in aqueous solution. Because
of excellent solubility and relatively non-toxic nature of
histidine at optimal concentrations, it may be useful
clinically.
Protection strategies
Elimination of free radicals - Extensive experimental
evidence proves the importance of free radical induced
myocardial injury in a variety of model systems.
Unfortunately, the ability to translate this into clinical benefit
via elimination of these destructive species has been
difficult. Several approaches have been employed with a
variety of enzymatic scavengers of .O? and HrOr (SOD and
catalase). During ischaemidreperfusion, the substrates for
these enzymes increase dramatically so that the need far
Oxygen free radicals and inyocardial inju p
Control
Ischaemia/reperfusion
-m
I
C
Ischaemia/reperfusionwith 25 mM histidine
t
h
Reperfusion
30
60
65
&-
75
Time (min)
Figure 7 Typical ECG and ventricular pressure recordings from
hearts subjected to 30 min ischuemiu und 20 rnin reperficsion and
protective effect of 2.5 mM histidine: panel A represents control
perfused heart; B: heart subjected to 30 nzin of ischuemia followed
by 20 nzin or reperfusion; C: heart subjected to ischaemia and
reperfusion in presence of 25 mM histidine. Histidine was perfused
throughout the experimental period. Note thut there was immediate
recover?, of contractile function und reduction in arrhythmia
following reperfusion in the presence of histidine.
surpasses the quantity. Thus supplying more of these
scavenging enzymes should, theoretically, break down both
.02 and HzOr. Another approach to blocking formation of
oxygen free radicals is via inhibiting the enzymes used in
their production by the alternative pathways employed
during ischaemidreperfusion, namely by using the xanthine
oxidase inhibitor, allopurinol, a structural analogue of
hypoxanthine. This compound competitively inhibits
xanthine oxidase catalysed urate production with its
subsequent formation of the .OI anion. Desfemoxamine is a
strong chelatingagent of iron, which is the rate limiting step
in the Haber-Weiss reaction. Chelation of iron would prevent
ferric iron from undergoing the cyclic reduction and
oxidation required to catalyse .OH f0rmati0n.l~~In the
absence of metal ions, the rate constant for the Haber-Weiss
reaction in aqueous solution is virtually zero.
649
Desferrioxamine has been used to examine the role of .OH
in reperfusion injury for several reasons: it is known to have
high affinity (Kd I 0-31)
for ironlJh;it reacts quickly with .OH;
and it inhibits iron dependent lipid peroxidation and iron
dependent .OH generation from Hz02 in most systems."
Desferrioxamine and other chelators have been reported to
IJX IJ'J
limit creatine kinase release,
preserve myocardial
phosphocreatine content,"" lower vascular resistence,14') 151
and preserve myocardial ultrastructure'Jq and/or enhance
recovery of left ventricular developed
in
isolated perfused hearts subjected to ischaemia-reflow. Not
all studies, however, have yielded positive results. Maxwell
et alls3 found that doses of 30 mg.kg-' subcutaneously +
I - 100 rng.kg-'K' intravenously had no effect on infarct size
in rabbits subjected to 45 min of ischaemia and 3 h
reperfusion. It is proposed that desferrioxamine may exert a
biphasic antioxidandpro-oxidant effect: that is, it may
paradoxically amplify oxidative damage in the presence of
reducin agents (such as .OZ) in a dose dependent
fashion.5.54
Free radical mediated injury may also be attenuated by
preventing the secondary amplification of injury by the
neutrophil. This may be accomplished either by inhibiting
NADPH oxidase or preventing neutrophil adhesion.
Adenosine has also been shown to be protective in the
ischaemic heart when it is given prior to reperfusion."' In
addition to its vasodilator effect, adenosine has been shown
to inhibit .Or radical production by stimulated neutrophils
via a receptor mediated mechanism.15h Monoclonal
antibodies (60.3, 904) have also been raised against the
CD I I/CD I8 complex, a neutrophil membrane-glycoprotein
complex mediating adherence to endothelial cell^."^^'^'
These monoclonal antibodies inhibit neutrophil chemotaxis
and adherence.
Antioxidants, either natural or synthetic, can interrupt
peroxidation by a number of different mechanisms and thus
prevent the production of oxygen free radicals. In view of its
antioxidant capabilities, vitamin E (a tocopherol) has been
used by several investigators as a cardioprotective agent in
IhOklhJ
a variet of animal models,
while others question its
IhX16h
effect.
Many commonly used cardiovascular drugs
readily incorporate into cell membranes and thus may affect
their sensitivity to free radical injury.lh7 The
blocker
propranolol is one such drug. Indeed, Mak et a1 showed that
the membrane antiperoxidative properties of propranolol
could be translated into cytoprotective effects at the
myocellular level.'6XCalcium channel blockers have also
shown promise as antioxidants.'" I h X Their antioxidant effect
is thought to be mediated via neutralising the lipid peroxy
radicals in a similar fashion to a tocopherol, that is, the
cardioprotective effect is mostly related to .OH radical
Ih9 I70
scavenging by sulphydryl containing agents.
Captopril is a sulphydryl containing antioxidant and an
angiotensin converting enzyme inhibitor (ACE) receivin
some attention lately for its anti-inflammatory action, 171 14
antiarrhythmic effects,'" and attenuation of reperfusion
induced depression of myocardial function.'" Both Wegliki
et al and Mak et a1 have shown that captopril exhibits dose
dependent inhibition of cellular lipid peroxidation in cultured
endothelial and smooth muscle cells when exposed to the .OZ
and .OH generating systems.170 173 Bagchi et d17J
have
shown that captopril was as effective as dimethylthiourea
(DMTU) in scavenging .OH. On the other hand, Kukreja et al
found that captopril did not inhibit .OI dependent
cytochrome c or nitroblue tetrazolium reduction, therefore
ruling out a direct scavenging of .02 radical^.'^' Lastly,
650
Kukreja, Hess
nafazatrom, a compound with antithrombotic and antimetastatic activity, has been shown by electron spin resonance
experiments to be an extremely reactive scavenger of free
r a d i ~ a 1 s . lThe
~ ~ antithrombotic effects are postulated to be
related to the abilit of the drug to stimulate prostacyclin
(PGI?) p r o d ~ c t i o n . ’ ~has
~ I t also been shown to behave as a
lipoxygenase inhibitor and thus would be able to limit lipid
peroxidation. 17’
In evaluating the likelihood that a compound acts as an
antioxidant, it is essential to know whether the rate at which
it reacts with biologically important reactive species would
allow it to compete with biological molecules in vivo for
such species.’79Therefore there is no clear consensus for any
one of these agents as to a true clinical benefit. While several
studies may have shown preservation of myocardial function
by reducing the presence of reduced oxygen intermediates,
others have refuted this. This disparity is often attributed to
difference in model systems andor experimental design.
Ischaemic preconditioning - Another area which has
generated significant interest is “preconditioning” protection
of reperfusion injury. Preconditioning describes a condition
in which brief periods of ischaemia increase the tolerance of
the myocytes to subsequent ischaemic insults. In dogs
subjected to four 5 min coronary occlusions separated by 5
min of reperfusion, the amount of necrosis produced b a
IB(k18Y
subsequent 40 min occlusion is dramatically reduced.
In
rats, a single coronary occlusion lasting 3-5 min markedly
reduces the incidence of reperfusion induced arrhythmia
following a subsequent ischaemic episode.lB1 Myocardial
cells therefore appear to be able to adapt rapidly to brief
ischaemic stresses in a manner that makes them remarkably
resistant to subsequent stresses. The exact mechanism of this
effect is unknown. However, recent studies have revealed
several important metabolic features of preconditioned myocardium. Warner er a1’” reported that tissue acidosis during
the ischaemia was attenuated following preconditioning.
Jennings, Reimer and their group have shown that the
depletion of myocardial ATP level during the ischaemia was
slow and accumulation of toxic metabolites such as lactate
1x3 1x4
was less in the preconditioned myocardium.
The
mechanism of alteration of these metabolic changes induced
by preconditioning is unknown. Murry et a1’” proposed an
interesting hypothesis that oxygen free radicals generated
during the preconditioning procedure may contribute to its
cardioprotective effects. This hypothesis was based on the
fact that administration of SOD and catalase partially blocked
the infarct size limiting effect of preconditioning. In contrast,
Iwamoto et allg6presented evidence against the participation
of oxygen radicals in the cardioprotective effect of
preconditioning in a rabbit model.
Downey’s group has proposed that adenosine released
during the preconditioning occlusions might be the mediator
of c a r d i o p r o t e ~ t i o nThis
. ~ ~ ~hypothesis was based on the fact
that adenosine is released by the ischaemic rnyocyteslx8and
in turn stimulates cardiac AI receptors. Their studies showed
that the preconditioning protection could be blocked by two
dissimilar adrenoreceptor blocking agents, SPT and PD
115,199. Also, a 5 rnin intracoronary infusion of either
adenosine or the AI specific agonist R-PIA triggered a long
lasting change within the myocardium that caused it to be
more resistant to the ischaemic insult. It appears therefore
that it is possible to maintain the heart in a preconditioned
state indefinitely with the administration of an A I receptor
agonist.
Myocardial infarction is often preceded by multiple
episodes of angina pectoris. With recent advances in
thrombolytic therapy and coronary angioplasty. it is of
clinical interest whether preceding ischaemic episodes alter
the time course of myocardial injury in sustained final
ischaemia in humans. Whether preconditioning can occur in
a heart with totally occluded vessels or in already ischaemic
myocardium is uncertain. The diversity of clinical pathology
makes generalisations nearly impossible until considerably
more research is completed.
Heat shock proteins - Recent studies have also shown that
a heat shock pretreatment improves gostischaemic recovery
in isolated perfused heart models.’ This phenomenon is
known as thermotolerance and seems to be mediated by a
group of proteins, the heat shock proteins. Stress or heat
shock proteins were originally identified because of their
increased synthesis by many cell types after exposure to
raised temperatures. A 70 kD heat shock protein has been
identified in neonatal and adult heart tissue of several
species, including dog, rat, and rabbit, and synthesis of
this protein is increased by exposure to increased temperatures.”) Other stressful stimuli, eg, ischaemia,’” hypoxia,”’
lschaemialreperfusion
0 2
strategies
Hz02
Haber-Weissl
Fenton reaction
Q--+
‘OH/’07
Amino acids
damage
Protein damage
I
Myocardial injury
Figure 8 Schematic presentation of the various novel und
classical protective strategies in reducing ischaeinia/reper~tsion
induced myocardial injury. Ischaernia followed by reperjiusion
generates .OT which rapidly dismutates to H202. I n the presence of
iron, .OH radical is ,formed via iron cutulvsed Haber-Weiss
reaction, which initiates lipid peroxidation in the cell membranes.
Since lipid peroxides are extreme1.v unstable. thev tend to break
down rapidly to form lipid peroxyl rudicals. Singlet oxygen is
generated as a result of self reaction of peroxyl radicals in the
termination step of lipid peroxidation. These two species are known
to be highly reactive and directly damage amino acids and
membrane proteins including Ca2’-ATPase, Na’K‘-ATPase, Na’Ca” exchanger; and receptors located on ,sarcoplasn~icreticulum
and sarcolemma. Amino acids appear to protect by scavenging
singlet oxygen and .OH radical and providing a “pool” for the
replenishment of damaged amino acids. Traditional antioxidants,
which either scavenge oxyradicals or prevent their formation, are
not always effective because of their limited accessibility to the
site of damage. Another protective strategy is ischaemic
preconditioning, whereby brief periods of ischaemia increase the
tolerance of the myocardium to subsequent insults. Ischaemic
preconditioning is also accompanied by synthesis of so called “heat
shock proteins”. The heat shock proteins appeur to protect against
myocardial injury by acting as “molecular chaperones” to the cell
membrane proteins which are necessary for maintaining the normal
contractile function of the rnyocyte.
Oxygen free radicals and myocardial injury
transition metals,"' and pressure or volume overload,'" were
shown to induce increased synthesis of these proteins.
Closely related or identical proteins have been found in
normal cells. It is now widely believed that heat shock
proteins play an essential part in normal cells and in the
cellular responses to stress.'95 The common feature of the
stress proteins is their ability to bind denatured or malfolded
proteins during a period of stress. Studies in different species
have shown that increased heat shock protein ex ression
19pl97
may protect the heart against subsequent damage.
Currie et allg6 showed that exposure of rats to raised
temperature, with consequent cardiac heat shock protein
induction, resulted in an improved recovery of contractile
function after subsequent ischaemia and reperfusion.
Reperfusion damage, as measured by creatine kinase release,
was significantlf reduced in the heat shocked hearts. Yellon
and Latchman' * obtained similar results. The increased
concentrations of hsp 70 was accompanied by increases in
the corresponding mRNA and increase in activity of
~ata1ase.l~~
A possible hypothesis on protection afforded by
ischaemic preconditioning via heat shock protein synthesis is
presented in fig 8. An attractive proposal is that heat shock
proteins and antioxidant enzymes may mediate protection
against oxidative stress by two separate or overlapping
mechanisms. One hypothesis could be that both families of
proteins actually collaborate to protect the cell against
oxidative stress. For example, heat shock protein may bind
and protect the different subunits of catalase during
ischaemidreperfusion injury. This could explain the
observed increase in catalase activity. In view of this, it is
tempting to consider the possible development of a nonnoxious drug that would increase the exlression of the heat
shock proteins in the heart and serve as a therapeutic agent
against myocardial ischaemia. However, Thornton et a12"
have reported that ischaemic preconditioning may occur via
a mechanism that does not require stress protein synthesis.
The involvement of heat shock proteins in myocardial
protection appears to be controversial at this time and needs
to be critically tested.
Conclusions
There are currently abundant data to indicate that free
radicals and their metabolites derived from molecular
oxygen contribute to myocardial dysfunction during the
syndrome of ischaemia and reperfusion. Oxygen radicals and
their metabolites generated during postischaemic reperfusion
are thought to be responsible for much of this injury. Several
studies have shown reduction of reperfusion injury in
myocardial tissue by treatment with the oxygen free radical
scavengers. This hypothesis proposes that large quantities of
.O;anion are formed during ischaemia and reperfusion, and
as a result of divalent reduction, generate .OH radicals via
iron catalysed Fenton's and Haber-Weiss reactions. The .OH
radical in turn reacts with biological membranes forming the
carbon centered radicals (R-free radical; RO-alkoxyl
radical, and ROO-peroxy radical). Direct evidence for the
generation of these carbon centred free radicals has been
provided by electron spin resonance techniques. However we
must take into consideration the fact that generation of these
radicals is not enough to account for all the damaging effects
in the cells. It is now widely believed that .O; is a low
reactive species. Hydrogen peroxide is reactive but only at
non-physiological concentrations. The greatest danger with
the cellular accumulation of .O; and H202 is that these are
65 1
the precursors for the formation of the .OH radical. Part of
the reactivity of the .OH radical is attributed to its capability
to initiate lipid peroxidation, and the interaction of unstable
lipid free radicals generate '02. Studies from our laboratory
have shown that ' 0 2 severely inhibits sarcoplasmic reticular
function, causes aggregation of Ca2'-ATPase enzyme,
inhibits sarcolemmal Na+K+-ATPaseactivity, and generates a
negative inotropic effect on isolated papillary muscle.
Scavengers of ' 0 2 , particularly histidine, afford significant
protective effects on the subcellular damage initiated by
ischaemidreperfusion injury. Further evidence for the role of
' 0 2 in myocardial injury is provided by the demonstration
that it is able to produce significant reperfusion arrhythmias
in the normoxic rat heart. It therefore appears that ' 0 2 may
be one of the most important members of the toxic oxygen
species and deserves special attention in future
investigations. Polymorphonuclear leucocytes also play a
pivotal role with their ability to generate oxygen radicals,
their metabolites, and long acting oxidants. These oxidant
species generated from neutrophils include the .OI, H202, the
-OH radical, ' 0 2 , HOCl, and NH2Cl. Indirect proof of this
hypothesis comes from the demonstration that, in vivo,
activation of neutrophils has been shown to produce
significant myocardial and peripheral vascular depression by
a free radical mechanism and inhibition of neutrophil
function and/or generation of free radicals protect the
jeopardised myocardium. Based on this line of evidence, it
now appears that the oxygen free radical system plays a
pivotal role during the course of myocardial ischaemia,
reperfusion, and inflammation. Since the first report by Jolly
et all2' in 1984 at least 40 studies of infarct size limitation
by antioxidant therapy have been published. As recently
commented by Bolli,20' rarely in the history of basic
cardiology has so much work been done in so little time.
Unfortunately, despite such an impressive amount of work
(or perhaps because of it), this remains one of the most
controversial, confusing, and frustrating areas of research,
with 20 studies claiming reduction of infarct size and 20
failing to confirm this finding.' Failure of SOD to limit infarct
size has been attributed to several possibilities including
inappropriate dosing,12' the scheduling of SOD administration,
or premature withdrawal during the reperfusion period.
However, the mechanistic studies reveal differential
susceptibility of subcellular organelles to different species of
free radicals. Therefore there may be a need to develop a
special "cocktail" of scavengers which can eliminate all the
toxic oxygen species. One such possibility is the use of
histidine in ischaemidreperfusion protocols, because it will
eliminate ' 0 2 in addition to the .OH radical. Our in vivo
studies have shown that histidine significantly reduced
arrhythmias and infarct size in rats subjected to regional
ischaemia and reperfu~ion.'~
Thus there is the possibility of
developing histidine and other amino acids to serve as
effective antioxidants in ischaemidreperfusion injury.
Recent studies from Stadtman's group suggest that amino
acids inhibit the iron catalysed Haber-Weiss reaction due to
their susceptibility to oxidation by H202 and their ability to
sequester metal ions.''' Indeed, the 1990s will witness many
such new approaches to the effective management of oxygen
radical induced tissue injury.
Received 16 January; accepted 27 April 1992
Key terms: oxygen free radicals; myocardial injury; ischaernial
reperfusion injury
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