BIOHEART E-JOURNAL
BIOMOLECULAR AND BIOCHEMICAL RESPONSE OF MYOCARDIAL CELL TO ISCHEMIA AND
REPERFUSION IN COURSE OF HEART SURGERY
G.G.Corbucci
Institute of Anaesthesia –
Resuscitation University of Cagliari – Italy
Summary
Objective: Previous
studies have shown that biomolecular and biochemical adaptive changes antagonize oxidative
damage due to hypoxia and ischemia in myocardial cell. Aim of our study was to verify in
human ischemic and reperfused cardiac tissue the relationship between the mitochondrial
enzyme activities and the activation of HSP70 and c-fos synthesies in the context of a
cytoprotective mechanism. Nitric oxide (NO) modulating effects on mitochondrial
respiratory chain enzyme activities in ischemic and reperfused tissue were investigated
(preliminary report).
Methods: During elective coronary
artery bypass grafting, in 30 consecutive patients ventricle samples were taken one before
aortic clamping the second after 55± 8 min. ischemic period and the third 34± 5 after
final reperfusion. Coronary sinus blood samples were taken in parallel to assess free
radical release measured by malonaldehyde (MDA) levels. In a small number of patients
(N=5) nitric oxide tissue levels were analyzed.
Results: When compared with
normoxic tissue, a significant decrease in cytochrome coxidase (COX) and succinate Cyt-c
reductase (SCR) activities in ischemic and reperfused samples were observed. The
activation of HSP70-72 and c-fos transcription factor was evident in courses of ischemia
and reperfusion. Blood MDA levels undeline the concept that oxyradical generation
characterize the peroxidative damage in reoxygenated myocardial tissue while adaptive
changes which occur in ischemic cell seem antagonize the oxyradical injury.
Conclusions: In course of heart
surgery the myocardial cell seems to prevent the ischemic damage activating some peculiar
biomolecular and biochemical adaptive changes which permit the reversibility of the
oxidative injury. In contrast appears evident that massive and rapid reoxygenation of the
cardiac tissue leads to a peroxidative damage due to oxyradical generation. The nitric
oxide seems to play a crucial role in the cellular adaptation to ischemia even if further
studies will be needed to elucidate these findings.
From the data obtained in this work we
cannot draw certain conclusions in terms of human cardiac cell adaptation to ischemia
whereas it seems convincible that reoxygenation, as actually employed in clinical
practice, compromise the integrity of the cells.
KEY WORDS: Ischemia/Reperfusion -
Mitochondria – Heat Shock Proteins - Nitric Oxide.
Introduction
A growing body of evidence
indicates that in response to hypoxia and/or ischemia, mammalian cell activate several
protective mechanisms which antagonize the deleterious effects of low cellular oxygen
tension1-2. The mechanism underlying this adaptive process is not clearly
understood, but available evidence suggests that changes in mitochondrial respiratory
chain enzyme activities play a pivotal role, and that endogenous nitric oxide (NO) has a
modulating effect on mitochondrial respiration3-4-5. Studies on intact tissue
and on subcellular fractions have shown that the suppression of mitochondrial respiration
by NO is rapidly reversible upon restoration of cellular oxygen restoration4-6
while reperfusion of hypoxic/ischemic tissue is necessary to restore normal function, it
is well know that the so called "reperfusion syndrome" is characterized by
impaired mitochondrial function and increased production of O2- 7-8.
The potential roles of oxygen radical species such as O2-and ONOO-
in the process of cellular dysfunction that accompanies hypoxia/reoxygenation and
ischemia / reperfusion remain to be elucidated.
The generation of oxyradicals seems to be
related to a primary imbalance between the decreased oxidative capacity and the rapid
restoration of oxygen in the cell. In this regard, byproducts of lipid peroxidation or
depletion of endogenous antioxidants are often used as indirect markers for free radical
generation9. Malondialdehyde (MDA), a 3-carbon compound, reflects both
auto-oxidation and oxygen radical-mediated peroxidation of polyunsaturated fatty acids,
thus representing a suitable index of lipid peroxidation in ischemic and reperfused tissue9-10.
In the present study, we have used MDA blood levels, in correlation with other biochemical
and biomolecular parameters, as indirect markers for oxyradical activity.
If we assume that the adaptive changes in
mitochondrial respiratory chain enzyme activities during ischemia serve to antagonize
irreversible peroxidative injury, it also becomes important to investigate the role of
Heat Shock Proteins (HSP) in this pathologic process. It is well documented that in
several animal species including humans, members of the HSP 70/72 family are upregulated
during ischemia, functioning as molecular chaperones and facilitating protein folding,
assembly, transport and translocation11-12-13. HSP 70 are proteins induced by
diverse stress stimuli and their upregulation is often concomitant with the expression of
the c-Fos transcriptional factor14. During cardiac surgery the HSP induction is
widely referred to so called "ischemic preconditioning"11-15-16 and
seems of some interest to analyze HSP generation in the context of cytoprotective
mechanism against ischemia in the human cardiac cell.
The aim of this work is to investigate the
interrelation between mitochondrial oxidative metabolism and peroxidative damage and the
expression of HSP in myocardial ischemia and reperfusion in order to elucidate the roles
played by these adaptive processes in the protection of the cell.
Subjects and methods
Informed consent was obtained from all
patients before they were included in the study. The research and surgical protocols were
approved by the Ethical Committee of the University of Cagliari, Italy. Thirty consecutive
patients undergoing elective coronary artery by-pass grafting were studied and their
admission was determined on the basis of haemodinamic and clinical criteria. The surgical
outcome was uneventful in all patients and there were no complications. The ischemic time
in all patients was 55± 8 minutes, followed by aortic declamping and myocardial
reperfusion. The hearts were maintained in normoxic (PO2 = 140 mmHg) and
normothermic (34°C body temperature) conditions for the entire duration of ischemia. As
described by Calafiore17 and Doyle18 all patients received blood
cardioplegic solution plus KCL at 10 min. time intervals following this scheme:
I° dose: 600 cc (normoxic and normothermic
blood) + KCL (18 mEq/l)
II° dose: 400 cc + KCL (10 mEq/l)
III° and IV° doses: 400 cc + KCL (10 mEq/l)
V° dose: 400 cc + KCL (7 mEq/l)
Three left ventricle samples (30/40 mg)
were taken, one before aortic clamping, the second at the end of the ischemic period, and
the third 34± 5 minutes after final reperfusion. The ventricular biopsies were
snap-frozen in liquid nitrogen. Samples coronary sinus blood for the determination of MDA
were taken in parallel with the muscle biopsies.
Northern Blot
Analysis
Total cellular RNA was isolated and
equal amounts of RNA were electrophoresed under denaturing conditions19. To
confirm that each lane contained equal amounts of total RNA, the content of ribosomal RNA
in each lane was estimated in ethidium bromide-stained gels. RNA was transferred to
Hybond-N filters (Amersham), which were sequentially hybridized with the following
ß-labeled DNA probes: human pURHS70 cDNA for hsp70 (kindly provided by J.R. Nevins,
New York), murine pc-fos-3 clone, (kindly provided by F. Colotta, Milan). For quantitative
determination, autoradiographic bands in the linear range were scanned with a
densitometer/phosphor-imager, and the values were calculated after normalization to the
amount of ribosomal RNA.
Mitochondrial enzyme
activities
Tissue samples were homogenized
at 4°C in a medium containing 0.3 M sucrose, 10 mM KH2PO4 and 1
mg/ml bovine serum albumin (pH 6.50) (medium A). Mitochondria were immediately isolated
and mitochondrial protein was measured. Mitochondrial enzyme assays were performed in a
DWS (Beckman DU 640, USA). To convert absorbance to specific activity (expressed in nmol
min-1 mg protein-1), we used the equation (?A/?t) x V
x 106 (Ptotal x b x ?, where ?A is the
increase in absorbance at 550 nm (absolute value), ?t is the reaction time (min), V
is the volume (in litres) of the cuvette, Ptotal is the total amount of
mitochondrial protein in the cuvette (mg), b is the width of the cuvette (cm) and ?
is the extintion coefficient for cytochrome c (19.1 mmol-1 cm-1).
All assays were performed at 37°C in 1 ml of medium A. We calculated the cytochrome c
oxidase (COX) and succinate cyt. c reductase (SCR) activity from the initial pseudo-linear
rate of cytochrome c oxidation at 550 nm minus 580 nm. Finally, all experiments
were performed using different amounts of mitochondria (10, 5, 2 and 1 mg of mitochondrial
protein)20. A series of five assays with mitochondria from the same sample was
performed for each experiment.
Malondialdehyde
determination
We measured MDA by
high-performance liquid chromatography (HPLC) following the method described by Wong et Al21.
Statistical analysis
Statistical analyses were
carried out in cooperation with the Statistical Department of the University of Cagliari
and the data were analyzed with Stat View V2.0 on an Apple Macintosh IICI computer. The
data are expressed as mean ± standard deviation.
Results
We studied the activities of
cytochrome c oxidase (COX) and succinate cyt. c reductase (SDH) in mitochondrial in
response to tissue oxygen deprivation. In conditions of altered oxygen concentration it is
well known that oxyradical intermediates are produced by the cyt. c oxidase reaction22.
An additional important source of oxyradicals seems localized at Coenzyme Q reaction23.
Therefore, the succinate dehydrogenase reaction in ischemic and reperfused tissues is also
of crucial importance in oxyradical generation.
The significant decrease of COX and SCR
activities in myocardial cell (see Tab. I) is evident as response to ischemic injury.
In respect to ischemia in our patients no
further alteration in these activities was appreciated in reperfused tissue (see Tab. I).
This seems in agreement with current opinion which indicates an enzymic damage associated
with pre-formed oxygen-derived free radicals recirculation, even if the analysis of our
results presumes a different pathogenic evaluation as specified below.
The data regarding coronary sinus blood MDA
levels show a peculiar cellular response to ischemia and to reperfusion (see Tab. II).
This different response to ischemia and to reperfusion appears confirmed by the analysis
of gene reprogramming (see Fig. 1). As well documented in mammalian experimental models,
in our patients HSP 70 mRNA is constitutively expressed and shows a small, but definite,
increase in amount already during ischemia: the increase is maintained during reperfusion
that follows continuous ischemia.
Northern blot analysis reveals steady state
concentrations of mRNA but does not give indications about the mechanism involved in the
possible increases of these concentrations. It is extremely unlikely that the synthesis of
new mRNA molecules can occur under conditions of curtailed energy supply typical of
ischemic tissues: therefore the increase in HSP mRNA concentration in ischemic samples
most probably depends on increased stability of mRNA concentration in ischemic samples,
which is then maintained in the reperfused tissue. Analogous considerations can be applied
to results obtained with c-fos mRNA, which is not expressed at a detectable level in the
control and is a rapidly turning-over molecule, stabilized during ischemia and slowly
decreasing during post-ischemic reperfusion. The lower part of the figure, showing
ribosomal RMA, demonstrates equal loading of all the lanes.
Discussion
The most widely accepted
hypothesis on the pathophysiology of ischemia/reperfusion-induced damage can be summarized
as follows: in ischemic tissues, the generation of reactive oxygen species (ROS) triggers
a cascade of deleterious alterations, which become especially evident during reperfusion,
when molecular oxygen and pre-existing oxyradicals are reintroduced into the tissue8-10-24-25.
These biochemical events may, in turn, cause the hemodynamic and clinical complications
that are often observed postoperatively and, in general, in post-ischemic situations8.
The data obtained in our patients lead us
to propose a different pathway based on two stages: a series of adaptive changes occurring
in ischemic cells, followed by a phase of oxyradical generation and peroxidative damage
triggered by the rapid and massive tissue reoxygenation. The only moderate increase in MDA
blood levels that we found after complete and prolonged ischemia is in keeping with this
hypothesis and suggests that peroxidative injury does not represent the main damaging
process. In contrast, enzyme analyses showed a marked decrease of both succinate
dehydrogenase and cytochrome oxidase activities, which fell to about 25% of those in
normoxic tissue. Considering that these biochemical changes are fully reversible, the
impaired mitochondrial respiratory chain function can be viewed as a transient adaptation
to lower oxygen tension rather than the consequence of oxyradical damage.
In agreement with this concept, the
upregulation of HSP 70/72 and the induction of c-Fos could also contribute to cell
protection through their roles in enzyme protein refolding and assembly15-26-27-28.
Our results are compatible with such a role of HSP 70/72 in reverting cellular oxidative
damage, probably as part of a more complex cytoprotective system. In fact, c-Fos and HSP
70/72 participate in the refolding of enzyme proteins in conditions of cellular energy
depletion and altered oxidative activity14-16-26. Our results in ischemic heart
tissue confirm previous reports and document that HSP 70/72 are expressed during the
adaptation process as peroxidation antagonizing agents27-28.
The investigation on gene reprogramming in
post ischemic heart was undertaken on the basis of evidence emerging from experimental
models: the data that have been obtained seem to suggest that analogous events occur in
cases of clinical relevance, but the limitations in the amount of tissue and in the period
that is ethically allowed for investigation do not permit a full reproduction of the
results obtained with experimental models. However, the increased steady state levels of
mRNAs for HSP 70 and c-fos mRNAs indicate that the adaptation to post-ischemic reperfusion
involves a small but definite reprogramming in gene expression which seems to start
already during ischemia. The proteins synthesized in the later periods of reperfusion ,
not covered by the present investigation, i.e. the HSP 70 molecular chaperones and FOS
proteins building the AP-1 transcription factor are involved in the correct folding of new
protein molecules and in many other processes necessary for the restoration of the
function of cardiac cells.
The sequence of biochemical events observed
in reperfused hearts can be summarized as follows: the enzyme activities of the
mitochondrial respiratory chain are no different from those in ischemic tissue, whereas
MDA levels in blood show a significant increase. As suggested by most recent studies on
reperfusion syndrome, our data also indicate that the myocardial damage accompanying
massive reoxygenation is highly dependent on oxyradical overproduction24. In
other words, supplementation of large amounts of oxygen in a short time leads to
hyperoxemia, which, in turn, sharply elevates the amount of oxygen available in tissues
for oxidative metabolism. However, the oxidative capacity of the heart remains low, still
reflecting the adaptive changes to ischemia. The imbalance between oxygen tension and
oxidative capacity could further increase oxyradical generation by the respiratory chain.
This interpretation agrees with clinical experience that "controlled
reoxygenation" is beneficial in patients subjected to surgical ischemia or affected
by myocardial dysfunction and treated with extracorporeal membrane oxygenation29-30.
Further evidence for ROS toxicity in these conditions comes from the protective effect of
antioxidant administration, which minimizes reperfusion injury in hypoxic or ischemic
human hearts29-31-32.
In conclusion, our data support the
apparent paradox that ischemic injury is less harmful to myocardial cells than rapid and
massive reoxygenation. If, in fact, the drop in oxidative activity favors cell survival by
adapting oxidative metabolism to ischemia, how is this process initiated and controlled? A
plausible explanation is provided by recent studies of nitric oxide (NO) metabolism both
in endothelial cells and in mitochondria3-5-6-33-34. The vascular endothelium
participates actively in the regulation of vascular tone by releasing vasoactive
substances, such as NO34. In oxidative stress due to hypoxia or ischemia, the
endothelial NO may function as an "oxygen sensor". Indeed, generation of
endothelial NO seems to be inversely proportional to changes of blood PO234-35.
The gaseous nature of NO facilitates its diffusion into the cell and may explain its rapid
effect on the mitochondrial respiratory chain. Cleeter et al. first reported that NO
generation can reversibly inhibit mitochondrial respiration, and suggested that cytochrome
oxidase was the primary target of NO inhibition36. In agreement with this
hypothesis, recent findings have documented that NO inhibits O2 consumption and
that, in the presence of O2, this inhibition is reversed in a time-dependent
fashion37-38. It was also shown that NO binds rapidly to binuclear (heme a3-CIIb)
site, in competition with O2 and other ligands39-40. Thus, it is
conceivable that mitochondrial adaptation to hypoxia or ischemia may involve modulation of
cytochrome oxidase by NO in order to adjust the oxidative level to the lower O2
tension. The assumption that this mechanism is fully reversible once normoxia is restored
implies that the marked decrease of cytochrome oxidase activity in ischemic myocardial
cells is, in fact, an adaptive change rather than merely the result of damage41.
In contrast to this scenario, prolonged exposure of cells to NO or to peroxidative injury
(with consequent antioxidant depletion) causes irreversible inhibition of cellular
respiration: in these conditions, NO would be converted from a physiological mediator into
a pathological factor. This Dr. Jekyll/Mr. Hyde transformation could occur in reoxygenated
tissues, where, as mentioned above, oxyradical generation dominates cell metabolism.
To investigate these postulated opposite
roles of NO in human cardiac cells in ischemia and reperfusion, we have initiated studies
of NO generation in five patients. The concentrations of NO were measured in normoxic,
ischemic, and reperfused hearts using a microelectrode specific for NO detection.
Preliminary unpublished data seem to confirm a strong correlation between NO generation
and respiratory chain activities during ischemia. In reperfused hearts, we found that
peroxynitrate produced by rapidly restored O2 tension had deleterious effects
on respiratory chain activities.
In summary, we found that human myocardial
cells appear to be able to adjust their oxidative metabolism to ischemic conditions
whereas reoxygenation, which is actually employed in clinical practice, compromises the
functional integrity of these cells. These findings call for a re-evaluation of the
clinical management of cardiac hypoxia and ischemia and, in particular, suggest a novel
therapeutic approach in terms of cytoprotection of ischemic and reperfused heart.
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Tab. I – Mithocondrial respiratory
chain enzyme activities.
