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From the Departments of Cardiothoracic Surgery,*
and
Cardiac Medicine,
National Heart and Lung
Institute, Imperial College School of Medicine, London; and the
Department of Neurochemistry,
Institute of
Neurology, Queen Square, University College London, London,
United Kingdom
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Delayed myocardial tolerance to ischemia reperfusion injury can be increased by heat stress that results in de novo synthesis of heat shock proteins (Hsps)7 and antioxidant enzymes, such as manganese superoxide dismutase.8 Hsps are a family of highly conserved cytosolic proteins, transiently expressed after exposure of the cell to sublethal environmental stimuli such as hyperthermic and oxidative stress. Several studies have shown that controlled in vivo hyperthermic preconditioning resulting in elevated Hsp levels can improve myocardial functional recovery in isolated ischemic heart models.9 Each Hsp group has a specificity of function and cellular compartmentation. Cardiac heat-shock protein research, which has focused primarily on the abundant 70-kd group, has identified three main classes: the constitutive 73-kd isoform (Hsc 70), physiologically expressed at high levels in the cytosol and involved in protein-folding functions; an inducible nuclear orientating 72-kd isoform; and a mitochondrial-specific Hsp 75 isoform that is up-regulated after hypoxia.10-12 Hsps 60 and 10 have similarly been shown in combination to form a mitochondrial-located chaperonin complex conferring protection to ischemia-challenged myocytes.13 Equally pertinent to myocardial protection, is the 32-kd protein (Hsp 32) an inducible isoform of the free heme-metabolizing enzyme, heme oxygenase (HO-1). HO-1 catalyzes the cleavage of heme rings to release the vasodilator neurotransmitter carbon monoxide,14 ferrous iron, and biliverdin. This last product, biliverdin is rapidly metabolized by biliverdin reductase to form the cytosolic antioxidant bilirubin.15 Heme oxygenase-mediated antioxidant defenses are further extended in ischemia compromised tissues by the capacity of HO-1 to export the iron byproduct extracellularly thus reducing the potential for Fe2+-mediated hydroxyl formation.16
Increased expression of cytosolic heat-shock proteins has been associated with improved myocardial recovery after reperfusion.17,18 We hypothesized that the heat-stress response in the heart may increase mitochondrial respiratory complex activity, thereby protecting integrated mitochondrial and hence myocardial function. As some heat Hsps are known to be mitochondrial chaperones, we also measured the expression of several Hsps with a view to assessing the relationship to changes in mitochondria. The present study was designed, using a clinically relevant model of cardioplegic arrest, to determine the effect of heat stress applied in vivo on myocardial functional recovery after ischemia and its relation to mitochondrial respiratory chain function. Having verified up-regulation of Hsps 72, 60, and 32 in myocardium of heat-stressed animals, we investigated the capacity of heat shock to improve: 1) myocardial function recovery, 2) mitochondrial function (oxygen consumption), 3) mitochondrial respiratory-chain enzyme activities, and 4) cellular and mitochondrial morphology in the isolated rat heart model subjected to 6 hours of cardioplegic arrest.
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Materials and Methods |
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All animal procedures were performed in accordance with the "Animal (Scientific Procedures) Act (U.K.), 1986" and the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication no. 85-23, revised 1996). The study was approved by the National Heart and Lung Institute’s ethics committee on animal research. Animals were allowed standard laboratory rat diet and water ad libitum.
Induction of Heat Stress
Sprague-Dawley rats (male, 280 to 320 g) were anesthetized using sodium pentobarbitone (50 mg kg-1, i.p.). In heat-treated experimental groups, the rats were then placed in a whole-body temperature-controlled heating blanket (IMS K-Temp control unit; IMS, Cheshire, U.K.) and body temperature was raised to 41.8 to 42.0°C. Body temperature was monitored using a rectal temperature probe and maintained for 25 minutes as described previously.9 Sham-treated groups were similarly anesthetized but not heat stressed. On recovery, animals were rehydrated with normal saline (10 ml kg-1 i.p.) and allowed to recover for 24 hours.
Heart Perfusion
Sham-treated and hyperthermically treated rats were anesthetized with diethyl ether and sodium heparin (1000 IU/kg) injected via the femoral vein. Hearts were rapidly excised and immersed momentarily in ice-cold filtered (0.45-µm pore size) Krebs-Henseleit (KH) buffer containing (in mmol/L): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.23 KH2PO4, 24 NaHCO3, 11.1 glucose, and 1.2 CaCl2, pH 7.4. The hearts were cannulated in the Langendorff mode and perfused with KH buffer at 37°C, oxygenated (95%O2/5%CO2) at a constant pressure of 1,000 mm H2O.19 Hearts were not paced during the entire protocol.
Myocardial Function Assessment
Mechanical function was assessed using an intraventricular balloon inserted via the mitral orifice into the left ventricle to determine systolic pressures, as previously described.20 The balloon was inflated with water to produce a baseline end diastolic pressure of 10 mmHg and connected to a pressure transducer to record left ventricular pressures and heart rate. Coronary flow was measured using a Skalar electromagnetic MD1401 meter (Skalar, Delft, The Netherlands). Analogue signals were continuously recorded using a Biopaq AcqKnowledge Aquisition MP100 System (Linton Instruments, Norfolk, UK). To characterize cardiac function, left ventricular-developed pressure (LVDP) was assessed from the peak systolic pressure minus end diastolic pressure. Recovery of cardiac function was expressed using the relative recovery of post-ischemic versus pre-ischemic LVDP and pre- and post-ischemic coronary flows at the time points stated.
Mitochondrial Isolation Analysis
After the isolated perfusion period, the rat hearts were immersed in ice-cold isolation medium containing (in mmol/L) 225 mannitol, 75 sucrose, 10 Tris, 2 EGTA, pH 7.2, minced and digested using the protease enzyme, Nagarse (1.5 mg/ml) to enable recovery of both sarcolemmal and interfibrillar mitochondria. Nagarse was removed after an 8-minute period by washing (4x) in isolation medium and the tissue homogenized using four strokes of a Potter-Elvehjem homogenizer. Mitochondria were then isolated using differential centrifugation from the homogenate at 3,500 x g (3 minutes) and pelleted at 11,500 x g (2x 12 minutes) as described previously.21,22 Mitochondrial protein was measured using a modified Lowry method23 (Lowry D.C. kit; Biorad, UK).
Mitochondrial oxygen consumption was measured as previously described6,22 using a water-jacketed Clark-type oxygen electrode (World Precision Instruments, Hertfordshire, UK) and recorded using Biopaq AcqKnowledge software. All experiments were performed at 30°C in 350 µl of respiration medium containing (in mmol/L): 100 KCl, 75 mannitol, 25 sucrose, 10 Tris-HCl, 10 KH2PO4-Tris, and 0.05 ethylenediaminetetraacetic acid (dipotassium salt), pH 7.4. Incubations were performed using 0.5 mg of mitochondrial protein and 0.125 mg of fat-free bovine serum albumin. State 4 respiration was initiated using 5 mmol/L of glutamate plus 5 mmol/L of malate or 5 mmol/L of succinate plus 1 µmol/L of rotenone. State 3 respiration was initiated by the addition of ADP. Respiratory control indices (RCI) were calculated as state 3 rate/state 4 rate. Mitochondrial complex activities were measured in mitochondrial samples lysed by three cycles of freeze thawing. Assays for mitochondrial complex I (NADH-ubiquinone oxidoreductase, EC 1.6.99.3), complex II/III (succinate-ubiquinone/ubiquinol-cytochrome c reductase, EC 1.8.3.1), complex IV (cytochrome c oxidase, EC 1.9.3.1), complex V (ATPase, EC 3.6.1.3), and citrate synthase (EC 4.1.3.7) were performed essentially as described,24 except that the volumes were scaled down to a final reaction volume of 250 µl. Assays were performed using a SpectraMax-Plus 96-well spectrophotometer (Molecular Devices, Crawley UK) operating in kinetic mode at 30°C. After correction for optical path length in the microplate, results were expressed as nmol/min/mg protein for complexes I, II/III, V and citrate synthase and as the first order rate constant k/min/mg protein for complex IV.22,25
Stress Protein Expression
Levels of Hsp 32, 60, and 72 were determined by Western immunoblotting, as previously described.26 Briefly, 50 µg of protein of whole heart homogenates or purified mitochondrial pellets were separated on 10% T (total concentration of acrylamide plus bis-acrylamide in g/100 ml) sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to Hybond C Super membrane at 500 mA for 1 hour. Blots were probed with antibodies specific to Hsp 32 (OSA-111), Hsp 60 (SPA-806), or inducible Hsp 70 (SPA-820) (Bioquote Ltd., York, UK).
Reactivity of antibodies against the Hsps was visualized using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK). After enhanced chemiluminescence exposure, primary and secondary antibodies were stripped from blots by incubation at 50°C for 30 minutes in a solution of 2% w/v sodium dodecyl sulfate, 62.5 mmol/L of Tris-HCl, pH 6.7, and 100 mmol/L of 2-mercaptoethanol. Membranes were reprobed for the cytosolic marker ß-tubulin using a monoclonal antibody. Levels of Hsp 32, 60, and 70 levels were scanned densitometrically on enhanced chemiluminescence films using Quantity One software (P.D.I., NY) and normalized against respective ß-tubulin levels.
Transmission Electron Microscopy
The effects of heat stress on ultrastructural morphology were assessed by standard thin-section electron microscopy in pre-ischemic and reperfused myocardium. Heat-stressed and sham-treated hearts were subjected to nonischemic perfusion (35 minutes at 37°C) or to the cardioplegic arrest and reperfusion procedures as previously described. At the end of these procedures, hearts were immediately perfusion-fixed via the aorta with 2.5% glutaraldehyde in KH buffer for 5 minutes.27,28 Left ventricular tissue was dissected out and sections (1 x 1 x 1 mm) were further fixed for 2 hours in 2.5% glutaraldehyde. Tissue samples were postfixed in 2% osmium tetroxide, en bloc stained with uranyl acetate in 50% ethanol, dehydrated through an ethanol series, and embedded in araldite. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined using a Philips EM301 electron microscope.
Experimental Protocols
To determine the effect of elevated heat-shock protein expression with the protection of cardiac mitochondrial and myocardial mechanical function in isolated perfused hearts, two major experimental groups were used. Hearts derived from heat-stressed and sham-treated groups were subjected to the ex vivo ischemic procedures described and compared to appropriate pre-ischemic controls. Cardiac mitochondrial and myocardial mechanical function changes were studied in sham-treated and heat-stressed hearts before, and after 6 hours of cardioplegic arrest at 4°C. After an initial 30-minute normoxic perfusion at 37°C, pre-ischemic cardiac function was evaluated using heart rate, LVDP, and coronary flow. Hearts from each pre-ischemic group were also collected for mitochondrial function.
Subsequent to the initial perfusion, hearts in the postischemic groups were arrested by infusion of 4°C St. Thomas cardioplegic solution at a constant pressure of 60 mmHg for 2 minutes. Hearts were maintained for 6 hours immersed in St. Thomas cardioplegic solution at 4°C. After the 6-hour ischemic period, hearts were reperfused with oxygenated KH buffer at 37°C for 60 minutes. Post-ischemic cardiac function and mitochondrial parameters were evaluated at the end of the reperfusion period.
Statistical Analyses
Values are presented as group means ± SEM of six animals. Statistical analysis was performed using StatStat V2.03 (SPSS Inc, Erkrath, Germany). Comparison between groups was performed using a one-way analysis of variance for repeated measures followed by Bonferroni test to indicate individual significant differences. Normalized differences between sham- and heat-treated group means were assessed using the nonparametric Mann-Whitney test. P values less than 0.05 were considered significantly different.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1, A and B
, illustrates
alterations in Hsp expression in the myocardium of sham- and
heat-treated rats. Densitometric analysis of stress proteins confirmed
that exposure (25 minutes) to a hyperthermic episode resulted in a
significant elevation after 24 hours in the expression of myocardial
Hsps 72 and 32 as compared to untreated myocardium. Constitutive Hsp 60
levels were also enhanced (P < 0.05) in the
whole heart samples and in purified cardiac mitochondrial pellets
(Figure 1C)
derived from heat-treated rats. Anti-ß-tubulin antibody
did not react with mitochondrial proteins demonstrating that no
cytosolic contamination was present in the purified cardiac
mitochondrial pellets.
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The maximally attained percent recovery of functional values in
post-ischemic reperfused myocardium are presented in Table 1
. No significant differences were seen
under baseline conditions in pre-ischemic LVDP, heart rate, or coronary
flow between the two groups (sham-treated versus
heat-stressed groups).
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indicate that heat-stress treatment
provided significantly (P < 0.05) improved
functional recovery in comparison to the sham-treated group. During
reperfusion, the post-ischemic heat-stressed group attained recovery
coronary flows of 61.9 ± 3.8%, heart rates of 93.7 ±
2.9%, and LVDP levels of 49.5 ± 3.7% of pre-ischemic baseline
values. In the post-ischemic sham-treated group, coronary flow, heart
rate, and LVDP recovered to only 33.6 ± 2.3%, 81.4 ± 2.5%
and 29.6 ± 2.9%, respectively, of the pre-ischemic baseline
values. Recovery of Cardiac Mitochondrial Respiratory Function
Pre-ischemic state 4 and 3 mitochondrial respiratory rates and RCI values calculated in heat-stressed myocardium show no significant change from values obtained in sham-treated rats. These results confirm that the hyperthermic protocol used in this study did not affect any of the cardiac mitochondrial parameters considered in this study.
Figure 2
summarizes the respiratory data
in mitochondria isolated from the two groups of sham-treated and
heat-stressed animals. Mitochondria isolated from reperfused control
hearts subjected to 6 hours of cardioplegic ischemia showed, as
expected, a significantly (P < 0.05) decreased
state 3 respiratory rate with increased (P <
0.05) state 4 rate, resulting in a decline in RCI. As can be seen
however, reperfused cardiac mitochondrial respiratory parameters were
substantially salvaged by the hyperthermic treatment protocols used in
this study. Both glutamate plus malate (NAD+)-
and succinate (FAD)-driven state 4 rates were significantly
(P < 0.05) reduced in the reperfused
heat-stressed hearts from the values obtained in the sham-treated group
(Figure 2, A and B)
. At the same time, heat stress significantly
(P < 0.05) ameliorated the reperfusion-mediated
fall in malate NAD+-linked state 3 rate seen in
the sham-treated hearts (Figure 2, C and D)
. RCI values were observed
(Figure 2, E and F)
to recover significantly (P
< 0.05) in heat-stressed myocardium as a consequence of preserving
state 3 and 4 rates.
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Hyperthermic treatment raised individual mitochondrial
respiratory chain enzyme activities (Figure 3)
. Although, only complex I, IV, and V
activity were shown to be significantly (P <
0.05) increased from the sham-treated values, complex II-III
respiratory enzymes showed a slight increase of activity, which was not
statistically significant (P < 0.06).
Reperfusion of cardioplegically arrested myocardium after 6 hours of
cold cardioplegic storage significantly (P <
0.05) reduced mitochondrial enzyme activities (Figure 3)
. The results
obtained in this study showed that, whereas the complex activities were
all reduced at 1 hour after reperfusion, complex I (NADH-CoQ reductase)
and complex V (oligomycin-sensitive ATPase) decreased
markedly. Significantly, all of the complexes in the hyperthermic group
studied showed higher (P < 0.05) enzyme
activity levels after 6 hours of storage and reperfusion myocardium
than shown in the sham-treated group under identical conditions. It is
possible that complex IV activities in vivo may have been
decreased as a consequence of heme oxygenase-derived CO
binding.29
Any effects of CO binding on complex IV
activity measured in our spectrophotometric assay may be difficult to
ascertain however, because of CO being driven off the complex
IV-binding site by light from room light sources.
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. Unlike the membrane-bound
complexes, no significant increase in citrate synthase activity was
observed in heat-treated pre-ischemic myocardium.
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Sham-treated, pre-ischemic heart contained abundant
mitochondria in rows alongside the myofibrils (Figure 5A)
. Myofibril appearance was regular and
transverse tubules distinct. Where atypical structure was observed in
occasional mitochondria of control tissues, this was attributed to
suboptimal fixation. Reperfusion of the sham-treated myocardium after
cold ischemia (6 hours) resulted in severe disruption of the myofibrils
and disorganization of mitochondrial distribution (Figure 5B)
. This
appearance was representative of a large proportion (>80%) of the
fields examined. Heat-stress pretreatment produced no overall change in
the normal ultrastructural appearance of pre-ischemic myocardium
(Figure 5C)
, and post-ischemic pretreated hearts gave a very similar
ultrastructural appearance (Figure 5D)
to pre-ischemic myocardium.
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. In
pre-ischemic controls, mitochondria exhibited intact double membranes
with tightly packed, parallel lamellar cristae, characteristic of
normal ultrastructural morphology (Figure 6A)
. Mitochondria of hearts
subjected to cold ischemia and reperfusion, were severely disrupted,
swollen, and empty in appearance (Figure 6B)
. In contrast, the
appearance of the reperfused heat-stressed myocardium closely resembled
that of the pre-ischemic group in that mitochondrial structures (Figure 6C)
were preserved in the majority of fields examined.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mitochondrial dysfunction has been documented in various models of ischemia-reperfusion in relation to the content and activity of proteins involved in ATP production by oxidative phosphorylation.6,33 Reperfusion of sham-treated myocardium after exposure to cold cardioplegic ischemia for 6 hours consistently decreased state 3 rate and uncoupled mitochondrial respiration using both NAD+- and FAD-linked substrates. State 4 respiration rate was simultaneously raised, suggestive of an increased membrane proton leak.34 These data together with the loss of citrate synthase activity from the mitochondrial fraction confirm an increase in membrane permeability with diminished phosphorylation capacity in the reperfused myocardium.35 This result is supported by the post-ischemic ultrastructural appearance of the mitochondrial organelles in situ, in heat-stressed and sham-treated myocardium. Our study shows that recovery of RCI (consistent with ATP restoration after reperfusion) is much greater in heat-stressed than in the sham-treated hearts. The increase in pre-ischemic level however, of mitochondrial protein activity was not reflected in state 3 and 4 rates. Oxygen consumption and phosphorylation rates during physiological respiration state 3, have previously been shown to be controlled by a combination of respiratory chain enzyme and ATP synthase activity.24 Studies using specific inhibitors of the individual mitochondrial complexes demonstrate that the activities of several of the complexes can be in excess of that needed for maximal rates of fully integrated mitochondrial oxidative phosphorylation.36 This excess enzyme capacity may account for the lack of effect of whole-body hyperthermia on respiration states 3 and 4 in pre-ischemic myocardium.
Mitochondrial oxidative phosphorylation is dependent on the integration
of the polypeptide complexes of the respiratory chain with ATP synthase
assembled on the inner membrane (Figure 7)
. The data collected in this study
shows that, post-ischemic mitochondrial function is protected in
myocardium expressing high levels of Hsp 72, 60, and 32. We
suggest that this protected mitochondrial function arises as a
consequence of elevated pre-ischemic mitochondrial enzyme activities.
The relationship between Hsp expression and mitochondrial complex
activity presented here supports previous studies,10,37
showing that up-regulation of molecular chaperone complexes (Hsp72 or
Hsp 60/10) in the myocardium contribute to the preservation of
mitochondrial integrity. This may be achieved primarily through
facilitation of nuclear-encoded protein import and assembly in the
mitochondrial matrix.38
Indeed, translocation of the
ß-subunit of the F1F0
ATPase (complex V) into mitochondria of Saccharomyces
cerevisiae has been shown to be dependent on the presence of
cytosolic Hsp72 (Figure 7)
.39
Consequently, mitochondrial
enzyme activity may be increased at the translational level. Therefore,
up-regulation of inducible Hsp 70 expression should effectively sustain
state 3 respiration rates. Previous reviews have also postulated an
involvement of Hsp 72 in preventing electron leak between complexes III
and IV by binding and reducing cytochrome c loss from
mitochondrial membranes (Figure 7)
, thereby averting an increase in
state 4 respiration rates and induction of cytochrome c
linked apoptosis.40,41
Certainly, Hsp 60/10
over-expression and stress-induced chaperonin-like molecules such as
Tcm62p have been shown to markedly protect both nuclear and
mitochondrial encoded proteins including primary dehydrogenases from
ischemia damage.13,42
However, protection of mitochondrial
proteins from oxidative stress need not necessarily stem exclusively
from a chaperoning action of Hsps. Mitochondrial enzyme activity may be
also be simultaneously protected by antioxidant systems derived from
the activation of heat-stress-mediated scavenging mechanisms. Under
normal physiological conditions, superoxide anions
(O2-·), are generated by
the mitochondrial respiratory chain as a consequence of electron leak
onto molecular oxygen.24,43
Ischemic damage to the
myocardium simultaneously results in increased nitric oxide production
and exacerbation of O2-·
radical production with consequent increases in peroxynitrite and
hydroxyl radical formation. As mitochondrial enzyme complexes consist
of polypeptides encompassing iron-sulfur clusters, they can become
subject to oxidative deactivation by reactive oxygen and nitrogen
species, thereby reducing enzyme activity.44,45
By
up-regulating HO-1 (Hsp 32) expression and activity, levels of the
antioxidant bilirubin, known to be a peroxynitrite
scavenger,46
and shown to be cytoprotective at nanomolar
concentrations, are increased.15,47
Additionally,
oxidative stress-induced changes in cellular glutathione redox status
have recently been demonstrated to be responsible for the induction of
Hsp 70.48
Consequently, we propose that levels and
activity of respiratory enzymes may be augmented in this model of
normoxic and ischemic-reperfused myocardium by an Hsp-mediated
up-regulation of protein production and simultaneous dissipation of
free radicals. Recent studies by our group pursuing this theme, have
confirmed that selective viral-mediated Hsp 72 gene transfection of
transplanted hearts,49
results in elevated myocardial
complex I activities (Jayakumar J, Suzuki S, Sammut I, et al, submitted
for publication). The two phenomena of Hsp up-regulation and elevated
mitochondrial enzyme activities are therefore closely associated.
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In summary, this study provides the first evidence, confirming that in vivo heat stress enhances mitochondrial energetics, while protecting myocardial function against ischemia-reperfusion injury. Heat-stress mediated enhancement of mitochondrial enzyme activity and prevention of the loss of mitochondrial potential to drive oxidative-phosphorylation may confer resistance to mitochondrial-linked apoptosis. This energetic enhancement would also support a protection of cardiac high-energy phosphate supply. The present findings offer the exciting possibility that pharmacological modulation54,55 of heat-stress proteins in organ grafts may play a central role in the future of clinical transplantation.
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Acknowledgements |
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Footnotes |
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Supported by British Heart Foundation Grant PG99173. R.T.S. is a Visiting Professor at the Medical University of Gdansk, Poland..
Accepted for publication February 5, 2001.
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