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1 Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6; and 2 Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In view of the critical role of sarcoplasmic reticular (SR) Ca2+ release and the Ca2+ pump in cardiac contraction-relaxation, this study was undertaken to assess the status of SR function, protein content, and gene expression in isolated rat hearts subjected to global ischemia for 30 min followed by 60 min of reperfusion (I/R). Attenuated recovery of contractile function in the I/R hearts was associated with reduced SR Ca2+ uptake, Ca2+ release, and ryanodine-binding activities. mRNA levels and protein contents for SR Ca2+ pump ATPase and Ca2+ release channels were markedly depressed in the I/R hearts. Perfusion of hearts with superoxide dismutase plus catalase, well-known scavengers of oxyradicals, prevented the I/R-induced alterations in cardiac function and partially prevented SR Ca2+ transport activities and mRNA abundance. In hearts perfused with xanthine plus xanthine oxidase or H2O2, changes similar to those in the I/R hearts were observed. These results indicate that oxyradicals may participate in depressing the SR Ca2+ handling and gene expression in the I/R heart. It is suggested that treatment of hearts with antioxidants may improve the recovery of cardiac function by preserving the SR function and partially protecting the SR gene expression.
ischemia-reperfusion injury; cardiac contractile function; cardiac sarcoplasmic reticulum gene expression; cardiac sarcoplasmic reticulum calcium transport; oxygen free radicals
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THERE HAS BEEN a great deal of interest in understanding the contractile dysfunction that occurs under different pathological conditions such as acute myocardial infarction and ischemic heart disease as well as after cardiac bypass surgery, heart transplantation, and coronary angioplasty (3). A common feature among these pathologies and clinical maneuvers is the occurrence of reperfusion after an episode of myocardial ischemia (3). Although early restoration of blood flow after coronary occlusion has been reported to improve heart function and reduce infarct size (15), there is evidence that reperfusion, if not instituted within a certain time period of the ischemic insult, may induce cardiac contractile dysfunction, ultrastructural damage, and changes in myocardial metabolism (8, 25). Recent studies have shown proteolysis (29) and DNA fragmentation (11) during the postischemic reperfusion period. Intracellular Ca2+ overload (13), as a consequence of abnormalities in Ca2+ handling by cardiomyocytes, has also been suggested to explain the adverse effects of ischemia-reperfusion (I/R) in the heart (8). On the other hand, release of oxygen-derived free radicals within the first few minutes of reperfusion has been proposed to explain the I/R-induced contractile changes in the heart (32). In fact, exposure of the heart to different species of oxyradicals has been shown to cause structural (4) and functional (2) alterations in the heart that are similar to those seen in I/R; these changes have been demonstrated to be due to abnormalities in Ca2+ handling by the sarcoplasmic reticulum (SR) (23) and sarcolemma (14).
There is an increasing body of evidence to suggest the beneficial effects of antioxidants and oxyradical scavengers in ischemic heart disease. Earlier studies have shown that treatment of the heart with a combination of superoxide dismutase (SOD) and catalase (CAT) improved myocardial functional recovery after I/R (26, 32). SOD + CAT has been observed to protect the oxyradical-induced changes in membrane lipids, partially attenuate the ionic imbalance, and significantly reduce the ventricular fibrillation in the I/R hearts (26, 27). A recent study also reported an improvement in the functional recovery of the heart after I/R when the extracellular SOD was overexpressed in transgenic mice (5). Although SR Ca2+ uptake and release activities have been reported to be depressed in I/R hearts (8, 9), the role of oxyradicals in these defects has not been examined. Likewise, an enhanced gene expression of SR Ca2+-regulatory proteins in stunned pig heart has been reported (10), but the significance of this observation in terms of SR function in this condition is not clear. Furthermore, very little is known about the status of content and gene expression of SR proteins in the I/R hearts with or without scavenging of the oxyradicals. Thus the objective of this study was to investigate the involvement of reactive oxygen species as a possible mechanism underlying alterations in cardiac SR function due to I/R. Changes in mRNA levels and protein contents of the SR membrane were studied in isolated rat hearts subjected to I/R in the absence or presence of SOD + CAT, a potent scavenger system for different oxygen species. In addition, alterations in cardiac performance, SR function, protein content, and gene expression were monitored in hearts perfused with xanthine (X) and xanthine oxidase (XO), an oxyradical-generating system, or H2O2, an active species of oxygen and a potent oxidant, to provide further evidence for the role of oxyradicals in SR changes. To the best of our knowledge, this is the first report showing deleterious effects of reactive oxygen species in the heart as well as the protective effects of SOD + CAT treatment in I/R at the level of SR function and gene expression.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Heart perfusion and experimental protocol.
Male Sprague-Dawley rats (250-300 g) were anesthetized with a
mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). The hearts were
rapidly excised, cannulated to the Langendorff apparatus, and perfused
with Krebs-Henseleit solution (37°C), gassed with a mixture of 95%
O2-5%
CO2, pH 7.4, containing (in mM)
120 NaCl, 25 NaHCO3, 11 glucose,
4.7 KCl, 1.2 KH2PO4,
1.2 MgSO4, and 1.25 CaCl2. The hearts were
electrically stimulated at a rate of 300 beats/min (Phipps and Bird,
Richmond, VA), and the perfusion rate was maintained at 10 ml/min. A
water-filled latex balloon was inserted into the left ventricle and
connected to a pressure transducer (model 1050BP, Biopac System,
Goleta, CA) for the left ventricular systolic and diastolic pressure
measurements; the left ventricular developed pressure (LVDP) was the
difference between systolic and diastolic pressures. The left
ventricular end-diastolic pressure (LVEDP) was adjusted at 10 mmHg at
the beginning of the experiment, and the left ventricular pressures
were differentiated to estimate the rate of ventricular pressure
development (+dP/dt) and the rate of
ventricular pressure decline
(
dP/dt) with Acknowledge 3.03 software for Windows (Biopac System). All hearts were stabilized for 30 min before use in this study and were maintained at a constant temperature (37°C) throughout the experiment. The control hearts were perfused for 30, 60, and 90 min, and because no significant difference (P > 0.05) with respect
to each parameter was observed between these hearts, the values were
grouped together.
70° C for 2-3 days before use. SOD (from
bovine erythrocytes, ~3,500 U/mg) and CAT (from bovine liver,
~25,000 U/mg) were purchased from Sigma-Aldrich (Oakville, ON,
Canada). For studying the effects of oxyradicals and oxidants, the
hearts were perfused in the absence (control) and presence of the X + XO system or
H2O2
for 20 min; the protocol of this set of experiments is shown in Fig. 1.
The final concentrations of X, XO, and
H2O2
were 2 mM, 0.03 U/ml, and 300 µM, respectively. X and XO were
purchased from Sigma-Aldrich;
H2O2
was purchased from Fisher Scientific (Nepean, ON, Canada). The
selection of different concentrations of X, XO,
H2O2,
SOD, and CAT was based on our previous experience with these agents
(14, 20, 21). Furthermore, preliminary experiments revealed that SOD + CAT was required for preventing the X + XO- and
H2O2-induced
changes completely as well as for obtaining maximal beneficial effects
against I/R-induced cardiac changes reported in this study.
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SR preparation. SR vesicles were obtained by a method described previously (1, 31) with slight modifications. Briefly, ventricular tissue was pulverized and homogenized in a mixture of (in mM) 10 NaHCO3, 5 NaN3, and 15 Tris · HCl, pH 6.8 (10 ml/g tissue), with a Polytron homogenizer (Brinkmann, Westbury, NY) at a setting of 5. The homogenate was then centrifuged for 20 min at 9,500 rpm to remove cellular debris. The supernatant was further centrifuged for 45 min at 19,000 rpm (model JA 20.0, Beckman), the pellet was suspended in 8 ml of a mixture of 0.6 M KCl and 20 mM Tris · HCl, pH 6.8, and centrifuged for 45 min at 19,000 rpm. The final pellet was suspended in 1 ml of 250 mM sucrose and 10 mM histidine, pH 7.0. All solutions contained 0.1% phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor. The purity of the membrane preparation was determined by measuring the activities of marker enzymes such as ouabain-sensitive Na+-K+-ATPase (sarcolemmal marker), cytochrome c oxidase (mitochondrial marker), glucose-6-phosphatase (SR marker), and rotenone-insensitive NADPH cytochrome c reductase (SR marker) according to methods described earlier (1). These marker studies revealed that SR preparations from control and experimental hearts contained negligible (2-4%), but to an equal extent, cross contamination by other subcellular organelles.
Measurement of Ca2+ uptake. Ca2+ uptake activity of SR vesicles was measured by a procedure described previously (12). Briefly, a total volume of 250 µl contained 50 mM Tris maleate (pH 6.8), 5 mM NaN3, 5 mM ATP, 5 mM MgCl2, 120 mM KCl, 5 mM potassium oxalate, 0.1 mM EGTA, 0.1 mM 45CaCl2 (20 mCi/l), and 25 µM ruthenium red. Ruthenium red was added as an inhibitor of the Ca2+ release channel under the assay conditions mentioned above. The reaction at 37°C was initiated by adding SR vesicles (10 µg protein) and terminated after 1 min by filtering a 200-µl aliquot of the incubation mixture through 0.45-µm Millipore filters. The latter were washed with 5 ml of washing buffer, dried at 60°C for 1 h, and then counted in a liquid scintillation counter. As described earlier (1), the Ca2+ uptake reaction was linear during 2 min of the incubation period.
[3H]ryanodine-binding assay. [3H]ryanodine binding with SR preparations was estimated by a procedure described elsewhere (31). Briefly, SR membranes (0.1 ml of 0.5 mg/ml) were incubated at 37°C for 60 min in a total volume of 1 ml containing 25 mM imidazole (pH 7.4), 1 M KCl, 1-40 nM [3H]ryanodine, 0.95 mM EGTA, and 1.013 mM CaCl2 (free Ca2+ concentration was 20 µM). The reaction was terminated by filtering 0.3 ml of the reaction mixture through 0.45-µm filter paper (type HA, Millipore), presoaked in the washing buffer (25 mM imidazole and 1 M KCl), and then washed twice with 5 ml of the washing buffer. Each sample was added to a vial containing 8 ml of scintillation liquid and counted in the Beckman liquid scintillation counter for 5 min. The nonspecific binding was determined in the presence of 10 µM ryanodine. Data of the specific binding were analyzed using the Ligand software for calculating the maximal binding activity (Bmax) and rate constants (Kd).
Measurement of EGTA-induced Ca2+ release and ryanodine-sensitive Ca2+ release. The Ca2+ release activity of SR vesicles was measured by a procedure described previously (19). Briefly, the SR fraction (62 µg protein) was suspended in a total volume of 625 µl of loading buffer containing (in mM) 100 KCl, 5 MgCl2, 5 potassium oxalate, 5 NaN3, and 20 Tris · HCl (pH 6.8). After incubation with 10 µM 45CaCl2 (20 mCi/l) and 5 mM ATP for 45 min at room temperature, EGTA-induced Ca2+ release was carried out by adding 1 mM EGTA to the reaction mixture. The reaction was terminated at 15 s by the Millipore filtration technique. Radioactivity in the filter was counted in 10 ml of scintillation fluid. For Ca2+-induced Ca2+ release, 1 mM EGTA + 1 mM CaCl2 was added to the reaction mixture; the rest of the procedure was the same as described for EGTA-sensitive Ca2+ release. The Ca2+-induced Ca2+ release was completely prevented (95-97%) by treatment of SR preparations with 20 µM ryanodine.
Western blot analysis. The protein content of Ca2+ pump ATPase [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a)], ryanodine receptor (RyR), phospholamban (PLB), and calsequestrin (CQS) was determined according to methods described by other investigators (18). Protein samples (20 µg of total protein/lane) were separated by electrophoresis through a 10% mini-SDS-PAGE in 5% (for RyR), 10% (for SERCA2a), 12% (for CQS), and 15% (for PLB) gels. Samples for SERCA2a, PLB, and CQS were transferred to polyvinylidene difluoride membranes; the RyR sample was transferred to a nitrocellulose membrane. The membranes were probed with monoclonal anti-SERCA2a (1:1,400; Affinity Bioreagents, Golden, CO), monoclonal anti-RyR (1:1,400), monoclonal anti-PLB (1:2,000), or polyclonal anti-CQS (1:2,000) antibodies. The antibodies for RyR, PLB, and CQS were purchased from Upstate Biotechnology (Lake Placid, NY). For SERCA2a and PLB, a peroxidase-linked anti-mouse IgG was used as a secondary antibody (1:5,000); biotinylated anti-mouse IgG antibody (1:2,500; Amersham Life Science, Oakville, ON, Canada) was used for RyR and CQS. The membranes for RyR and CQS were incubated with enhanced chemiluminescence (ECL) streptavidin-conjugated horseradish peroxidase (1:5,000; Amersham Life Science, Oakville, ON, Canada). Antibody-antigen complexes in all membranes were detected by the ECL kit (Amersham Life Science). Protein bands were then visualized on Hyperfilm-ECL. An imaging densitometer (model GS-670, Bio-Rad, Hercules, CA) was used to scan the protein bands; these were quantified using the Image Analysis Software (version 1.3). A linear relationship between the density of blots and protein load was observed when 5, 10, 20, and 30 µg of membrane protein were used per lane.
RNA isolation and Northern blot RNA-DNA hybridization analysis. Total RNA was extracted from the ventricular tissue by the guanidinium thiocyanate method (6). Samples normalized to 20 µg of total RNA were denatured with formaldehyde and electrophoresed in a 1% agarose-formaldehyde gel. The fractionated mRNA transcripts were transferred to a charge-modified nylon filter (Nytran Maximum Strength Plus, Schleicher and Schuell, Keene, NH) for 24 h. The membrane was then ultraviolet cross-linked (UV Stratalinker 2400, Stratagene). Blots were prehybridized at 42°C overnight with use of an Innova 4080 incubator (New Brunswick Scientific, Edison, NJ) oscillating at a rate of 65 rpm. Labeled probes were added to the prehybridization solution and left overnight under the same conditions. The hybridized blots were exposed to X-ray film (Kodak-X-OMAT). The radiolabeled mRNA bands were scanned using an imaging densitometer (model GS-670, Bio-Rad, Mississauga, ON, Canada) and quantified with Image Analysis software.
Inserts were separated from recombinant plasmids and used as probes as follows: a 0.762-kb cDNA fragment from rabbit heart for SERCA2a (courtesy of the laboratory of Dr. A. K. Grover, McMaster University, Hamilton, ON, Canada); a 2.2-kb cDNA fragment from rabbit heart for the Ca2+ release channel or RyR and a 0.153-kb cDNA fragment from rabbit heart for PLB (generous gifts from Dr. D. H. MacLennan, University of Toronto, Toronto, ON, Canada); a 2.50-kb cDNA fragment of rabbit cardiac CQS for CQS (a gift from Dr. A. Zilberman, University of Cincinnati, Cincinnati, OH); and a 24-base oligonucleotide probe of rat ribosomal RNA for 18S. The cDNA used to hybridize specific mRNA transcripts was prepared and autoradiographed using a Random Primer DNA labeling system (New England Nuclear, Boston, MA) radiolabeled with d-[
-32P]CTP.
Statistical analysis. Values are means ± SE and were statistically evaluated by one-way ANOVA. P < 0.05 was considered the threshold for statistical significance between the control and the experimental groups.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiac performance, SR function, and SR protein levels in
myocardial I/R.
Cardiac function in isolated hearts was monitored by measuring LVDP,
LVEDP, +dP/dt, and
dP/dt, whereas
Ca2+ uptake,
Ca2+ release, and
[3H]ryanodine-binding
activities were studied in the isolated SR preparations to assess SR
function. Hearts subjected to global ischemia for 30 min failed
to generate LVDP, +dP/dt, and
dP/dt but showed a marked
increase in LVEDP (Fig. 2,
A-D). Reperfusion of the
ischemic hearts for 60 min recovered the contractile function, as
represented by LVDP, +dP/dt, and
dP/dt, by 20-25% of the
respective preischemic values, but LVEDP was further increased markedly
(Fig. 2, A-D). The recovery of
contractile activity in I/R hearts was markedly improved by SOD + CAT
treatment; this was reflected by 69% recovery of LVDP as well as 72 and 83% recovery in +dP/dt and
dP/dt, respectively, in
comparison to the preischemic values in addition to a marked reduction
in LVEDP in comparison to the I/R group (Fig. 2,
A-D). SOD + CAT treatment did
not affect cardiac contractile activity in the control heart
preparations.
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1 · min1,
respectively, vs. 24.71 ± 1.95 nmol · mg
protein1 · min1
for the control group (Fig. 2E). On
the other hand, with SOD + CAT treatment,
Ca2+ uptake recovered to the
control values (22.4 ± 2.81 nmol · mg protein1 · min1,
P < 0.05). Although the
Ca2+ uptake values for the control
group in this study are relatively low compared with those reported by
us earlier (8, 19), the pattern of changes observed in the ischemic or
I/R hearts is similar to that seen previously (8, 19). To rule out the
possibility that the observed change in SR
Ca2+ uptake was not a test tube
phenomenon, SR preparations from control, ischemic, I/R, and SOD + CAT-treated I/R hearts (n = 2 in each group) were isolated in the presence of a cocktail of different protease inhibitors such as leupeptin (1 µM), pepstatin (1 µM), and
PMSF (100 µM). Results in this experiment were similar to those
obtained with SR preparations isolated from different groups in the
presence of PMSF alone. In another set of experiments using four hearts
in each group, oxalate-supported
Ca2+ uptake activity was measured
in the heart homogenate according to the procedure described earlier
(19). The Ca2+ uptake values for
homogenates from control, ischemic, I/R, and SOD + CAT-treated I/R
hearts were 11.92 ± 0.9, 4.2 ± 0.41, 5.8 ± 0.75, and 10.5 ± 1.90 nmol
Ca2+ · mg
protein1 · min1, respectively.
No change in the dissociation constant
(Kd) for
ryanodine binding was observed between the experimental groups and the
control (1.57 ± 0.24 nM). The density of ryanodine-binding sites
(Bmax) was reduced significantly
in ischemia (1.4 ± 0.05 vs. 2.4 ± 0.11 pmol/mg for the
control group) and further decreased on reperfusion (0.8 ± 0.02 pmol/mg; Fig. 2F); this change was
prevented by SOD + CAT treatment (1.8 ± 0.09 pmol/mg,
P < 0.05). Ryanodine-sensitive Ca2+-induced
Ca2+ release (inhibited by 20 µM
ryanodine) and EGTA-sensitive Ca2+
release were significantly decreased in the ischemic and I/R hearts
(Table 1) compared with controls, but SOD + CAT treatment markedly improved the SR
Ca2+ release in the I/R hearts.
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Cardiac performance, SR function, and SR protein levels in hearts
perfused with reactive oxygen species.
To test whether the I/R effects are simulated by some reactive oxygen
species, hearts were perfused for 20 min with X + XO or
H2O2,
and changes in cardiac function as well as SR
Ca2+ uptake and
Ca2+ release were assessed. The
LVDP decreased by 6- and 3.5-fold (Fig.
4A),
whereas the LVEDP increased by 11- and 6.9-fold (Fig. 4B) in X + XO- and
H2O2-perfused
hearts, respectively, compared with control hearts. Depressed cardiac
function was accompanied by a marked decrease in the rates of
contraction and relaxation; X + XO decreased
+dP/dt by 18.5-fold and
dP/dt by 11-fold (Fig. 4,
C and
D), whereas
H2O2
decreased +dP/dt and
dP/dt by 3.7- and 5.8-fold,
respectively.
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1 · min1)
when hearts were perfused with X + XO or
H2O2
(9.28 ± 0.85 and 13.97 ± 1.45 nmol · mg
protein1 · min1,
respectively; Fig. 4E). As for
[3H]ryanodine binding,
the control Kd
value (1.68 ± 0.21 nM) was not significantly different from that in
the ischemia or I/R groups. However, the
Bmax value was 2.3 ± 0.1 pmol/mg in the control group but decreased significantly in X + XO- and
H2O2-perfused
hearts (1.0 ± 0.05 and 0.9 ± 0.04 pmol/mg, respectively; Fig.
4D). X + XO- and
H2O2-perfused
hearts showed significant reduction in ryanodine-sensitive
Ca2+-induced
Ca2+ release as well as
EGTA-induced Ca2+ release (Table
1). In preliminary experiments, perfusion of hearts with X or XO alone
exerted no effect on cardiac performance or SR function. X + XO reduced
the protein levels of SERCA2a, RyR, and PLB by 68.94, 45.24, and
30.04%, respectively, compared with controls (Fig.
5,
A-D), whereas
H2O2
depressed only the SERCA2a protein content by 41.9% (Fig. 5,
A-D). In the case of CQS, the results were similar to those observed in I/R, inasmuch as the protein
levels increased by 76.7 and 84.1% in X + XO- and
H2O2-perfused hearts, respectively, above the controls (Fig.
5E).
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Gene expression in myocardial I/R and in hearts perfused with
reactive oxygen species.
For studying the possible mechanism of functional impairment as
observed in the stunned myocardium over a prolonged period due to I/R,
we examined changes in gene expression of SR proteins. Northern blot
analysis showed that ischemia reduced the level of SERCA2a mRNA
by 16%, RyR mRNA by 33.5%, and CQS mRNA by 21% in comparison to the
controls, whereas the level of PLB mRNA remained unaltered (Fig.
6). Reperfusion after ischemia
significantly decreased the mRNA abundance of SERCA2a by 37.7%, RyR by
56%, PLB by 28.7%, and CQS by 46.4% compared with controls (Fig. 6).
Compared with I/R, treatment of the I/R hearts with SOD + CAT improved
the level of SERCA2a mRNA by 24.5%, RyR mRNA by 33.7%, PLB mRNA by
35.2%, and CQS mRNA by 27.4%.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Depressed cardiac function as a consequence of oxyradical generation (30) has been reported to occur in clinical and diseased conditions when the heart is reperfused after an episode of ischemia (3). Our results showing depression of cardiac function in hearts subjected to I/R or perfused with X + XO as well as H2O2, which are known to generate different reactive oxygen species, are in agreement with previous reports (17, 26). The improvement of cardiac function in the I/R hearts treated with SOD + CAT further supports the involvement of oxyradicals in I/R injury (26). Our data are also consistent with other studies that have reported a decrease in SR Ca2+ uptake (9, 19, 31), Ca2+-induced Ca2+ release (19), and [3H]ryanodine-binding sites (Bmax) without any change in the Kd values (31). Because we have observed a decrease in the protein content of SERCA2a and RyR in ischemic and I/R hearts, the depressed SR functions due to ischemia and reperfusion may be the result of decreased protein content of SERCA2a and RyR in the SR membrane. Proteolytic degradation may account for the decrease in protein content in ischemia and reperfusion, since the activation of an endogenous cytosolic protease (30) and subsequent degradation of high-molecular-weight proteins such as RyR (22) have been reported in ischemia and reperfusion (29). PLB protein contents were unaltered in ischemic hearts and slightly decreased in I/R hearts, whereas CQS protein contents were increased under these conditions. The exact reasons for such differential changes in SR protein contents are not clear. Although SR RyR, SERCA2a, and PLB contents were decreased in hearts perfused with oxyradical-generating systems, the depression in these SR protein contents due to I/R was not prevented by SOD + CAT; the mechanism for such a change remains to be investigated.
In this study we have observed the beneficial effects of treatment with SOD + CAT on the depressed SR Ca2+ uptake, Ca2+ release, and ryanodine-binding capacity in I/R hearts. Unlike the SR function, the decrease in protein contents for SERCA2a, RyR, and PLB due to I/R was not prevented with antioxidant treatment. In this regard, Davies and Goldberg (7) reported that different antioxidants protect erythrocytes against lipid peroxidation but do not prevent proteolysis when exposed to oxidative stress. Because oxyradicals have been shown to promote lipid peroxidation in SR membranes (16), it is possible that the protective effect of SOD + CAT treatment in the I/R hearts with respect to changes in SR function may be due to the prevention of SR lipid peroxidation. Furthermore, on the basis of the ability of oxyradicals to modify the SR Ca2+ pump activity and sulfhydryl groups (28), it is likely that the I/R-induced changes in SR function may be due to their effect on the sulfhydryl groups of the SR proteins. Therefore, in view of the protection of SR function with SOD + CAT treatment and the critical role of SR Ca2+ release and Ca2+ uptake processes in cardiac contraction and relaxation, the improved recovery of heart function observed in SOD + CAT-treated I/R hearts may partly be due to improved SR Ca2+ handling. It has been well documented that reactive oxygen species such as superoxide radical, H2O2, and hydroxyl radical are implicated in the pathogenesis of I/R injury with respect to heart function and structure. In this report the role of oxyradicals and reactive species of oxygen was confirmed when hearts perfused with X + XO or H2O2 showed depressed SR Ca2+ uptake, Ca2+ release, and [3H]ryanodine-binding capacity similar to that observed in I/R. Protection of hearts subjected to I/R by the antioxidant treatment and the damage induced by direct perfusion with the reactive oxygen species further support the role of oxidative stress in SR dysfunction. Because SOD + CAT proved to be beneficial in improving cardiac performance and SR function of I/R hearts, it may be suggested that antioxidant treatment may be beneficial for the short-term recovery of the I/R myocardium.
Although the precise mechanisms by which oxidative stress may induce functional damage to the stunned heart for a prolonged period are not clear, the contribution of changes in the SR gene expression cannot be ignored. There is sufficient evidence in the literature suggesting multiple mechanisms by which oxidative stress may contribute to the alterations in mRNA levels; these include fragmentation of nucleosomal DNA in myocardial I/R (12) and alterations in nucleocytoplasmic transport activity (24). Our results showed that a 30-min period of ischemia was sufficient to significantly reduce the mRNA abundance of SERCA2a, RyR, and CQS. Reperfusion for 1 h after 30 min of global ischemia resulted in a further decrease in mRNA abundance of SERCA2a, RyR, and CQS from the ischemic level. In addition, there was a reduction in the mRNA of PLB that was not observed in ischemia. Exposure of hearts to X + XO as well as H2O2 depressed mRNA levels of all the SR proteins similar to those observed in I/R, corroborating the role of oxidative stress. Because mRNA expression of SR proteins was depressed in ischemia and I/R, it may be suggested that a reduction in SR protein content as a consequence of this change may occur in prolonged functional impairment similar to that observed in myocardial stunning. Because SOD + CAT treatment was found to protect against changes in SR protein gene expression in I/R hearts, it may be suggested that antioxidant treatment may be beneficial for the long-term recovery of the I/R myocardium. This effect of SOD + CAT on gene expression may not contribute to changes in SR function in the I/R heart under acute conditions, because when I/R hearts were treated with SOD + CAT, a significant recovery in the SR protein gene expression was observed without any protection at the protein level. These results, showing the SOD + CAT treatment-induced recovery of SR gene expression, suggest that antioxidant treatment may render protection of the ischemic myocardium at the pre- and/or transcriptional level. Because the recovery of the I/R-induced changes in SR Ca2+ uptake and Ca2+ release activities and gene expression was only partial on treatment with SOD + CAT, mechanisms other than oxidative stress for the observed changes in SR function in the I/R hearts cannot be ruled out.
In summary, this study provides direct and indirect evidence for the role of oxidative stress in depressed SR gene expression and SR dysfunction in the I/R hearts. Antioxidant treatment with an oxyradical scavenger system such as SOD + CAT may provide short- and long-term cardioprotection. The short-term beneficial effects may be related to protecting SR function, most probably by preventing lipid peroxidation of the SR membrane, whereas the long-term beneficial effect may be a consequence of preventing changes in SR protein gene expression. Our results suggest that supplements of antioxidants during surgical procedures and interventions would go a long way in the clinical management of ischemic heart disease.
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ACKNOWLEDGEMENTS |
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This study was supported by a grant from the Medical Research Council (MRC) of Canada (MRC Group in Experimental Cardiology). R. M. Temsah was a predoctoral fellow of the University of Manitoba; N. S. Dhalla held the MRC/Pharmaceutical Manufacturers Association of Canada Chair in Cardiovascular Research supported by Merck Frosst Canada.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Taché Ave., Winnipeg, MB, Canada R2H 2A6 (E-mail: cvso{at}sbrc.umanitoba.ca).
Received 13 July 1998; accepted in final form 31 March 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Afzal, N.,
and
N. S. Dhalla.
Differential changes in left and right ventricular SR calcium transport in congestive heart failure.
Am. J. Physiol.
262 (Heart Circ. Physiol. 31):
H868-H874,
1992
2.
Blaustein, A. S.,
L. Schine,
W. W. Brooks,
B. L. Fanburg,
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P < 0.05 vs. ischemia;
# P < 0.05 vs. reperfusion.
