Vol. 278, Issue 4, H1084-H1090, April 2000
Dietary coenzyme Q10 supplement renders swine
hearts resistant to ischemia-reperfusion
injury
Nilanjana
Maulik,
Tetsuya
Yoshida,
Richard M.
Engelman,
Debasis
Bagchi,
Hajime
Otani, and
Dipak K.
Das
Department of Surgery, University of Connecticut School of
Medicine, Farmington, Connecticut 06030-1110; Department of
Surgery, Baystate Medical Center, Springfield, Massachusetts 01199;
and Creighton University School of Pharmacy and Allied Health
Professions, Omaha, Nebraska 68178
 |
ABSTRACT |
To
examine whether nutritional supplementation of coenzyme Q10
(CoQ10) can reduce myocardial ischemia-reperfusion
injury, a group of swine was fed a regular diet supplemented with
CoQ10 (5 mg · kg
1 · day1)
for 30 days. Another group of pigs that were fed a regular diet supplemented with placebo served as a control. After 30 days, isolated
in situ pig hearts were prepared and hearts were perfused with a
cardiopulmonary pump system. Each heart was subjected to 15 min of
regional ischemia by snaring of the left anterior descending coronary artery, followed by 60 min of hypothermic cardioplegic global
ischemia and 120 min of reperfusion. After the experiments were
completed, myocardial infarct size was measured by triphenyltrazolium chloride staining methods. Postischemic left ventricular contractile function was better recovered in the CoQ10 group than in
the control group of pigs. CoQ10-fed pigs revealed less
myocardial infarction and less creatine kinase release from the
coronary effluent compared with control pigs. The experimental group
also demonstrated a smaller amount of malonaldehyde in the coronary
effluent and a higher content of the endogenous antioxidants ascorbate
and thiol. Significant induction of the expression of ubiquitin mRNA
was also found in the hearts of the CoQ10-fed group. The
results of this study demonstrate that nutritional supplementation of
CoQ10 renders the hearts resistant to
ischemia-reperfusion injury, probably by reducing the oxidative stress.
ubiquitin; ubiquinone; oxygen free radicals; oxidative stress
| |
INTRODUCTION |
A LIPID SOLUBLE BENZOQUINONE, coenzyme Q10
(CoQ10), is an essential component for electron transport
in oxidative phosphorylation of mitochondria. Also called
ubiquinone, its principal function is to act as an electron carrier
between the NADH and succinate dehydrogenases and the cytochrome system
(35). During mitochondrial electron transport, ubiquinone also occurs
as semiquinone and ubiquinol, the fully reduced form of ubiquinone.
Semiquinone has a role in the generation of superoxide anions during
mitochondrial respiration (22), whereas ubiquinol functions as an
intracellular antioxidant, presumably by preventing both the initiation
and propagation of lipid peroxidation (13).
CoQ10 appears to be involved in the coordinated regulation
between oxidative stress and antioxidant capacity of heart tissue. When
the heart is subjected to oxidative stress in various pathogenic conditions (9, 23), the amount of CoQ10 is decreased, which triggers a signal for increased CoQ10 synthesis. It has
been reported that in patients with cardiac disease such as chronic
heart failure, the myocardium becomes deficient in CoQ10
and CoQ10 reductase (35). CoQ10 level is also
reduced in other cardiovascular diseases such as cardiomyopathy (28).
CoQ10 can protect human low-density lipoprotein (LDL) from
lipid peroxidation, suggesting its role in atherosclerosis (33).
Several reports exist in the literature indicating cardioprotective
effects of CoQ10 against ischemia-reperfusion injury (15, 28, 34). However, none of these studies attempted to
evaluate the mechanism(s) of CoQ10-mediated
cardioprotection, and none demonstrated whether postischemic
improvement of myocardial function was caused by the improvement of an
endogenous defense system.
In the present study, the mechanism of action of CoQ10 was
examined by feeding pigs CoQ10 for 1 mo in an attempt to
increase the CoQ10 content of the heart. The results of
this study demonstrated that CoQ10-fed pigs were resistant
to myocardial ischemia-reperfusion injury. The hearts of
CoQ10-fed animals had higher levels of CoQ10, higher levels of the intracellular antioxidants ascorbate and thiol,
and an increased amount of ubiquitin gene expression, which may be
attributed to its ability for being resistant to ischemic injury.
| |
MATERIALS AND METHODS |
Measurement of myocardial CoQ10.
For CoQ10 assay, the left ventricular (LV) biopsies from
the ischemic region were quickly frozen in liquid nitrogen. Frozen tissues were stored at 
70°C for subsequent determination of
CoQ10. At a later date, the tissues were homogenized and
treated with 2% ferric chloride to convert the reduced form of
ubiquinone into the oxidized form, which was extracted with
n-hexane. The solvent was evaporated at 30°C under
nitrogen. The residue was dissolved in isopropyl alcohol and analyzed
by HPLC (11). CoQ10 was eluted with methanol-ethanol (1:1
vol/vol) by being passed through a C18 column. Ultraviolet absorbance
of the elute was monitored at 275 nm.
Treatment with CoQ10.
Male Yorkshire pigs weighing 18-25 kg were used in this study.
Animals received humane care in compliance with the "Principles of
Laboratory Animal Care" formulated by the National Society for
Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Research Council (Revised 1996). Twelve
pigs were fed coenzyme Q10 (5 mg · kg1 · day1;
UAS Laboratories, Minnetonka, MN) supplemented with regular laboratory
diet for 30 days. Twelve age-matched pigs that were fed regular diets
plus placebo served as controls.
Experimental preparation.
At the end of 30 days, each pig was tranquilized with ketamine (20 mg/kg body wt) and anesthetized with pentobarbital sodium (25 mg/kg
body wt). Endotracheal intubation was performed, and ventilation was
maintained by a volume respirator with room air. A median sternotomy
was performed, and the azygos vein was ligated (25). The pericardium
was incised and suspended in a pericardial cradle. After heparinization
with heparin sodium (300 U/kg body wt), an arterial cannula was placed
in the ascending aorta through the right carotid artery and a venous
cannula was placed in the right atrium. A cannula was also placed in
the left atrium through the appendage to control preload for
measurements of ventricular function. Sonometric dimension crystals
(diameter 6 mm) made of 3-MHz piezoelectric crystals (Triton
Technologies, San Diego, CA) were placed at the endocardial surface
across the anteroposterior minor axis, septal-free wall minor axis, and
base-apex major axis of the left ventricle. The anteroposterior
crystals were placed adjacent to the anterior and posterior descending
coronary arteries. The septal-free crystals were located one-half the
distance from the apex to the base. The base crystal was placed into
the left ventricle adjacent to the origin of the left circumflex
coronary artery, and the apex crystal was placed into the LV apex.
Cardiopulmonary bypass with a membrane oxygenator was initiated, and
blood was collected from the pig in a reservoir. The heart was then
isolated in its own perfusion circuit by completely cross-clamping the ascending aorta just distal to the right brachiocephalic artery distal
to the arterial inflow and ligating both superior and inferior vena
cavae. The main pulmonary trunk was drained. This achieves complete
cessation of systemic circulation while coronary perfusion is maintained.
On stable bypass, the left anterior descending coronary artery (LAD)
was then snared just distal to the first diagonal branch. After 15 min
of normothermic regional ischemia, the aorta was clamped and
the ligature removed from the LAD. Normothermic cardioplegic arrest was
initiated. Initial high-K+ blood cardioplegic solution
(K+ 20-24 meq/l, 20 ml/kg) was administered through
the carotid cannula into the coronary circulation at a flow rate that
insured a perfusion pressure of 50-75 mmHg. This
high-K+ blood cardioplegic solution arrested each heart
promptly. Additional low-K+ blood cardioplegic solution
(K+ 8-12 meq/l, 10 ml/kg) was administered every 15 min (at 15, 30, and 45 min) for a total of 60 min of arrest.
After 60 min of cardioplegic arrest, the heart was reperfused on
cardiopulmonary bypass. The snare of the LAD was released just before
reperfusion. Normothermia was maintained with a heat exchanger, and
reperfusion was continued for a total of 180 min. Coronary perfusion
was maintained at 
75 mmHg. Defibrillation was applied when the heart
suffered ventricular fibrillation during reperfusion. The heart was
atrially paced at 120 beats/min. No cardiotonic or antiarrhythmic drugs
were administered during the experiment. Blood samples were taken
through the pulmonary arterial cannula (coronary sinus blood) at 0 min
(baseline) and during the reperfusion for malonaldehyde (MDA) and
creatine kinase (CK) measurements. Before regional ischemia and
at the end of experiment, LV biopsies were taken for biochemical determination.
Functional data were obtained by adding 60 ml of saline through the
left atrial cannula to raise the LV end-diastolic pressure, with
subsequent withdrawal of the saline while maximal developed pressure
and end-diastolic pressure were measured before ischemia (control) and during reperfusion. The first derivative of LV pressure (LV dP/dt) was calculated as a polynomial approximation from
the digital LV pressure signal. LV dP/dtmax was
obtained when the point of the LV pressure-volume loop showed peak LV
developed pressure (LVDP).
Estimation of CK release from heart.
CK was quantified from 0.5 µl of plasma by the enzymatic assay method
using a CK assay kit (Sigma Diagnostics, St. Louis, MO). The absorbance
was read at 340 nm using a Beckman DU-8 spectrophotometer. The enzyme
activity was expressed in units per milliliter.
Assessment of myocardial infarction.
After 2 h of reperfusion, triphenyltetrazolium chloride (TTC; 10 ml,
1% solution) in phosphate buffer preheated to 37°C was injected
directly into the coronary arteries through the ascending aorta at
70-75 mmHg after the distal ascending aorta was clamped. We did
not attempt to measure the area of risk because it is well known that
pig hearts have little collateral coronary circulation. However, the
area of risk was identical in the two groups when the LAD was clamped
just distal to the first diagonal branch. After a 10-min incubation,
noninfarcted myocardium was stained red and infarct area was unstained.
The heart was stored at 20°C, and frozen slices
were analyzed with sections from apex to base. The infarct area was
calculated as a percentage of the total LV myocardium.
Thin sections were placed between plates and scanned using NIH Image
processing software. Each digitized image was subjected to background
subtraction and contrast enhancement. The total left ventricle and the
area of infarction were traced and the respective areas calculated in
terms of pixels. The infarct volume was calculated, and the sum of all
slices was used to compute a ratio of percent infarct to total LV area.
Measurement of MDA formation.
MDA was assayed as described previously to estimate the lipid
peroxidation (5). Plasma (1.5 ml) was mixed with an equal volume of
20% TCA and 5.3 mM sodium metabisulfite. Protein was precipitated on
ice for 10 min. Two milliliters of supernatant were derivatized with
2,4-dinitrophenylhydrazine and extracted with pentane. MDA formation
was then measured using HPLC (5).
Determination of antioxidant reserve.
The antioxidant reserve (12, 33) of each heart was measured on the
basis of its ability to reduce the phenoxyl radical of a hindered
phenol,
4'-dimethyl-epipodophyllotoxin-9-(4,6-O-ethylidene-
-D-glucopyranoside) (VP-16), generated as an intermediate in the oxidation of phenoxyl radical by tyrosinase (12). Reduction of the generated VP-16 phenoxyl
radical by endogenous antioxidants results in a delay (lag period) in
the appearance of the characteristic electron spin resonance (ESR)
signal of the VP-16 radical.
Hearts were homogenized in ice-cold 0.1 M phosphate buffer (iron-free),
pH 7.4, using a Polytron homogenizer. ESR measurements were performed
with an ESR spectrometer using a sample volume of 0.1 ml containing 500 µM VP-16, 3 U/µl tyrosinase, 25 µl of heart homogenate (20 mg
protein/ml), and 100 µM deferoxamine. The lag period was determined
as the time interval elapsed from the beginning of the measurement to
the appearance of VP-16 phenoxyl radical signal, as described
previously (12, 27).
Assessment of ubiquinone gene expression.
LV biopsies obtained for ubiquitin mRNA determination were immediately
frozen in liquid nitrogen and stored at 70°C for subsequent mRNA determination. Total RNA was extracted from the heart by the
acid-guanidinium-thiocyanate-phenol-chloroform method as previously described by Maulik et al. (26). For Northern blot
analysis, total RNA was electrophoresed in 1%
agarose-formaldehyde-formamide gel and transferred to Gene Screen Plus.
After prehybridization, membranes were hybridized with a 780-bp
(Hind IIV/BamH I) cDNA insert encoding porcine
ubiquitin (32). Each hybridization was repeated at least three times
using different membranes. After each hybridization, ubiquitin cDNA was
removed and rehybridized with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA probe, the results of which served as loading controls.
The autoradiographs were quantitatively evaluated by a computerized
beta scanner. The results of densitometric scanning were normalized
relative to the signal obtained with GAPDH cDNA probe.
Statistical analysis.
The results are expressed as means ± SE. An ANOVA was carried out
first to compare variance between groups. Differences between groups
were analyzed by a two-tailed Student's t-test. A P
value <0.05 was considered statistically significant.
| |
RESULTS |
Effects of CoQ10 feeding on myocardial CoQ10
content.
To ensure the bioavailability of CoQ10 in the heart, the
amount of CoQ10 was estimated in the hearts of both
CoQ10-fed and non-CoQ10-fed groups. Hearts of
the CoQ10-fed group showed significantly higher
CoQ10 content (28 ± 0.5 µg/g heart) compared with
controls (21 ± 0.7 µg/g heart) (Fig.
1). After ischemia, control hearts demonstrated a lower CoQ10 content (19.2 ± 0.9 µg/g
heart) than the baseline values. In contrast, the amount of
CoQ10 remained unaltered in the CoQ10-fed
hearts (27.3 ± 0.8 µg/g heart). Two hours of reperfusion did not
alter these values significantly in either group of hearts.
View larger version (30K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of coenzyme Q10 (CoQ10) feeding on
CoQ10 content of heart. Myocardial content of
CoQ10 was estimated in control (filled bars) and
CoQ10-fed pigs (hatched bars) as described in
MATERIALS AND METHODS. CoQ10 content was
measured in left ventricular (LV) biopsies at baseline, after
ischemia, and after reperfusion. Results are expressed as means ± SE of 6 animals/group. *P < 0.05 compared with
control.
|
|
LV function.
Hemodynamic parameters with a load of 60 ml of saline in control and
treatment groups are shown in Fig. 2. The
CoQ10-fed group consistently demonstrated higher recovery
of postischemic LV systolic function. For example, LVDP was lowered
after 30 min of reperfusion in the non-CoQ10-fed group to
142 ± 9.5 mmHg compared with 165 ± 7.2 mmHg in the
CoQ10-fed group. The same trend persisted up to 120 min of
reperfusion, when LVDP in the control group was 92 ± 3.9 mmHg
compared with 131 ± 4.2 mmHg in the CoQ10-fed group. LV
dP/dtmax followed a similar pattern. Thus, at the
end of reperfusion, these values were 1,110 ± 98 mmHg/s for the
control group versus 1,976 ± 85 mmHg/s for the CoQ10-fed
group.
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of CoQ10 feeding on LV functions. LV developed
pressure (LVDP) (A) and maximal first derivative of LV pressure
(LV dP/dtmax) (B) were measured in control
(filled bars) and CoQ10-fed pigs (hatched bars) as
described in MATERIALS AND METHODS. Hearts of
CoQ10-fed pigs demonstrated significantly better LV
function compared with hearts of corresponding control pigs at 60, 90, and 120 min of reperfusion (R). Results are expressed as means ± SE
of 6 animals/group. *P < 0.05 compared with control.
 P < 0.05 compared with baseline.
|
|
Tissue injury.
CK release from the heart truly reflects cellular injury and tissue
necrosis. As shown in Fig. 3, CK release
increased steadily and progressively in both groups. However, the
amount of CK release in the hearts of the CoQ10-fed group
was consistently lower than that in hearts of the
non-CoQ10-fed group. For example, after 30 min of
reperfusion, the amount of CK release from the control hearts was 112 ± 5.8 IU/l compared with 72 ± 5.6 IU/l from the CoQ10-fed hearts. At the end of 2 h of reperfusion, CK
release from the control heart was 212 ± 5.9 IU/l compared with only
115 ± 6.9 IU/l for the CoQ10-fed pig hearts.
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of CoQ10 feeding on creatine kinase (CK) release
from heart. CK release in CoQ10-fed pigs ( ) is
significantly lower than that in control pigs ( ). Results are
expressed as means ± SE of 6 animals/group. *P < 0.05 compared with control.
|
|
Infarct size for each heart was expressed as (sum of infarct area of
each slice/sum of total LV area of each slice) × 100. The mean
value of infarct size in the CoQ10-fed group was
significantly smaller than that in the non-CoQ10-fed group
(Fig. 4). For example, infarct size for the
CoQ10-fed group was only 19.5 ± 2.0% compared with 35.0 ± 2.8% for the non-CoQ10-fed group of hearts. Our
results thus indicate that CoQ10 feeding resulted in
reduced myocardial tissue damage as documented by less CK release and
smaller myocardial infarction.
View larger version (10K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of CoQ10 feeding on myocardial infarct size.
Infarct size (expressed as %LV) was estimated in control and
CoQ10-fed pigs (Q10) at end of each experiment
as described in MATERIALS AND METHODS. Hearts of
CoQ10-fed pigs showed a significantly lower amount of
infarct size than did hearts of control pigs. Results are expressed as
means ± SE of 6 animals/group. *P < 0.05 compared with
control.
|
|
Reduction of oxidative stress by CoQ10.
The amount of oxidative stress in the heart was determined by examining
the free radical-lipids interaction product, lipid peroxidation, by
monitoring the amount of MDA content in the coronary effluent (3, 5).
An increased amount of MDA was found in all groups during the early
reperfusion, as expected. However, as shown in Fig.
5, the amount of MDA was significantly
lower in the CoQ10-fed group than in the
non-CoQ10-fed group at each time point tested, suggesting
that CoQ10 resulted in the reduction of oxidative stress
that is developed during ischemia and reperfusion. At 3 min of
reperfusion following ischemia, the MDA concentration was 110 ± 6 pmol/ml for the control group versus 52 ± 4.8 pmol/ml for
CoQ10-fed pig hearts. Even after 2 h of reperfusion, the
amount of MDA released from the control hearts was 62 ± 5 pmol/ml
compared with 30 ± 4.5 pmol/ml from the CoQ10-fed hearts.
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of CoQ10 feeding on malonaldehyde (MDA) content of
heart. MDA content was measured as described in MATERIALS AND
METHODS. Hearts of CoQ10-fed pigs () showed a
lower amount of MDA formation compared with hearts of control pigs
(), indicating a smaller degree of oxidative stress in hearts of
CoQ10-fed pigs. Results are expressed as means ± SE of 6 animals/group. *P < 0.05 compared with control.
|
|
Effects of CoQ10 on antioxidant reserve of the heart.
The VP-16 phenoxyl radical formed as an intermediate in the VP-16
oxidation by tyrosinase was detected. In the presence of heart
homogenates, the characteristic ESR signal of the VP-16 phenoxyl
radical could be observed only after a lag period. Heart homogenates
from the non-CoQ10-fed animals showed a significantly lower
amount of lag period for the appearance of the tyrosinase-induced VP-16
phenoxyl radical ESR signal (Fig.
6). A significantly greater amount of lag period was found for CoQ10-fed heart
homogenates, suggesting greater antioxidant reserve in the hearts of
CoQ10-fed pigs.
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of CoQ10 feeding on antioxidant reserve of heart.
Estimation of lag period of VP-16 phenoxyl radical generation by
tyrosinase-catalyzed VP-16 oxidation in presence of heart homogenate
from control (filled bars) and CoQ10-fed pig hearts
(hatched bars) is shown at baseline, after ischemia, and after
reperfusion. Results are expressed as means ± SE of 6 animals/group. *P < 0.05 compared with control.
P < 0.05 compared with baseline.
|
|
Ubiquitin gene expression.
The ubiquitin gene was ubiquitously present in the hearts of all
animals. The transcripts UbB and UbC were detected in the hearts of
non-CoQ10-fed animals at 2.5 and 3.5 kb, respectively (Fig.
7). Induction of the expression of UbB and
UbC was increased significantly in the CoQ10-fed hearts.
Additionally, an induction of the expression of UbA was also prominent
in the hearts of the CoQ10-fed group only.
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of CoQ10 feeding on ubiquitin gene expression.
Upregulation of ubiquitin gene expression is shown in
CoQ10-fed pig hearts. Sizes of 3 different transcripts are
1.3, 2.5, and 3.5 kb for UbA, UbB, and UbC, respectively. C, control;
Q10, CoQ10-fed pig; G3PDH,
glyceraldehyde-3-phosphate dehydrogenase.
|
|
| |
DISCUSSION |
In the present study, we demonstrated an ~30% increase in the
myocardial content of CoQ10 after 30 days of feeding. There are a number of data showing that the tissue level of CoQ10
increases after the administration of exogenous CoQ10 in
mammals, including humans (17). Exogenously administered
CoQ10 is nonspecifically incorporated into the cell
membranes and into various subcellular fractions and organelles, such
as mitochondrial membranes and sarcoplasmic reticular membranes (29).
Ferrara et al. (15) reported that after 4 wk of dietary supplementation
with CoQ10, tissue concentration of CoQ10 was
elevated by 22% and oxidative stress was significantly suppressed. In
our study, we showed a 30% increase in myocardial CoQ10
content after 1 mo of CoQ10 feeding. After ischemia
and reperfusion were completed, the average myocardial CoQ10 content in the treatment group was still higher than
that in the control group by 37%. These results corroborated with the findings that total antioxidant reserve in the heart was higher and the
amount of oxidative stress was lower in the CoQ10-fed group
compared with the non-CoQ10-fed group of hearts. In
concert, CoQ10 feeding resulted in higher postischemic
ventricular recovery, lower CK release from hearts, and reduced infarct
size, suggesting that CoQ10 might be instrumental in the
reduction of myocardial ischemia-reperfusion injury. We
measured LVDP and LV dP/dtmax after 30, 60, 90, and
120 min of reperfusion. CoQ10-fed animals showed
significantly higher levels of LV dP/dtmax at each
time point, whereas higher levels of LVDP were found only at 60, 90, and 120 min of reperfusion. Although at 30 min of reperfusion the LVDP
of CoQ10-fed pigs was higher than that of controls, the difference was not significant.
A significant number of reports exist in the literature to support a
role of oxygen free radicals and oxidative stress in ischemia-reperfusion injury (6, 24). The presence of reactive oxygen species has been detected directly using ESR and HPLC techniques and indirectly from the extent of increased lipid peroxidation and DNA
and protein damage (3, 4). A variety of antioxidants and antioxidant
enzymes such as Mn-superoxide dismutase, catalase, and
glutathione-peroxidase were shown to protect cells from oxidative stress and reduce myocardial ischemia-reperfusion injury (8). It was reported that activity of endogenous antioxidants decreased after ischemia and reperfusion (7, 19). In addition to the role
of CoQ10 as a component of the mitochondrial respiratory chain, its role as an intracellular antioxidant has gained attention in
recent years. However, the precise mechanism of action is not yet well
understood. In vitro study demonstrated that the reduced form of
CoQ10 protects membrane phospholipid and serum LDL from lipid peroxidation (2). In vivo study reported that CoQ10
reduces myocardial ischemia-reperfusion injury induced by
oxidative stress through the suppression of the formation of reactive
oxygen species (28, 35). Ferrara et al. (15) showed that long-term
CoQ10 supplementation renders protection against oxidative
stress induced by ischemia-reperfusion. On the other hand, Hano
et al. (18) showed that a beneficial effect is not observed when
CoQ10 is added at the onset of reperfusion. Our study
showed that long-term administration might enable hearts to increase
CoQ10 content, which may be beneficial in protecting the
heart from ischemia-reperfusion injury.
In the present study, we used the cardiopulmonary bypass model, which
utilizes an isolated in situ heart and mimicks the in vivo
ischemia-reperfusion injury. This animal model also simulates human open-heart surgery, because the time course is consistent with
human surgery and the heart is subjected to reperfusion with blood. We
also adopted hypothermic intermittent blood cardioplegia to protect
globally ischemic myocardium. In this model, whole blood was used as a
perfusate and the main component of cardioplegia. Whole blood carries
leukocytes and oxygen that might be an additional source of free
radicals. For example, activated neutrophils during cardiopulmonary
bypass generate free radicals (1). Thus the heart was subjected to
additional oxidative stress (like in a real situation of open-heart
surgery) from blood perfusion in addition to
ischemia-reperfusion. Sarcolemmal phospholipids are potential
targets for reactive oxygen species, and interaction gives rise to
membrane lipid peroxidation. Several aldehydes and ketones such as MDA
are the breakdown products of spontaneous fragmentation (-cleavage)
of peroxides derived from the free radical-polyunsaturated fatty acid
interactions. In this study we measured serum MDA, and
CoQ10-fed hearts showed a significantly reduced amount of
MDA in serum after reperfusion. A significantly higher amount of MDA
was found in the control hearts after 3 min of reperfusion, which was
quite as expected because free radicals are generated at the onset of
reperfusion. CoQ10 blocked this early increase of MDA and
maintained lower MDA throughout the reperfusion period. Under
normal conditions, free radicals and lipid peroxidation products cannot
accumulate in the heart because they are promptly removed by the
endogenous protecting system such as antioxidants and antioxidant
enzymes that may include endogenous CoQ10.
In this study we adapted a previously described technique for measuring
intracellular antioxidants that truly reflects the interactions between
the two most important intracellular antioxidants, ascorbate and thiol,
in their natural environments (12, 27). To determine tissue antioxidant
activity toward phenoxyl radical, we quantitated the ability of heart
homogenates from non-CoQ10-fed and CoQ10-fed
pigs to reduce an in vitro generated phenoxyl radical. The
ESR signal with characteristic features of VP-16 phenoxyl radical was
apparent before the addition of tissue homogenates that immediately
quenched this signal. As described in earlier reports (12, 27), the
kinetics of the regeneration of the phenoxyl radical ESR signal
following the addition of heart homogenates provides an estimate of two
endogenous antioxidants in the hearts: ascorbate and thiol. The results
for antioxidant reserve corroborated with the results of MDA formation,
demonstrating higher antioxidant reserve and lower oxidative stress in
the hearts of CoQ10-fed animals.
In mitochondria, CoQ10 acts as a mobile distributor of
reducing equivalents among NADH dehydrogenase, succinate dehydrogenase, and the cytochrome b-c1 segment of the
electron transport chain and as a participant in the protonmotive Q
cycle responsible for the transfer of protons across the coupling
membrane (14). In biological systems, CoQ10 functions as a
potent antioxidant, and in its reduced form, ubiquinol, it acts as a
free radical scavenger (36). Two different mechanisms of
CoQ10 antioxidant function are known to exist: 1)
it may act independently as a chain-breaking antioxidant, providing
hydrogen atoms to reduce peroxyl and/or alkoxyl radicals; or 2)
a redox interaction may exist between CoQ10 and another
lipid-soluble antioxidant, such as 
-tocopherol, in its one-electron
oxidized form, vitamin E phenoxyl radical (16).
Ubiquitin is a protein of 76 amino acids found in all eukaryotic cells
(20). It has been implicated in ATP-dependent proteolysis and exhibits
remarkable evolutionary sequence conservation from animal to animal
(21). It has been characterized as a heat shock-induced protein in
humans, chickens, pigs, and yeast as well as other organisms (10).
During the initial periods of heat shock, the protein-ubiquitin
conjugation undergoes rapid and pronounced changes, presumably because
of deubiquitination of histone H2A and subsequent accumulation of
aberrant proteins. The altered transcription of the ubiquitin gene
seems to be essential for restoration of normal activities (30). In the
present study, upregulation of ubiquitin gene expression was observed
in the hearts of CoQ10-fed pigs. UbA transcript was not
found in the hearts of non-CoQ10-fed pigs, whereas UbB and
UbC transcripts were identified in these hearts. The levels of UbB and
UbC were significantly enhanced, and UbA was induced, in the hearts of
pigs fed CoQ10. In recent studies (31, 32), a coordinated
expression pattern of ubiquitin and heme oxygenase genes was shown in
the same porcine model in which the LAD was occluded for 10 min and
reperfused for 30 min and after a second occlusion of 10 min followed
by 210 min of reperfusion. Given the fact that ubiquitin plays an
essential role for degradation of many proteins, the significant
induction of ubiquitin gene expression suggests that CoQ10
may be involved in antioxidant adaptation on both degradation of
nonessential harmful proteins and induction of new proteins essential
for survival in the new environment.
In conclusion, this study demonstrated that nutritional supplementation
with CoQ10 provides cardioprotection against
ischemia-reperfusion injury. Reduction of oxidative stress in
conjunction with increased antioxidant reserve in CoQ10-fed
hearts suggests that CoQ10 functions through the
improvement of antioxidant reserve of the heart. The present study
utilized pig hearts in a cardiopulmonary bypass model. The hearts were
subjected to 15 min of LAD occlusion followed by 60 min of hypothermic
cardioplegic arrest and 2 h of reperfusion. Infarct size was determined
in the hearts subjected to 2 h of reperfusion, whereas the standard
practice for TTC infarct size estimation in in vivo preparations is a
minimum of 3 h of reperfusion.
| |
ACKNOWLEDGEMENTS |
This study was supported by National Heart, Lung, and Blood
Institute Grants HL-22559, HL-34360, HL-33889, and HL-56803 and by a
Grant-in-Aid from the American Heart Association.
| |
FOOTNOTES |
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: D. K. Das,
Cardiovascular Division, Dept. of Surgery, Univ. of Connecticut School
of Medicine, 263 Farmington Ave., Farmington, Connecticut 06030-1110 (E-mail: ddas{at}neuron.uchc.edu).
Received 21 July 1999; accepted in final form 13 October 1999.
| |
REFERENCES |
1.
Bagchi, D.,
Das D. K.,
Engelman R. M.,
Prasad M. R.,
and
Subramanian R.
Polymorphonuclear leucocytes as potential source of free radicals in the ischaemic-reperfused myocardium.
Eur. Heart J.
11:
800-813,
1990[Abstract/Free Full Text].
2.
Cadenas, E.,
Hochstein P. P.,
and
Ernster L.
Pro- and antioxidant functions of quinones and quinone reductases in mammalian cells.
Adv. Enzymol. Relat. Areas Mol. Biol.
65:
97-147,
1992[ISI][Medline].
3.
Cordis, G. A.,
Bagchi D.,
Maulik N.,
and
Das D. K.
High-performance liquid chromatographic method for the simultaneous detection of malonaldehyde, acetaldehyde, formaldehyde, acetone and propionaldehyde to monitor the oxidative stress in heart.
J. Chromatogr. A
661:
181-191,
1994[ISI][Medline].
4.
Cordis, G. A.,
Maulik G.,
Bagchi D.,
Riedel W.,
and
Das D. K.
Detection of oxidative DNA damage to ischemic reperfused rat hearts by 8-hydroxydeoxyguanosine formation.
J. Mol. Cell Cardiol.
30:
1939-1944,
1998[ISI][Medline].
5.
Cordis, G. A.,
Maulik N.,
and
Das D. K.
Detection of oxidative stress in heart by estimating the dinitrophenylhydrazine derivative of malonaldehyde.
J. Mol. Cell Cardiol.
27:
1645-1653,
1995[ISI][Medline].
6.
Das, D. K.,
Engelman R. M.,
Liu X.,
Maity S.,
Rousou J. A.,
Flack J.,
Laksmipati J.,
Jones R. M.,
Prasad M. R.,
and
Deaton D. W.
Oxygen derived free radicals and hemolysis during open heart surgery.
Mol. Cell. Biochem.
111:
77-86,
1992[ISI][Medline].
7.
Das, D. K.,
Engelman R. M.,
Rousou J. A.,
Breyer R. H.,
and
Lemeshow S.
Pathophysiology of superoxide radical as a potential mediator of reperfusion injury in pig heart.
Basic Res. Cardiol.
81:
155-166,
1986[ISI][Medline].
8.
Das, D. K.,
and
Maulik N.
Antioxidant effectiveness in ischemia-reperfusion tissue injury.
Methods Enzymol.
233:
601-610,
1994[ISI][Medline].
9.
Das, D. K.,
and
Maulik N.
Protection against free radical injury in the heart and cardiac performance.
In: Exercise and Oxygen Toxicity, edited by Sen C. K.,
Packer L.,
and Hänninen O.. Amsterdam, The Netherlands: Elsevier Science, 1995, p. 359-388.
10.
Das, D. K.,
and
Maulik N.
Role of small HSP gene expression in myocardial ischemia and reperfusion.
In: Heat Shock Proteins and the Cardiovascular Systems, edited by Knowlton A. A.. Norwell, MA: Kluwer Academic, 1997, p. 159-181.
11.
Edlund, P. O.
Determination of coenzyme Q10, alpha-tocopherol and cholesterol in biological samples by coupled-column liquid chromatography with coulometric and ultraviolet detection.
J. Chromatogr.
425:
87-97,
1988[ISI][Medline].
12.
Engelman, D. T.,
Watanabe M.,
Engelman R. M.,
Rousou J. A.,
Kisin E.,
Kagan V. E.,
Maulik N.,
and
Das D. K.
Hypoxic preconditioning preserves antioxidant reserve in the working rat heart.
Cardiovasc. Res.
29:
133-140,
1995[ISI][Medline].
13.
Ernster, L.
Ubiquinol as a biological antioxidant.
In: Active Oxygens, Lipid Peroxides and Antioxidants, edited by Yagi K.. Tokyo: Japan Sci. Soc, 1993, p. 1-38.
14.
Ernster, L.,
and
Dalner G.
Biochemical, physiological and medical aspects of ubiquinone function.
Biochim. Biophys. Acta
1271:
195-204,
1995[Medline].
15.
Ferrara, N.,
Abete P.,
Ambrosio G.,
Landino P.,
Caccese P.,
Cirillo P.,
Oradei A.,
Littarru G. P.,
Chiariello M.,
and
Rengo F.
Protective role of chronic ubiquinone administration on acute cardiac oxidative stress.
J. Pharmacol. Exp. Ther.
274:
858-865,
1995[Abstract/Free Full Text].
16.
Frei, B.,
Kim M. C.,
and
Ames B. N.
Ubiquinol-10 is an effective lipid-soluble antioxidant at physiological concentrations.
Proc. Natl. Acad. Sci. USA
87:
4879-4883,
1990[Abstract/Free Full Text].
17.
Hanaki, Y.,
Sugiyama S.,
Ozawa T.,
and
Ohno M.
Ratio of low-density lipoprotein cholesterol to ubiquinone as a coronary risk factor.
N. Engl. J. Med.
325:
814-815,
1991[ISI][Medline].
18.
Hano, O.,
Thompson-Gorman S.,
Zweier J.,
and
Lakatta E.
Coenzyme Q10 enhances cardiac functional and metabolic recovery and reduces Ca2+ overload during postischemic reperfusion.
Am. J. Physiol. Heart Circ. Physiol.
266:
H2174-H2181,
1994[Abstract/Free Full Text].
19.
Haramaki, N.,
Stewart D. B.,
Aggarwal S.,
Ikeda H.,
Reznick A. Z.,
and
Packer L.
Networking antioxidants in the isolated rat heart are selectively depleted by ischemia-reperfusion.
Free Radic. Biol. Med.
25:
329-339,
1998[ISI][Medline].
20.
Hershko, A.,
and
Ciechanover A.
The ubiquitin system for protein degradation.
Annu. Rev. Biochem.
61:
761-807,
1992[ISI][Medline].
21.
Jentsch, S.,
Seufert W.,
Sommer T.,
and
Reines H. A.
Ubiquitin-conjugating enzymes: novel regulators of eukaryotic cells.
Trends Biochem. Sci.
15:
195-198,
1990[ISI][Medline].
22.
Kagan, V. E.,
Arroyo A.,
Tyurin V. A.,
Tyurina Y. Y.,
Villalba J. M.,
and
Navas P.
Plasma membrane NADH-coenzyme Q10 reductase generates semiquinone radicals and recycles vitamin E homologue in a superoxide-dependent reaction.
FEBS Lett.
428:
43-46,
1998[ISI][Medline].
23.
Konishi, T.,
Nakamura Y.,
Konishi T.,
and
Kawai C.
Accentuated negative inotropism of verapamil after ischaemic intervention in isolated working rat heart.
Cardiovasc. Res.
19:
38-43,
1984.
24.
Kramer, J. H.,
Misik V.,
and
Weglicki W. B.
Lipid peroxidation-derived free radical production and postischemic myocardial reperfusion injury.
Ann. NY Acad. Sci.
723:
180-196,
1994[Abstract].
25.
Liu, X.,
Engelman R. M.,
Moraru I. I.,
Rousou J. A.,
Flack J. E.,
Deaton D. W.,
Maulik N.,
and
Das D. K.
Heat shock: a new approach for myocardial preservation in cardiac surgery.
Circulation
86:
358-363,
1992.
26.
Maulik, N.,
Sharma H.,
and
Das D. K.
Induction of the haem oxygenase gene expression during the reperfusion of ischemic rat myocardium.
J. Mol. Cell. Cardiol.
28:
1261-1270,
1996[ISI][Medline].
27.
Maulik, N.,
Watanabe M.,
Engelman D. T.,
Engelman R. M.,
Kagan V. E.,
Kisin E.,
Tyurin V.,
Cordis G. A.,
and
Das D. K.
Myocardial adaptation to ischemia by oxidative stress induced by endotoxin.
Am. J. Physiol. Cell Physiol.
269:
C907-C916,
1995[Abstract/Free Full Text].
28.
Mortensen, S. A.
Perspectives on therapy of cardiovascular diseases with coenzyme Q10 (ubiquinone).
Clin. Investig.
71, Suppl 8:
S116-S123,
1993[ISI][Medline].
29.
Nakamura, T.,
Sanma H.,
Himeno M.,
and
Kato K.
Effects of exogenous ubiquinone-10 on endogenous ubiquinone-10 in canine plasma and on electron transport enzymes.
In: Biochemical and Clinical Aspects of Co-enzyme Q10, edited by Yamamura Y.,
Folkers K.,
and Ito Y.. New York: Elsevier/North Holland Biomedical, 1980, vol. 2, p. 3-14.
30.
Parag, H. A.,
Raboy B.,
and
Kulka R. G.
Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.
EMBO J.
6:
55-63,
1987[ISI][Medline].
31.
Sharma, H. S.,
Das D. K.,
and
Verdouw P. D.
Enhanced expression and localization of heme oxygenase-1 during recovery phase of porcine stunned myocardium.
Mol. Cell. Biochem.
196:
133-139,
1999[ISI][Medline].
32.
Sharma, H. S.,
Maulik N.,
Gho B. C. G.,
Das D. K.,
and
Verdouw P. D.
Coordinated expression of heme oxygenase-1 and ubiquitin in the porcine heart subjected to ischemia and reperfusion.
Mol. Cell. Biochem.
157:
111-116,
1996[ISI][Medline].
33.
Singal, P. K.,
and
Kirchenbaum L. A.
A relative deficit in antioxidant reserve may contribute in cardiac failure.
Can. J. Cardiol.
6:
47-49,
1990[ISI][Medline].
34.
Stocker, R.,
Bowry V. W.,
and
Frei B.
Ubiquinol-10 protects human low-density lipoprotein more efficiently against lipid peroxidation than does alpha-tocopherol.
Proc. Natl. Acad. Sci. USA
88:
1646-1650,
1991[Abstract/Free Full Text].
35.
Sunamori, M.,
Tanaka H.,
Maruyama T.,
Sultan I.,
Sakamoto T.,
and
Suzuki A.
Clinical experience of coenzyme Q10 to enhance intraoperative myocardial protection in coronary artery revascularization.
Cardiovasc. Drugs Ther.
5:
297-300,
1991.
36.
Szarkowska, L.
The restoration of NADPH oxidase activity by coenzyme Q (ubiquinone).
Arch. Biochem. Biophys.
113:
519-525,
1966[ISI][Medline].
Am J Physiol Heart Circ Physiol 278(4):H1084-H1090
0363-6135/00 $5.00
Copyright © 2000 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
