15-F2t-isoprostane exacerbates myocardial ischemia-reperfusion injury of isolated rat hearts

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15-F2t-isoprostane exacerbates myocardial ischemia-reperfusion injury of isolated rat hearts Authors: Zhengyuan Xia1,2, Kuo-Hsing Kuo1;3, David V. Godin1, Michael J Walker1 and Michelle C.Y. Tao1* , David M. Ansley1

Affiliations: 1Centre for Anesthesia & Analgesia, Dept. of Anesthesiology, Pharmacology & Therapeutics, The University of British Columbia, Vancouver, BC, V6T 1Z3, Canada; 2Anesthesiology research laboratory, Dept of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan, China; 3 Dept. of Anatomy and Histology, University of Northern British Columbia, Canada; *Summer student

Running title: Isoprostane exacerbates cardiac reperfusion injury Word counts: 3912 words (Text). Address correspondence and request for reprints to: Dr. David M. Ansley University of British Columbia Department of Anesthesiology Room 3200, 3rd Floor JPP 910 West 10th Ave Vancouver, BC Canada V5Z 4E3 Phone: 604-875-4575 Fax: 604-875-5344 Email: [email protected] Presented in part at the First Global Conference on Cardiovascular Clinical Trials and Pharmacotherapy incorporating the 2nd World Heart Federation Global Conference on Cardiovascular Clinical Trials and 13th International Society of Cardiovascular Pharmacotherapy Congress, Oct 1-3, 2004. Hong Kong, China. Published in abstract form in Cardiovasc Drugs Ther 2004;18(supplement 1): A63.

Articles in PresS. Am J Physiol Heart Circ Physiol (June 3, 2005). doi:10.1152/ajpheart.00042.2005

Copyright © 2005 by the American Physiological Society.

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Abstract Background: 15-F2t-isoprostane is a specific marker of in vivo lipid peroxidation induced by

reactive oxygen species (ROS) whose formation is increased after myocardial ischemia and

during the subsequent reperfusion. 15-F2t-isoprostane possesses potent bioactivity under

pathophysiological conditions. However, it remains unknown whether 15-F2t-isoprostane, by

itself, can influence myocardial ischemia-reperfusion injury (IRI).

Methods: Adult rat hearts were perfused by the Langendorff technique with Krebs-Henseleit

solution (KH) at a constant flow rate at 10 ml/min. 15-F2t-isoprostane 100 nM (IsoP), SQ 29548

1µM (SQ), a thromboxane receptor antagonist that can abolish the vasoconstrictor effect of 15-

F2t-isoprostane, or their combination (IsoP+SQ) in KH or KH alone (vehicle control) were

applied for 10 min before inducing 40 min global ischemia, followed by 60 min of reperfusion.

During ischemia, saline (control), IsoP, IsoP+SQ or SQ in saline was perfused through the aorta

at 60 µl/min. Either IsoP, IsoP+SQ or SQ in KH were infused during the first 15 min of

reperfusion. Results: Coronary effluent endothelin-1 (ET-1) concentrations in the IsoP group

were significantly higher than those in the control group during ischemia and also in the later

phase of reperfusion (P<0.05). Infusion of 15-F2t-isoprostane resulted in increased release of

cardiac-specific creatine kinase (CK-MB) and reduced cardiac contractility during reperfusion

and also increased myocardial infarct size relative to the control group. SQ 29548 abolished

the deleterious effects of 15-F2t-isoprostane. Conclusion: 15-F2t-isoprostane exacerbates

myocardial IRI, and may therefore act as a mediator of IRI. 15-F2t-isoprostane induced ET-1

production during cardiac reperfusion may represent a mechanism underlying the deleterious

actions of 15-F2t-IsoP.

Key words: 15-F2t-isoprostane; myocardial; ischemia-reperfusion injury

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Myocardial ischemia-reperfusion injury (IRI) and its sequelae, cardiac depression and

arrythmogenesis, have been shown experimentally to result, at least in part, from the

disruptive action of reactive oxygen species (ROS) on membrane lipids and intracellular

proteins required for cellular integrity and function. (12;15;26) The release of high levels of

ROS during ischemia-reperfusion can overwhelm endogenous antioxidant defenses, a crucial

event determining the onset of irreversible cellular necrosis secondary to extensive lipid

peroxidation.(17)

A recent advance in free radical biology has been the discovery of isoprostanes, which

are stable in vivo end products of arachidonic acid peroxidation.(22) Of the variety of

isoprostanes detected, 15-F2t-isoprostane (15-F2t-IsoP) (28) has been found to be a specific,

reliable marker of oxidative stress. This has facilitated investigation of the role of ROS in a

variety of disease states, most notably cardiovascular disease. Of interest, 15-F2t-IsoP

possesses potent biological activity, including vasoconstriction and platelet activation under

pathophysiological conditions.(21) 15-F2t-IsoP has no effect on coronary flow in the absence

of ischemia in the isolated rat hearts (up to a concentration of 256 nM), but significantly

reduces coronary flow in the hypoxic or post-ischemic reperfused rat heart (at 30 nM).(14)

Clinically we identified an inverse correlation between the speed of decay of plasma

15-F2t-IsoP concentrations during the early phase of reperfusion and post-operative cardiac

functional recovery in patients undergoing coronary artery bypass surgery utilizing

cardiopulmonary bypass (CPB).(1) The characteristics of 15-F2t-IsoP production and its

effects under conditions of ischemia and reperfusion are similar to those of endothelin-1 (ET-

1). Endothelin-1 is one of the most potent vasoconstrictors known, and it has been postulated

to contribute to post-ischemic myocardial dysfunction (25;27). ET-1 release has been shown

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to increase during and after myocardial ischemia (3) and its vasoconstrictor effect appears to

be potentiated during post-ischemic reperfusion in isolated hearts. (23;38)

We hypothesized that 15-F2t-IsoP can exacerbate myocardial ischemia-reperfusion

injury and that the mechanism of 15-F2t-IsoP action may involve the release and/or enhancing

the production of ET-1 during cardiac ischemia and reperfusion. Our hypothesis was tested in

an isolated rat heart model, using SQ 29548, a thromboxane A2 receptor (TXA2) antagonist

used to abolish the vasoconstrictive actions of 15-F2t-IsoP.(13)

Methods

Heart preparation

This study was approved by the Committee of Animal Care of the University of

British Columbia. Animals were cared for in accordance with the principles and guidelines of

the Canadian Council on Animal Care. Male Sprague-Dawley rats (280~320g) were

anesthetized with pentobarbital (70mg/kg intraperitoneally) and heparinized with sodium

heparin (1000 IU/kg, intraperitoneally). After median thoracotomy, hearts were quickly

excised and immersed in ice-cold Krebs-Henseleit (KH) solution to stop contractions. Hearts

were gently squeezed to remove residual blood to prevent clot formation. Hearts were

retrogradely perfused via the aorta in a non-working "Langendorff" preparation at a constant

flow rate of 10 ml/min using a peristaltic pump. The perfusion fluid (pH 7.4; temperature,

37°C) was KH solution that contained (in mM): NaCl 118; NaHCO3 24; KCl 4.63; MgCl2 1.2;

CaCl2 1.25; KH2 PO4 1.17; glucose 11. The perfusate was bubbled with a mixture of 95% O2

and 5% CO2. Temperatures of the perfusate solution and of the chamber in which the hearts

were rested were maintained at 37°C using a thermostatically controlled water circulating

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system. Coronary perfusion pressure (CPP) was measured via a side arm of the perfusion

cannula connected to a pressure transducer (Statham p23 ID, Gould Electronics, Cleveland).

A latex water-filled balloon fixed to a pressure transducer was inserted through the mitral

valve into the left ventricle for the determination of left ventricular (LV) developed pressure

(LVDP), which was calculated by subtracting end-diastolic pressure (LVEDP) from LV peak

systolic pressure (LVSP). LVEDP was adjusted to approximately 5 mmHg before the start of

the experiment by adjusting the volume in the intraventricular balloon.

Experimental Protocol

All hearts were initially equilibrated for 10 min (BS10). They then were randomly

assigned to a sham group or one of the four experimental groups (n=7 per group): ischemia-

reperfusion untreated control (control), 15-F2t-IsoP (IsoP), 15-F2t-IsoP plus SQ 29548 (IsoP-

SQ) or SQ 29548 (SQ) alone . After BS10, 15-F2t-IsoP 100 nM/L (IsoP), SQ 29548 1µM/L

(SQ) or 15-F2t-IsoP 100 nM/L plus SQ 29548 1µM/L (IsoP+SQ) were applied for 10 min

respectively in the corresponding groups, before global ischemia (40 min) was induced by

stopping perfusion. Control hearts underwent an additional 10 min period of equilibration

before global ischemia was induced. During ischemia, saline (control), 15-F2t-IsoP 100 nM/L

(IsoP), SQ 29548 1µM/L (SQ) or 15-F2t-IsoP 100 nM/L plus SQ 29548 1µM/L (IsoP+SQ) in

saline was perfused through the aorta at 60 µl/min using a mini-pump. KH was perfused

during 60 min of reperfusion in the control group. Either 15-F2t-IsoP, SQ 29548 or 15-F2t-

IsoP plus SQ 29548 in KH was perfused for the first 15 min of reperfusion. Hearts were

electrically paced at a rate of 300 beats/min, prior to and following, but not during the

ischemic period when hearts ceased to beat spontaneously.

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The perfusion flow rate was based on the result of a pilot study which showed that

sham isolated hearts perfused at 10 ml/min with KH beat well and remain hemodynamically

stable for 120 min (the duration of the experiment) in our experimental set-up. The

concentration of 15-F2t-IsoP applied in the current study was based on: 1) prior reporting that

15-F2t-IsoP 100 nM did not affect coronary flow in sham-perfused rat hearts but significantly

reduced coronary flow in ischemic-reperfused rat hearts (13); and 2) our own pilot study

which showed that 15-F2t-IsoP at 30 nM did not cause significant reduction in post-ischemic

LVDP (n=3) compared to control, but when 300 nM 15-F2t-IsoP was given in our

experimental set-up, hearts were not able to resume beating during reperfusion (n=2). Studies

have shown that SQ 29548, a thromboxane A2 (TXA2) receptor antagonist (24), abolished

15-F2t-IsoP (56 nM)-induced reduction in coronary flow in ischemic-reperfused rat hearts at a

concentration of 0.1µM (13) and abolished 15-F2t-IsoP (>300 nM) -induced reduction in

coronary flow in isolated perfused guinea pig heart at 1µM.(20) Therefore, 1µM SQ 29548

was applied in the current study to ensure blockade of 15-F2t-IsoP action in our model.

Effluent perfusate was sampled at BS10, the first 30 min of ischemia (isch) and at 1

(Re-1), 5 (Re-5), 30 (Re-30) and 60 (Re-60) min of reperfusion in the four experimental

groups or at the corresponding time points in the sham group. Aliquots of the effluent samples

were immediately stored at -70 °C until analysis for cardiac specific creatine kinase (CK-MB)

in all study groups and for 15-F2t-IsoP in the sham, control and SQ groups. Another portion of

the effluent sample was initially concentrated (see below) and then stored at -70 °C for

analysis of ET-1 concentration. At the end of the 60 min of reperfusion, 37ºC 1% 2,3,5-

triphenyltetrazolium in buffer (0.1 M phosphate buffer adjusted to pH 7.4) was pumped into

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the heart at 1 ml/gm/min for 15 min until the epicardial surface became deep red. The hearts

were then stored in 10% formaldehyde for later analysis of myocardial infarct size.

Measurement of Endothelin-1

Enzyme immunoassays (EIA) of ET-1 concentrations in the coronary effluent were

performed in duplicate according to the manufacturer’s instruction (human ET-1 EIA kit 900-

020, Assay Designs, Inc. Ann Arbor). The Assay kit detects ET-1 levels in biological fluids of

human, bovine, canine, murine, porcine and rat (32) samples. Based on pilot studies, ET-1

concentrations in our samples were often below the ET-1 sensitivity of the assay (0.14pg/ml).

Therefore effluent samples were concentrated 4-fold by evaporation of solvent (i.e. the KH

solution) at room temperature under a stream of dry nitrogen. ET-1 concentration was

calculated as 1/4 of the measured ET-1 level in the concentrated sample. The accuracy of this

approach was confirmed by prior testing using known ET-1 standards. The assays plates were

read at 450 nm and the values of the unknowns were expressed as picograms ET-1 per

milliliter effluent.

Measurement of CK-MB

Measurement of CK-MB was determined by enzyme immunoassay (Catalog number:

BC-1121, BioCheck, Inc, Burlingame, CA). The unknowns were expressed as nanograms

CK-MB per milliliter effluent.

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15-F2t-IsoP Assays

Enzyme immunoassay of free 15-F2t-IsoP was performed according to the methods

provided by the manufacturer (Cayman Chemical, Ann Arbor) as previously described

(33,34). The values of the unknowns were expressed as picograms 15-F2t-IsoP per milliliter

effluent.

Infarct size measurement

The measurement of infarct size was essentially identical to that described by Downey

(8) except for the method of quantification. After the 2,3,5-triphenyl-tetrazolium chloride

(TTC) reaction, the hearts were sectioned transaxially, and size of infarct was evaluated as

percentage of sectional area of infarcted tissue to the sectional area of the whole heart in 1

mm layers (five layers, LG scanner). Morphometric measurements of infarct size were

performed with a LG scanner and 6.0 CE software. The histogram counts of the red (viable

tissue) and white (infracted tissue) were recorded. The percent infarction was calculated as

white counts divided by the sum of the red plus white counts.

Statistical analysis

All data are presented as means ± SEM. Cardiac variables and chemical assay

parameters were compared by two-way analysis of variance (ANOVA) with repeated

measures. One-way ANOVA was used to test for differences in infarct size between groups.

The correlation between effluent ET-1 and 15-F2t-IsoP concentration was evaluated by the

Pearson test. P<0.05 was considered statistically significant.

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Results

Endothelin-1 release and its relation with 15-F2t-IsoP

Baseline effluent ET-1 concentrations did not differ among the experimental groups

(Fig 1A). Effluent ET-1 did not significantly change over time in the sham group (data not

shown). ET-1 increased in the control group during ischemia (Fig 1A, P <0.001 vs baseline)

and increased further in the Iso-P group compared to control (P<0.05). ET-1 increased by

approximately 20% at Re-1 and by 32.8±26.9% at Re-30 compared to baseline (BS10) in the

control group. These changes did not reach statistical significance (P>0.1). Effluent ET-1

concentration in the IsoP group was significantly higher than that in the control group (P<0.05)

at reperfusion 60min (Re-60). Effluent ET-1 concentrations in both the IsoP-SQ and the SQ

groups did not differ from those found in the control during ischemia and reperfusion. A weak

but statistically significant positive correlation (r = 0.77, P =0.04, Fig 1B) was noted between

effluent concentrations of 15-F2t-IsoP and ET-1 during ischemia, but not during reperfusion,

in the control (i.e., untreated) group.

15-F2t-IsoP generation during ischemia-reperfusion

Effluent 15-F2t-IsoP release in the sham group did not change over a 120 min

perfusion period (Fig 2). As shown in Fig 2, effluent 15-F2t-IsoP levels increased during

ischemia (P < 0.001 vs BS10) and remained elevated at Re-1 (P<0.05 or P<0.01 vs BS10) in

the control and SQ groups. Effluent 15-F2t-IsoP release during ischemia and reperfusion did

not significantly differ between the control and SQ groups.

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CK-MB release during ischemia-reperfusion

Baseline CK-MB release was detectable in this model and did not differ among groups

(Fig 3). Effluent CK-MB release did not significantly change over time in the sham group.

During ischemia, CK-MB increased significantly from baseline values only in group iso-P

(p<0.05).

During reperfusion, CK-MB increased gradually and was significantly higher than

baseline at Re-30 in the control group (P<0.01). Effluent CK-MB concentration in the IsoP

group increased more rapidly during reperfusion and was significantly higher at Re-5 than its

baseline value (P<0.05). This was also significantly greater than values measured in control

hearts at the same time interval (p<0.05). CK-MB levels in IsoP-SQ were similar to values

measured in untreated control hearts during ischemia and reperfusion. At Re-1, CK-MB level

in SQ was higher than in the control group and the IsoP group (<0.05) but then decreased

quickly thereafter.

Contracture development during ischemia

The LVEDP increased progressively during ischemia in the control group (Fig 4A).

LVEDP in the IsoP group increased more quickly. At 30 and 35 min of ischemia, the

magnitude of LVEDP in the IsoP group was significantly higher than that in the control group

(P<0.05). SQ 29548 attenuated the effect of 15-F2t-IsoP on ischemic contracture. LVEDP was

significantly lower in the IsoP-SQ group than the IsoP group at ischemia 30 min and onwards.

LVEDP in the IsoP-SQ and the SQ group did not differ from that measured in the control

group during ischemia.

Time to the onset of ischemic contracture was significantly shorter in the IsoP group

(11.4±1.9 min) compared to control (17.4±1.6 min, P <0.05, Fig 4B). The latency to ischemic

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contracture in the IsoP-SQ (20.0±1.5 min) and the SQ (18.1±1.5 min) groups was

significantly increased compared to that in the IsoP group (P<0.01 or P<0.05), but did not

differ from that in the control group (P>0.05, Fig 4B).

Functional response to ischemia/reperfusion

During reperfusion, LVEDP in the control group was significantly higher than that at

baseline (Fig 5A). 15-F2t-IsoP significantly augmented the increase of LVEDP during

reperfusion at Re-30 and Re-60 (P<0.01). SQ 29548 attenuated the 15-F2t-IsoP-induced

increase in LVEDP. The magnitude of LVEDP in the IsoP-SQ and the SQ groups did not

significantly differ from that in the control group during reperfusion.

The LVDP in the sham group did not change significantly over time during the

experimental period. The LVDP in the control group recovered to a maximum of 87.0±11.6 %

of its baseline value at Re-30 (P>0.05 vs BS10, Fig 5B) and decreased thereafter. The LVDP

in the IsoP group recovered to a maximum of 56.5±13.5% of its baseline value at Re-30

(P<0.05 vs BS10) and decreased quickly thereafter. At Re-60, LVDP in the IsoP group was

lower than that in the control group. The LVDP values in the IsoP-SQ and the SQ group did

not differ from those in the control group at Re-60. SQ 29548 exacerbated 15-F2t-IsoP

induced reduction in LVDP relative to control group at Re-10.

Coronary perfusion pressure (CPP)

Neither 15-F2t-IsoP, SQ 29548, nor their combination affected CPP before ischemia.

CPP did not increase significantly until after 60 min of reperfusion in the untreated control

group (80.4±11.0 mmHg at Re-60 vs 51.3±1.1 mmHg at BS10, P<0.05). CPP in the IsoP

group increased more quickly during reperfusion relative to the control group. At Re-30, the

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CPP value in the IsoP group (100.4±13.9 mmHg) was higher (P<0.05) than its baseline value

(52.1±3.9 mmHg) and higher (P<0.05) than the corresponding value in the control group

(65.6±4.6 mmHg). SQ 29548 did not significantly affect CPP as compared to the control

group. At Re-60, CPP values did not significantly differ among the control (80.4±11.0

mmHg), the IsoP (110.4±14.9 mmHg), the IsoP+SQ (115.4±14.9 mmHg) and the SQ

(84.8±13.5 mmHg) groups (P>0.05).

Myocardial infarct size

As shown in Figure 6, myocardial infarct size in the IsoP group is significantly larger

than that of the control (untreated) group (P<0.05). The myocardial infarct sizes in the IsoP-

SQ and SQ groups are significantly smaller than those in the IsoP group (P<0.05 or P<0.01).

Infarct sizes in the SQ group and IsoP-SQ groups were somewhat smaller than those in the

control group, but the differences did not attain statistical significance.

Discussion

This is the first study providing evidence that 15-F2t-IsoP exacerbates myocardial IRI

in isolated perfused rat hearts. Our findings include the following: (1) 15-F2t-IsoP did not

affect pre-ischemic cardiac mechanics and coronary perfusion pressure but did reduce cardiac

tolerance to ischemic insult, as manifested by an early onset and higher magnitude of

ischemic contracture; (2) 15-F2t-IsoP stimulated the release and/or production of ET-1 during

ischemia which was accompanied by an increased severity of myocardial cellular damage as

evidenced by increased CK-MB release; (3) 15-F2t-IsoP increased myocardial infarct size and

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exacerbated post-ischemic myocardial dysfunction, which may be attributable, in part, to

stimulation of ET-1 production and/or release during reperfusion.

Endothelin-1 has potent vasoconstrictor properties and is known to reduce myocardial

contractility and contribute to the progression of the heart failure.(27) Plasma levels of ET-1

increase during cardiac operations requiring cardiopulmonary bypass (CPB)(2;31). A high

plasma ET-1 level during the early postoperative period has been associated with prolonged

pharmacologic management (i.e., inotropic support), longer intensive care unit stay, and

complicated recovery.(2;7). The present study clearly demonstrates that 15-F2t-IsoP, whose

formation increased in the myocardium and coronary artery during CPB surgery,(18) can

increase the release and/or production of ET-1 during myocardial ischemia-reperfusion. This

might be a mechanism whereby 15-F2t-IsoP exacerbates myocardial IRI. The positive

correlation between effluent concentrations of 15-F2t-IsoP and ET-1 during ischemia in the

control (untreated) group suggests that endogenous 15-F2t-IsoP may act to stimulate increased

ET-1 release during ischemia.

We observed a reduction in ET-1 concentration at 30 min of reperfusion (Re-30), but a

statistically significant increase by 60 min of reperfusion (Re-60) in the IsoP group compared

to control (Fig 1A). 15-F2t-IsoP may have triggered an increased formation of ET-1 during

late reperfusion. In the IsoP group, the infusion of 15-F2t-IsoP was terminated at 15 min of

reperfusion. Sequestration of a significant amount 15-F2t-IsoP in the heart tissue 45 min after

the termination of exogenous 15-F2t-IsoP infusion is unlikely in this study, since the 15-F2t-

IsoP decay half-life in this model is about 4 min (data not presented). 15-F2t-IsoP stimulation

of ET-1 formation during late reperfusion could represent an important mechanism

responsible for post-ischemic myocardial dysfunction in the clinical setting. Whereas we

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previously found that plasma free 15-F2t-IsoP levels increased during ischemia-reperfusion for

approximately 30 min during cardiac surgery, the 15-F2t-IsoP decay pattern during early

reperfusion correlated with early postoperative cardiac recovery.(1) Plasma ET-1 levels may

remain elevated at least 24 hours after cardiac surgery.(35) Recent study conducted in isolated

rat cardiomyocytes has shown that 30 min of ischemia with or without 30 min of reperfusion

is sufficient to rapidly stimulate the gene expression of myocyte ET-1 as well as the ET-1

receptors. (9) It is possible that high levels of 15-F2t-IsoP during ischemia and/or early

reperfusion may have induced ET-1 gene expression resulting in increased ET-1 production

during late reperfusion. Further study is merited to address the underlying mechanism.

Based on our results we postulate that 15-F2t-IsoP may increase ET-1 release into the

coronary circulation relative to the myocardial tissue during ischemia. It has been previously

shown that the ratio of ET-1 secretion to the interstitial transudates versus secretion to

coronary effluent is about 6.6 at baseline in isolated perfused rat hearts (3). This ratio is

reduced to about 2.5 during the period of low-flow ischemia and the first 30 min of

reperfusion.(3) The relative reduction of ET-1 concentration observed in the IsoP group at 30

min of reperfusion, 15 min after the termination of 15-F2t-IsoP infusion, indicates 15-F2t-IsoP

may have primarily stimulated ET-1 release rather than its production during ischemia and

early reperfusion.

Despite 15-F2t-IsoP’s bioactivity as a vasoconstrictor,(21;29) reduction of coronary

flow is not likely a major mechanism of 15-F2t-IsoP action during myocardial IRI, at least in

this model. In the current study, hearts were perfused at a constant flow rate. In addition, CPP

at Re-60 did not differ significantly among experimental groups although LVDP in the IsoP

group was significantly lower than that in the control, IsoP-SQ and the SQ groups. It is

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possible that 15-F2t-IsoP aggravates myocardial IRI by a complex mechanism involving the

activation of Na+-H+ exchange indirectly through ET-1.(4;11). Alternatively, 15-F2t-IsoP may

act by reducing the intrinsic activity of nitric oxide,(19) an endogenous vasodilator. This may

explain why the CPP value at Re-30 was higher in the IsoP group relative to control

irrespective of the similar effluent levels of ET-1 at this time point.

Despite abolishing the deleterious effects of high concentration exogenous 15-F2t-IsoP,

SQ 29548 did not confer any beneficial effect in attenuating myocardial IRI compared to the

control in this model. This is in keeping with previous findings describing the effect of

exogenous 15-F2t-IsoP on the isolated guinea pig heart. (20) The relatively high concentration

of CK-MB at Re-1 in the SQ group is likely due to rapid release of CK-MB from the ischemic

tissue rather than the result of more intense tissue damage, since the infarct size of the SQ

group was comparable to that in controls. The inability of SQ 29548 to attenuate myocardial

ischemia-reperfusion injury in the isolated perfused heart model may suggest the following: 1)

endogenous 15-F2t-IsoP production in the myocardium during ischemia and reperfusion is

relatively low under the current experimental condition, and it is mainly a marker (i.e., the

result of lipid peroxidation) rather than a mediator of oxidative damage; 2) TXA2 may play

little role in myocardial IRI in rat, a finding similar to that found in gene knock-out mice. (37)

Given that TXA2 may stimulate rat heart smooth to generate ET-1(6), we can not exclude the

possibility that SQ 29548 blockade of endogenous TXA2 action may have contributed to the

decrease in ET-1 release at Re-60 seen in the isoP-SQ group. It seems apparent that, SQ

29548 blockade of 15-F2t-IsoP action rather than the blockade of TXA2 action represents a

major mechanism of myocardial protection seen in the current study.

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It is intriguing that SQ blocked the effects of 15-F2t-IsoP on LVEDP during ischemia

but not during reperfusion. It appears that the concentration of 15-F2t-IsoP may be a

determinant of the effectiveness of SQ. At ischemia 40 min (Fig. 4A), the magnitude of

LVEDP in group IsoP-SQ was not only significantly lower than that in group IsoP, but was

also about 40% lower than the corresponding values in the control or SQ groups (P=NS). The

effluent level of ET-1 during ischemia in the IsoP-SQ group was about 20% lower than the

corresponding values in the control or SQ groups. It is possible that this slight 20% difference

in ET-1 concentration during ischemia caused the 40% difference in the magnitude of LVEDP

mentioned above. Study has shown that ischemia may cause time-dependent externalization

of ET-1 receptor binding sites in rat cardiac membranes (16), which may sensitize and

exacerbate ET-1 deleterious effects, such as the exacerbation of ischemic contracture (4).

Further study is merited to address why SQ could act differently, during myocardial ischemia,

in the presence or absence of 15-F2t-IsoP.

It is should be noted that ET-1 has been shown to exert a cardioprotective or

preconditioning-like effect in both in vivo (10) and in vitro (5)models of myocardial ischemia-

reperfusion in the rat, when applied prior to ischemia. Of interest, our most recent study

suggests that ET-1 may confer post-preconditioning-like effect as well in the isolated

ischemic-reperfused rat hearts (36). We found that the ET-1 A and B receptor antagonist

bosentan, when applied during the first 15 min of reperfusion worsened post-ischemic

myocardial dysfunction in the rat heart, and unmasked any potential beneficial effects of ET-1

blockade during ischemia (36). However, when ET-1 receptor blockade was applied only

during later phase of reperfusion, post-ischemic myocardial infarct size was reduced. Hence,

as observed in the current study, an 15-F2t-IsoP induced ET-1 increase during ischemia, and

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especially during later reperfusion, may represent a major mechanism underlying the

deleterious actions of 15-F2t-IsoP.

In conclusion, our finding that 15-F2t-IsoP can increase myocardial infarct size and

exacerbate myocardial IRI may have important clinical implications. During cardiac surgery,

systemic production of ROS occurs during CPB and may exceed production arising from

reperfusion of the ischemic heart. The plasma level of 15-F2t-IsoP has been observed to

dramatically increase shortly after the start of CPB.(30) These high levels of 15-F2t-IsoP could

enter the heart either before aortic cross-clamping (the beginning of global myocardial

ischemia) or at the time of aortic declamping, triggering and/or exacerbating myocardial IRI.

The findings of the current study combined with our previous work on the effect of

antioxidant supplementation with propofol suggest that combined therapy with antioxidant

and 15-F2t-IsoP antagonism during ischemia and early reperfusion could offer a promising

approach to attenuate myocardial IRI.

Acknowledgements

This study is supported in part by internal department development funding (to D. Ansley) and

in part by the National Natural Science Foundation of China (No: 30471659, to Z. Xia). D. Ansley

received a Clinical Scholar Research Award from the International Anesthesia Research Society. Z.

Xia received a research fellowship from the Centre for Anesthesia & Analgesia, Dept. Pharmacology

& Therapeutics, The University of British Columbia, Canada.

18

Figure legends

Fig.1. A. Effluent Endothelin-1 (ET-1) concentrations during myocardial ischemia-

reperfusion. BS10 and isch indicate 10 min after stabilization and 30 min during global

myocardial ischemia, respectively; Re-1, Re-5, Re-30 and Re-60 indicate 1, 5, 30 and 60 min

after reperfusion, respectively. * P<0.001 vs BS10; # P<0.05 or P<0.01 vs control. (n=7 for

each group). B. Relationship between 15-F2t-isoprostane (15-F2t-isoP) and ET-1

concentration during the first 30 min of ischemia in the control group. ET-1 release is

positively correlated with 15-F2t-isoP concentration (r = 0.7695, 95% CI: 0.0389 – 0.9640,

n=7, P (two-tailed) = 0.043).

Fig. 2. Effect of SQ 29548 (SQ) on 15-F2t-isoprostane (15-F2t-isoP) release during myocardial

ischemia-reperfusion. BS10 and isch indicate 10 min after stabilization and 30 min during

global myocardial ischemia, respectively; Re-1, Re-5, Re-30 and Re-60 indicate 1, 5, 30 and

60 min after reperfusion, respectively. * P<0.001 or P< 0.05 vs BS10. # P< 0.001 vs control. P

>0.05 SQ vs control, n=7 per group.

Fig.3. Effluent CK-MB concentration during myocardial ischemia-reperfusion. BS10 and isch

indicate 10 min after stabilization and 30 min during global myocardial ischemia, respectively;

Re-1, Re-5, Re-30 and Re-60 indicate 1, 5, 30 and 60 min after reperfusion, respectively. *P<

0.05 vs BS10; #P<0.05 vs control; +P<0.05 vs isoP group. (n= 7 for each group)

19

Fig. 4. A: Development of left ventricular end-diastolic pressure (LVEDP), reflecting

myocardial contracture, during ischemia. B: Ischemic contracture onset time. # P<0.05 vs

control; +P<0.05 or P<0.01 vs IsoP (15-F2t-isoprostane) group; *P<0.05 or P<0.01 vs

ischemia 30 min within the same group (n=7 per group).

Fig. 5. A: Variations of left ventricular end-diastolic pressure (LVEDP), reflecting

myocardial stiffness, during reperfusion. BS10 and Pre-isch indicate 10 min after stabilization

and the time immediately prior to ischemia, respectively; Re-30 and Re-60 indicate 30 and 60

min after reperfusion, respectively. *P<0.05 or P<0.01 vs BS10; # P<0.05 or P<0.01 vs

control; +P<0.05 or P<0.01 vs IsoP (15-F2t-isoprostane) group. B: Recovery of left

ventricular developed pressure (LVDP), reflecting effective myocardial contractility, during

reperfusion. BS10 and Pre-isch indicate 10 min after stabilization and the time immediately

prior to ischemia, respectively; Re-30 and Re-60 indicate 30 and 60 min after reperfusion,

respectively. *P<0.05 vs BS10; # P<0.05 vs control; +P<0.05 or P<0.01 vs IsoP (15-F2t-

isoprostane) group (n=7 per group).

Fig. 6. Myocardial infarct size. Top: representative images showing myocardial infarction

(white) in the control (A), 15-F2t-isoprostane plus SQ 29548 (IsoP+SQ, C) and SQ 29548

(SQ, D) groups. Bottom: Percentage infarction (Mean ± SEM, n=7 per group): *P<0.05 vs

control; +P<0.05 or P<0.01 vs IsoP group.

20

BS10 isch Re-1 Re-5 Re-30 Re-600.00

0.25

0.50

0.75

1.00 ControlIsoPIsoP.SQ

A

*

#

* * SQ

*

Time during ischemia-reperfusion

ET-1

con

cent

ratio

n (p

g/m

l)

0 50 100 150 2000.00

0.25

0.50

0.75

1.00B

15-F2t-isoP release during ischemia incontrol group (pg/ml)

ET-1

rel

ease

dur

ing

isch

emia

in c

ontr

ol g

roup

(pg/

ml)

Fig. 1.

21

BS10 isch Re-1 Re-5 Re-30 Re-600

25

50

75

100

125 ControlSQ

* *

*

*

Sham

Time during ischemia-reperfusion

15-F

2t-is

oP (p

g/m

l)

Fig. 2.

22

BS10 isch Re-1 Re-5 Re-30 Re-600.0

0.2

0.4

0.6 ControlIsoPIsoP-SQSQ

*

+# *

# *+#

*

Time during ischemia-reperfusion

CK

-MB

con

cent

ratio

n(n

g/m

l)

Fig.3.

23

15 20 25 30 35 400

10

20

30ControlIsoPIsoP-SQSQ

##

++

*

+*

+

*+* *

*

A

Duration of ischemia (min)

LVED

P (m

m H

g)

Control IsoP IsoP-SQ SQ0

5

10

15

20

25ControlIsoPIsoP-SQSQ

B

#

++

Group

Con

trac

ture

ons

et ti

me

(min

)

Fig.4.

24

BS10 Pre-isch Re-30 Re-600

20

40

60

80ControlIsoPIsoP-SQ

*#

*

*+ *

*#

*

*+*

A

SQ

Time points before and after reperfusion

LVED

P (m

m H

g)

BS10 Pre-isch Re-30 Re-600

25

50

75

100ControlIsoPIsoP-SQSQ* + +

*+

B

*#

Time during ischemia-reperfusion

LVD

P (m

mH

g)

Fig. 5.

25

Control IsoP IsoP-SQ SQ05

1015202530354045 Control

IsoPIsoP-SQSQ

*

++

Groups

Infa

rct s

ize

(% o

f tot

al a

rea)

Fig 6.

26

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