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Ann Thorac Surg 2003;76:1623-1630
© 2003 The Society of Thoracic Surgeons

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Original article: cardiovascular

Effect of 11,12-epoxyeicosatrienoic acid as an additive to st. thomas’ cardioplegia and university of wisconsin solutions on endothelium-derived hyperpolarizing factor–mediated function in coronary microarteries: influence of temperature and time

^a Department of Surgery, Division of Cardiothoracic Surgery, Chinese University of Hong Kong, Hong Kong SAR, China
^b Providence Heart Institute, Albert Starr Academic Center, Department of Surgery, Oregon Health and Science University, Portland, Oregon, USA

Accepted for publication April 23, 2003.

^* Address reprint requests to Prof He, Department of Surgery, Block B, 5A, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
e-mail: gwhe{at}cuhk.edu.hk

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
BACKGROUND: We examined the effect of 11,12-epoxyeicosatrienoic acid (EET_11,12) added to St. Thomas’ Hospital (ST) solution or University of Wisconsin (UW) solution on endothelium-derived hyperpolarizing factor (EDHF)–mediated relaxation under clinically relevant temperature and exposure time.

METHODS: Porcine coronary microarteries (200 to 450 µm) were incubated with Krebs’ solution (control), ST with or without EET_11,12 (300 nmol/L) at 22°C for 1 hour as well as at 4°C for 1 or 4 hours, and UW with or without EET_11,12 at 4°C for 4 hours. The EDHF-mediated relaxation was induced by bradykinin (-10 to approximately -6.5 log M) in the precontraction evoked by U₄₆₆₁₉ (10 nmol/L) or U₄₆₆₁₉ (1 nmol/L) plus endothelin-1 (6 nmol/L).

RESULTS: The EDHF-mediated relaxation was reduced after exposure to UW (79.7% ± 4.6% versus 93.6% ± 2.8%, p = 0.01) at 4°C for 4 hours. One-hour exposure to ST under 22°C or 4°C decreased the relaxation (75.2% ± 7.6% versus 96.7% ± 1.6%, p < 0.05) or the sensitivity to bradykinin (-8.04 ± 0.15 versus -8.50 ± 0.20 log M, p < 0.05). The relaxation increased to 86.8% ± 5.3% by addition of EET_11,12 to ST (1 hour at 22°C, p < 0.05) but was unchanged when added to either ST or UW at 4°C for 1 or 4 hours.

CONCLUSIONS: As an additive to ST solution, EET_11,12 may partially restore EDHF-mediated endothelial function under moderate hypothermia but had no significant effect under profound hypothermia when added to either ST or UW solution. Further investigation is necessary to improve the effect.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
St. Thomas’ Hospital solution (ST) is widely used as cardioplegia in cardiac surgery. In addition, ST solution as well as University of Wisconsin (UW) solution are used for heart preservation in transplantation. Although these two solutions were designed in different formulae, both have been demonstrated to be effective in protecting the myocardium against ischemia [1–3]. As the common component, potassium at high concentration (hyperkalemia) contributes to cardiac protection by depolarizing the myocyte membrane and causing asystole, which avoids the depletion of the high-energy phosphate pool by ischemia and conserves the myocardial energy reserves [4].

Endothelium plays an important role in regulating the activity of the underlying vascular smooth muscle. Among the vasoactive agents released by endothelium, prostacyclin (PGI₂), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF) are responsible for the endothelium-dependent relaxation. Studies showed that direct contact with hyperkalemic solutions damages the endothelial function [5–7]. Our previous studies further demonstrated that in both large and microcoronary arteries, EDHF-mediated function is impaired by hyperkalemic solutions, either cardioplegia (ST) or organ preservation (UW) solution [8–12].

The chemical identity of EDHF has not been clarified despite the fact that it is a transferable factor [13]. Among the candidates for EDHF, epoxyeicosatrienoic acids (EETs), the cytochrome P-450 monooxygenase metabolites of arachidonic acid have been demonstrated to be EDHF in certain vasculatures [14, 15]. Epoxyeicosatrienoic acids originate from endothelium and hyperpolarize smooth muscle cells by increasing the open probability of calcium-activated potassium channels [14]. Our recent study indicated that in porcine coronary microarteries, although EET_11,12 may not be identical to EDHF, it partially mimics the EDHF function and addition of EET_11,12 to hyperkalemic (K⁺ 20 mmol/L) solution partially restores EDHF-mediated function reduced by hyperkalemia exposure [16]. We therefore hypothesized that the addition of EET_11,12 to clinically used cardioplegia or organ preservation solutions may also partially restore the EDHF-mediated function and this effect may depend on the composition of the solution and other factors such as temperature and exposure time.

The present study further examined the EDHF-mediated relaxation in porcine coronary microarteries preserved with integrated hyperkalemic cardioplegia or organ preservation solutions. The overall effect of EET_11,12 supplementation in ST and UW solutions on EDHF-related endothelial function was investigated under clinically relevant temperature and exposure time.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Fresh porcine hearts collected from a local slaughterhouse were placed in a container filled with cold Krebs solution and immediately transferred to the laboratory. Upon receipt of the heart, intramyocardial coronary microarteries (usually the tertiary branches of the left anterior descending artery, diameter 200 to 450 µm) were carefully dissected and removed under a microscope. The vessels were cleaned of fat and connective tissue and cut into cylindrical rings of 2-mm length under a microscope. The Krebs solution was aerated with a gas mixture of 95% O₂ and 5% CO₂ at 37°C and had the following composition (in mM): 144 Na⁺, 5.9 K⁺, 2.5 Ca²⁺, 1.2 Mg²⁺, 128.7 Cl^-, 25 HCO₃^-, 1.2 SO₄^2-, 1.2 H₂PO₄^-, and 11.0 glucose.

During the above procedure the endothelium was intentionally preserved by carefully dissecting and mounting the rings. After the rings were mounted in a four-channel myograph (model 610A; J.P. Trading, Aarhus, Denmark), a previously described method [8, 10] was used to normalize vascular rings under a condition simulating the transmural pressure encountered in vivo in the coronary microartery. Briefly the artery rings were progressively stretched until the passive transmural pressure reached 100 mm Hg. The internal circumference was then set to a normalized value, estimated to be equivalent to 90% of the circumference at a passive transmural pressure of 100 mm Hg. This pressure was maintained throughout the experiments.

All rings were equilibrated for 30 minutes before normalization. The following protocol was used.

Effect of UW solution on EDHF-mediated relaxation in microarteries
Coronary microarterial rings were incubated with Krebs or UW solution at 4°C for 4 hours followed by washout (n = 8 in each group). With inhibition of PGI₂ and NO by indomethacin (7 µmol/L), N^G-nitro-L-arginine (_L-NNA, 300 µmol/L), and oxyhemoglobin (HbO, 20 µmol/L), EDHF-mediated relaxation was induced by bradykinin (-10 to -6.5 log M) in the rings precontracted with U₄₆₆₁₉ (10 nmol/L, in the control group) or U₄₆₆₁₉ (1 nmol/L) plus endothelin-1 (6 nmol/L, in the UW group). The reason to precontract the arteries by U₄₆₆₁₉ plus endothelin-1 in the UW group is because in this group, even after a short-time washout, the arteries were still in plegic status without comparable contraction force to U₄₆₆₁₉ in the control group. We therefore did a group of pilot experiments that demonstrated that U₄₆₆₁₉ (1 nmol/L) plus endothelin-1 (6 nmol/L) in the UW group may evoke enough contraction force comparable to the force in the control group.

Effect of ST solution on EDHF-mediated relaxation in microarteries
Group IA: moderate hypothermic (22°c) exposure for 1 hour
Two coronary microarterial rings taken from the same artery were incubated with Krebs or ST solution at 22°C for 1 hour (n = 8 in each group). After being precontracted with U₄₆₆₁₉ (10 nmol/L), EDHF-mediated relaxation was induced by bradykinin with the presence of indomethacin, _L-NNA, and HbO.

Group IIA: profound hypothermic (4°c) exposure for 1 hour
Two rings taken from the same microartery were respectively exposed to ST or Krebs solution at 4°C for 1 hour (n = 7). With the presence of indomethacin, _L-NNA, and HbO, EDHF-mediated relaxation was induced by bradykinin in the U₄₆₆₁₉ (10 nmol/L) precontracted vessels.

Group IIIA: profound hypothermic (4°c) exposure for 4 hours
In this group of experiments, one arterial ring was exposed to ST solution at 4°C for 4 hours, the other was immersed in Krebs solution as control. The EDHF-mediated relaxation was established after U₄₆₆₁₉ (1 nmol/L) plus endothelin-1 (6 nmol/L) precontraction with the presence of all the inhibitors used in group Ia and IIa. The purpose of using endothelin-1 (6 nmol/L) as well as U₄₆₆₁₉ is to match the precontraction in the UW solution experiments (see below).

Addition of EET_11,12 to UW solution on EDHF-mediated relaxation
Two rings from the same microartery were allocated into two groups. One was exposed to UW solution at 4°C for 4 hours and the other was exposed to UW solution with addition of 300 nmol/L EET_11,12. After being precontracted with U₄₆₆₁₉ (1 nmol/L) plus endothelin-1 (6 nmol/L), bradykinin-induced, EDHF-mediated relaxation was established in the presence of indomethacin, _L-NNA, and HbO.

Addition of EET_11,12 to ST solution on EDHF-mediated relaxation
Group IB: moderate hypothermic (22°c) exposure for 1 hour
Two rings taken from the same microartery were exposed to ST or EET_11,12 (300 nmol/L) supplemented ST solution at 22°C for 1 hour. The indomethacin, _L-NNA, and HbO resistant relaxation to bradykinin was observed after U₄₆₆₁₉ (10 nmol/L) precontraction.

Group IIB: profound hypothermic (4°c) exposure for 1 hour
In this group, two rings were exposed to ST with/without EET_11,12 (300 nmol/L) at 4°C for 1 hour before contraction with U₄₆₆₁₉ (10 nmol/L). The EDHF-mediated relaxation was then established with the presence of the inhibitor of PGI₂ and NO, as in group Ib.

Group IIIB: profound hypothermic (4°c) exposure for 4 hours
The protocol was similar as in group IIb, except that the constrictor was U₄₆₆₁₉ (1 nmol/L) plus endothelin-1 (6 nmol/L) and the exposure time extended to 4 hours.

Data analysis
Relaxation is expressed as the percentage decrease in isometric force induced by U46619 or U46619 plus endothelin-1. The effective concentration of bradykinin that caused 50% of maximal relaxation was defined as EC₅₀. The EC₅₀ was determined from each concentration-relaxation curve by a logistic, curve-fitting equation: E = MA^P/(A^P + K^P), where E is response, M is maximal relaxation, A is concentration, K is EC₅₀ concentration, and p is the slope parameter. From this fitted equation, the mean EC₅₀ ± SEM was calculated for each group.

Data are expressed as mean ± SEM and were analyzed with paired t test, unpaired t test, or analysis of variance (ANOVA) followed by the Scheffé F test when appropriate. Values of p less than 0.05 were considered significant.

Drugs used and their sources are as follows: bradykinin, _L-NNA, indomethacin, HbO, and EET_11,12 (Sigma, St. Louis, MO); U₄₆₆₁₉ (Cayman Chemical, Ann Arbor, MI). The _L-NNA (dissolved in distilled water) and indomethacin (dissolved in ethanol) were stored at 4°C. The solutions of U₄₆₆₁₉, HbO, bradykinin, and EET_11,12 were held frozen until needed.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Resting force and precontraction
With regard to the resting force, there were no differences among the arteries incubated with Krebs, UW, and ST solution with or without EET_11,12 either at 4°C for 1 or 4 hours or at 22°C for 1 hour (p > 0.05, Table 1). The U₄₆₆₁₉-induced precontraction (12.0 ± 1.7 mN) was not significantly affected by moderate hypothermic exposure (22°C) to ST solution although there was a slight decrease (8.1 ± 1.1 mN, p = 0.09, 95% confidence interval [CI]: -8.4 to 0.6 mN). Exposure to ST solution at 4°C for 1 hour did not alter coronary contractility to U₄₆₆₁₉ (14.0 ± 2.0 versus 11.4 ± 1.7 mN, p = 0.154, 95% CI: -6.4 to 1.3). In contrast, after storage with UW at 4°C for 4 hours, U₄₆₆₁₉ failed to contract the arteries effectively even in high concentration (data not shown). However, the combination of U₄₆₆₁₉ (1 nmol/L) and endothelin-1 (6 nmol/L) caused marked contraction to the similar extent as in the control group (12.2 ± 1.5 versus 13.6 ± 1.5 mN, p = 0.568, 95% CI: -3.5 to 6.3 mN). Compared with the precontraction in UW-treated arteries, U₄₆₆₁₉ (1 nmol/L) and endothelin-1 (6 nmol/L) evoked more contraction in ST-preserved vessels after profound hypothermic exposure for 4 hours (19.1 ± 1.2 mN, p = 0.004, versus UW, unpaired t test, 95% CI: -11.1 to -2.6 mN) and the contraction was not different from the control (21.3 ± 1.0 mN, p = 0.213, 95% CI: -1.4 to 5.8 mN). Addition of EET_11,12 did not affect the contraction in the arteries incubated with UW solution (10.8 ± 1.1 versus 12.2 ± 1.5 mN, p = 0.348, 95% CI: -1.9 to 4.8 mN). Similarly, the vascular contractility was not changed by supplementation of EET_11,12 after exposure to ST solution at 22°C for 1 hour (7.4 ± 1.2 versus 8.1 ± 1.1 mN, p = 0.575, 95% CI: -2.3 to 3.8 mN) and at 4°C for 1 hour (13.0 ± 1.9 versus 15.2 ± 2.6 mN, p = 0.189, 95% CI: -1.5 to 6.0) as well as at 4°C for 4 hours (21.1 ± 1.1 versus 19.1 ± 1.2 mN, p = 0.267, 95% CI: -6.1 to 2.0 mN).

View larger version (14K): [in this window] [in a new window]   Fig 1. Concentration-relaxation curves for bradykinin in the U46619 (1 nmol/L) plus endothelin-1 (6 nmol/L)–precontracted microarteries (see Methods) with the presence of indomethacin (7 µmol/L), NG-nitro-L-arginine (300 µmol/L), and oxyhemoglobin (20 µmol/L) after exposure to (A) Krebs solution (control; solid circles) or University of Wisconsin (UW) solution (open circles) or (B) UW solution without (solid circles) or with (open circles) 11,12-epoxyeicosatrienoic acid at 4°C for 4 hours. Data are shown as mean ± SEM. *p less than 0.05, **p = 0.01 compared with the control (n = 8). Unpaired t test in (A) and paired t test in (B).

View larger version (15K): [in this window] [in a new window]   Fig 2. Concentration-relaxation curves for bradykinin in the U46619 (10 nmol/L)–precontracted microarteries (see Methods) with the presence of indomethacin (7 µmol/L), NG-nitro-L-arginine (300 µmol/L), and oxyhemoglobin (20 µmol/L) after exposure to (A) Krebs solution (control; solid circles) or St. Thomas’ Hospital (ST) solution (open circles) or (B) ST solution without (solid circles) or with (open circles) 11,12-epoxyeicosatrienoic acid at 22°C for 1 hour. Data are shown as mean ± SEM. *p less than 0.05 compared with the control in (A); **p less than 0.01 compared with ST in (B) (n = 8, paired t test).

View larger version (13K): [in this window] [in a new window]   Fig 3. Concentration-relaxation curves for bradykinin in the U46619 (10 nmol/L)–precontracted microarteries (see Methods) with the presence of indomethacin (7 µmol/L), NG-nitro-L-arginine (300 µmol/L), and oxyhemoglobin (20 µmol/L) after exposure to (A) Krebs solution (control; solid circles) or St. Thomas’ Hospital (ST) solution (open circles) or (B) ST solution without (solid circles) or with (open circles) 11,12-epoxyeicosatrienoic acid at 4°C for 1 hour. Data are shown as mean ± SEM (n = 7, paired t test).

View larger version (12K): [in this window] [in a new window]   Fig 4. Concentration-relaxation curves for bradykinin in the U46619 (1 nmol/L) plus endothelin-1 (6 nmol/L)–precontracted microarteries (see Methods) with the presence of indomethacin (7 µmol/L), NG-nitro-L-arginine (300 µmol/L), and oxyhemoglobin (20 µmol/L) after exposure to (A) Krebs solution (control; solid circles) or St. Thomas’ Hospital (ST) solution (open circles) or (B) ST solution without (solid circles) or with (open circles) 11,12-epoxyeicosatrienoic acid at 4°C for 4 hours. Data are shown as mean ± SEM (n = 8, paired t test).

Effect of EET_11,12 in ST solution on EDHF-mediated relaxation at 4°c exposure for 1 hour
Storage of coronary microarteries at 4°C for 1 hour with ST solution decreased the sensitivity of the arteries to the vasodilator bradykinin, indicated by the right-shifted EC₅₀ value. Addition of EET_11,12 to ST solution did not show any effect on the bradykinin-induced, EDHF-mediated relaxation, either on the maximal response (82.8% ± 3.3% versus 89.2% ± 1.6%, p = 0.144, 95% CI: -2.9% to 15.8%) or the EC₅₀ (-7.86 ± 0.24 versus -8.27 ± 0.15 log M, p = 0.181, 95% CI: -1.08 to 0.25 log M; Fig 3, B).

Effect of EET_11,12 in ST and UW solutions on EDHF-mediated relaxation at 4°c exposure for 4 hours
After exposure to UW solution at 4°C for 4 hours the EDHF-mediated relaxation was reduced and this relaxation was not altered by the addition of EET_11,12 (77.2% ± 3.1% versus 79.7% ± 4.6%, p = 0.660, 95% CI: -10.2% to 15.1%; Fig 1, B). Similarly EDHF-related function was not altered in the arteries immersed in EET_11,12-containing ST solution under this temperature and exposure time (67.2% ± 2.9% versus 66.1% ± 2.7%; Fig. 1Fig 4, B).

With regard to the EC₅₀ there were no differences between the groups with or without addition of EET_11,12. It was -7.89 ± 0.18 versus -7.91 ± 0.22 log M in the UW treatment (p = 0.959, 95% CI: -0.94 to 0.90 log M) and -7.69 ± 0.15 versus -7.71 ± 0.32 log M (p = 0.973, 95% CI: -0.86 to 0.84 log M) in the ST exposure.

	Comment

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The present study demonstrates in porcine coronary microarteries that (1) EET_11,12 as an additive to ST solution may partially restore EDHF-mediated endothelial function impaired by ST exposure at 22°C for 1 hour; and (2) under the condition of profound hypothermia (4°C), addition of EET_11,12 to either ST or UW solution does not significantly alter the EDHF-mediated relaxation.

Methodology
This study used coronary microarteries (diameter ranging from 200 to 450 µm) to examine the effect of preservation solutions on the resistance arteries and the effect of supplementation of EET_11,12, a candidate for EDHF, in these solutions on the endothelial function in the coronary microcirculation. The in vitro study used a normalization procedure to set the vessels at the optimal point of their own length-tension curves to simulate the in vivo physiologic pressure.

Both ST and UW solutions have been used to preserve the heart in cardiac transplantation. The most commonly adopted preservative temperature is 4°C and the storage period is 4 hours. In addition ST solution is widely used as a cardioplegic solution under either mild or moderate hypothermia for routine intraoperative myocardial protection. During such period, the myocardial temperature is moderate (22°C) or profound hypothermic. In the present study by mimicking the clinical settings for the donor heart preservation or cardiac surgery we explored the effect of ST and UW solutions as well as EET_11,12 supplementation in these solutions on the coronary vascular system, particularly the effect on EDHF-mediated endothelial function. With consideration that the first hour of reperfusion after cardiac arrest is a critical time for the recovery of myocardial function that is further hampered by coronary dysfunction, we strictly controlled the experimental protocol, and the time from washout to the end of bradykinin-induced response was within 50 minutes.

Contractility of coronary microarteries after ST or UW solution exposure
After exposure to UW solution at 4°C for 4 hours the contraction to U₄₆₆₁₉ was inhibited markedly. Even with increased concentrations of U₄₆₆₁₉ the contraction was still too low for subsequent relaxation studies. This result is in accordance with our previous work [12]. The reduced contractility to U₄₆₆₁₉ may be due to the partial plegic status of coronary artery immediately after exposure to UW and the decreased membrane depolarization by U₄₆₆₁₉ is likely involved [12]. In order to make the subsequent relaxation comparable we used U₄₆₆₁₉ plus endothelin-1 to obtain equal contraction to the control. In contrast in the arteries exposed to ST solution at 22°C for 1 hour the inhibition of U₄₆₆₁₉-induced contraction was slight, which may be explained by the little effect on the membrane potential change caused by U₄₆₆₁₉ [12]. The noninhibitory effect of ST solution on coronary contractility was further observed after profound hypothermic (4°C) exposure for either 1 hour or 4 hours. Our results clearly indicate that neither the contraction to U₄₆₆₁₉ nor that to U₄₆₆₁₉ plus endothelin-1 was depressed by ST exposure under this temperature.

EDHF-mediated relaxation after incubation with ST or UW solution
Although different in formula, the intracellular type of organ preservation solution UW and the extracellular type of solution ST have been demonstrated to be effective in cardiac protection. However recent studies have provided evidence of the impairment of these solutions on the endothelial function [6, 7, 17–19]. Pearl and associates [7] reported that the loss of endothelium-dependent relaxation after multidose of University of Wisconsin cardioplegia is related to the reduced NO release. Mankad and colleagues [17, 18] indicated the temperature-dependent endothelial dysfunction caused by UW solution. In addition regarding the use of ST solution some investigators found that intermittent infusion of ST solution through the ischemic period is superior to a single infusion in protecting endothelial function [20].

In the present study we investigated the effect of these two types of solutions on EDHF-related endothelial function under clinically relevant temperature and time. After exposure to UW/ST solution at 4°C for 1 or 4 hours or exposure to ST solution at 22°C for 1 hour the EDHF-mediated relaxation in coronary microarteries was induced by bradykinin with the presence of indomethacin, _L-NNA, and HbO to eliminate the effect of PGI₂ and NO [21]. The results from this study indicate that exposure to UW solution at 4°C for 4 hours or exposure to ST at 22°C for 1 hour reduced EDHF-mediated relaxation and this is consistent with our previous studies in porcine as well as in human coronary arteries [10–12]. The impairment of EDHF-related function also occurred after 1 hour of profound hypothermic (4°C) exposure to ST solution, which was marked by a right-shifted EC₅₀ value. However, there were no differences between the arteries exposed to either ST or Krebs (control) solution for a prolonged period (4 hours) at 4°C, both being reduced to approximately 70%. This reduced relaxation is most likely due to the stronger contraction induced by U₄₆₆₁₉ plus endothelin-1 that made the vessel more difficult to relax.

With regard to the mechanism of reduced EDHF-mediated relaxation after exposure to ST or UW solution, based on the fact that ST and UW solutions are hyperkalemic solutions (the potassium [K⁺] concentration is 20 mmol/L and 125 mmol/L, respectively) and based on our previous studies on the detrimental effect of hyperkalemia on the EDHF-mediated function [8, 9], we proposed that the endothelial damage of ST and UW solutions with regard to EDHF is related to the high K⁺ concentration that reduces the EDHF-mediated relaxation by affecting K⁺ channels and prolonging the membrane depolarization.

Effect of EET_11,12 added to ST or UW solution on EDHF-mediated relaxation
Since the discovery of EDHF several candidates for this relaxing factor have been proposed. These include anandamide, ATP, NH₃, citrulline, hydrogen peroxide, and EETs [14, 22, 23]. As one of the regioisomers of EETs, EET_11,12 was suggested to be EDHF in the porcine coronary artery [15, 24]. However, there is also a conflicting report regarding the role of EET_11,12 in this vasculature [25]. In our recent study [16] we found that in porcine coronary microarteries in the presence of indomethacin, _L-NNA, and HbO, bradykinin-induced maximal relaxation was much more than that induced by EET_{11, 12.} This finding suggests that EET_11,12 may not be identical to EDHF in the porcine coronary microcirculation.

On the other hand we demonstrated the favorable effect of EET_11,12 on the EDHF-mediated relaxation impaired by hyperkalemia (20 mmol/L) [16]. As a further step, in the present study we examined the effect of EET_11,12 on the vasorelaxation related to EDHF when added to clinically used hyperkalemic ST and UW solutions. To explore the potential use of this substance as an additive to organ preservation solutions or cardioplegia for endothelial protection, we studied the effect of EET_11,12 supplementation under the condition simulating the clinical setting. The results show that addition of EET_11,12 to ST solution may partially restore the EDHF-mediated relaxation in coronary microarteries but the favorable effect only occurred under moderate hypothermic (22°C) exposure. The benefit of EET_11,12 supplementation in this solution disappeared with lower temperature (4°C), either for a short (1 hour) or for a prolonged (4 hour) period. Similarly the function of EDHF was not improved by EET_11,12 after UW exposure at 4°C for 4 hours. Therefore the protective effect of EET_11,12 on EDHF is temperature- and exposure time-dependent.

Clinical implications
Hyperkalemic solutions are commonly used in cardiac surgery to protect the heart in order to produce better postoperative recovery of myocardial function. The perfect heart protection should include the preservation of cardiac myocytes as well as endothelium. Furthermore perfect preservation of the endothelial function should involve all three endothelium-derived relaxing factors (EDRFs). The present study indicates that under the moderately hypothermic (22°C) condition, addition of EET_11,12 to ST solution may partially restore the EDHF-mediated relaxation but the beneficial effect is eliminated with lowered temperature. The recovery of EDHF function in EET_11,12-containing ST solution supports the use of EET_11,12 as an additive to ST cardioplegia in cardiac surgery. Although the protection of EET_11,12 is limited the results from this study provide new insights into the further development of cardioplegia and organ preservation solutions.

In summary this study demonstrates a temperature- and exposure time-dependent effect of EET_11,12. Moderate hypothermic (22°C) exposure to ST solution impairs the endothelial function related to EDHF and the addition of EET_11,12 to ST solution may partially restore the EDHF-mediated relaxation in porcine coronary microarteries. In contrast under profound hypothermia (4°C) the addition of EET_11,12 to either ST or UW solution does not improve EDHF-mediated relaxation in this vasculature. Further investigation on other candidates of EDHF or substances such as magnesium [26] is necessary to improve the effect of preservation solutions on coronary endothelial function.

	Acknowledgments

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The work described in this paper was fully supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region (Project No. CUHK4127/01M), China, and the Providence St. Vincent Medical Foundation, Portland, Oregon.

	References

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