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Right arrow Cardiac - physiology

Ann Thorac Surg 2006;81:1708-1714
© 2006 The Society of Thoracic Surgeons

Original article: Cardiovascular

Effect of Hypoxia-Reoxygenation on Endothelial Function in Porcine Cardiac Microveins

Ying-Ying Dong, MD a , Min Wu, MD a , Anthony P.C. Yim, MD a , Guo-Wei He, MD, PhD a , b , c , *

a Department of Surgery, The Chinese University of Hong Kong, Hong Kong
b Providence Heart Institute, Albert Starr Academic Center, Department of Surgery, Oregon Health and Science University, Portland, Oregon
c Wuhan Heart Institute, The Central Hospital of Wuhan, Wuhan, China

Accepted for publication December 1, 2005.

* Address correspondence to Prof He, Department of Surgery, The Chinese University of Hong Kong, Block B, 5A, Prince of Wales Hospital, Shatin, N.T., Hong Kong SAR, China (Email: gwhe{at}cuhk.edu.hk).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: The cardiac venous system possesses up to 30% of total coronary vascular resistance and the effect of hypoxia-reoxygenation (H-R) and St Thomas (ST) cardioplegic solution on the vein is unknown. We investigated the effects of H-R, with or without ST, on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in porcine cardiac microveins under clinically relevant temperatures.

METHODS: The microveins (diameter 200 to 450 µM) mounted in a myograph were subjected to hypoxia (Po2 < 5 mm Hg) for 30 minutes in Krebs solution (n = 8) or for 60 minutes in Krebs (n = 8) or in ST at 37°C (n = 8) or 4°C (n = 8), followed by 30-minute reoxygenation. The microvein was precontracted with thromboxane A2 mimetic U46619 (–7 log M) and the EDHF-mediated relaxation was induced by bradykinin (–10 to –6 log M) in the presence of indomethacin, NG-nitro-L-arginine, and oxyhemoglobin before and after H-R.

RESULTS: The maximal EDHF-mediated relaxation was significantly reduced after 30-minute hypoxia (38.7 ± 2.0% vs 61.1 ± 2.3%, n = 8, p < 0.001) or 60-minute hypoxia in either Krebs or ST at 37°C (Krebs: 27.8 ± 1.2% vs 56.6 ± 2.5%, n = 8, p < 0.001; ST: 23.8 ± 4.1% vs 57.1 ± 1.5%, n = 8, p < 0.001). The relaxation was significantly less after prolonged H-R in Krebs (p < 0.001). Incubation in Krebs or ST at 4°C also reduced the EDHF-mediated relaxation (Krebs: 25.3 ± 3.3%, n = 8, p < 0.001; ST: 29.1 ± 4.4%, n = 8, p < 0.001) and there were no significant differences between Krebs and ST regarding the relaxation at either 37°C or 4°C (p > 0.05).

CONCLUSIONS: We conclude that (1) H-R impairs EDHF-mediated relaxation in the coronary microveins with more severe injury during prolonged H-R and (2) ST does not provide protection to the EDHF-mediated relaxation impaired by H-R at either 37°C or 4°C.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Hypoxia-reoxygenation (H-R) (or ischemia-reperfusion) injury in the heart involves both cardiac myocytes and coronary vessels (the vascular smooth muscle and the endothelium). The injury to the coronary vessels may change the coronary vascular tone and affect the coronary flow during the reperfusion period. Reduction of the coronary flow, in addition to the ischemia-reperfusion injury to the cardiac myocytes, may cause myocardial dysfunction, resulting in mortality and morbidity in cardiac surgery. In recent years studies have been focused on H-R injury to the coronary circulation [1, 2]. It has been demonstrated that both nitric oxide (NO) [3, 4] and endothelium-derived hyperpolarizing factor (EDHF) [5, 6] mediated relaxations may be affected by H-R injury in coronary arteries. However, the effect of H-R on endothelial function in the coronary venous system has not been studied.

Cardioplegia was designed to protect the heart from ischemia-reperfusion injury in combination with hypothermia or normothermia. Currently blood cardioplegia is the method most frequently used worldwide. Blood cardioplegia is composed of blood and crystalloid cardioplegic solution, usually St Thomas (ST) cardioplegic solution that is mixed in a 4:1 ratio with blood [7]. In addition, crystalloid cardioplegia such as ST is still used alone in certain units due to its effectiveness and to economic reasons. Further, ST has also been used in donor heart preservation under hypothermia for transplantation and has been demonstrated to be effective in protecting the myocardium against ischemia [8]. In ST and other depolarizing cardioplegic solutions, hyperkalemia (potassium at high concentrations) as the key component 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 [7]. However, studies also showed that cardioplegia damages coronary endothelial function [9, 10]. Our previous studies further demonstrated that in both large and micro coronary arteries the EDHF-mediated function is impaired by hyperkalemic solutions such as ST [11–14].

Endothelium-dependent relaxation is known to be due to a variety of different endothelium-derived relaxing factors. These include NO [15, 16], Prostacyclin (PGI2) [17], and EDHF. Both NO and PGI2 are able to hyperpolarize the membrane of the vascular smooth muscle cell through various mechanisms [18] but when the role of NO and PGI2 are completely inhibited, the endothelium-dependent hyperpolarization still exists [18–20]. This hyperpolarization is attributed to the third endothelium-derived relaxing factor; ie, EDHF. The nature of EDHF has not been identified [18, 19, 21, 22] although epoxyeicosatrienoic acids [14], anandamide, the potassium ion, hydrogen peroxide, citrulline, NH3, and nucleotides (adenosine triphosphate) [22] have been suggested to be EDHF. The NO and EDHF are thought to be the two major mechanisms in the endothelium-smooth muscle interaction, which is particularly important in maintaining adequate vascular tone. Further, it has been demonstrated that EDHF itself plays a role in blood flow homeostasis in both conduit and resistance vessels under the physiologic states, and may be a crucial compensatory or reserve mechanism for the maintenance of nutritive organ blood flow after inhibition or impairment of the NO/PGI2 pathway in the coronary microarteries [19, 21].

The coronary venous system (cardiac veins) collects one-third blood of the coronary circulation and is considered an important site for the blood-tissue exchange of water and nutrients, as well as a possible determinant of ventricular distensibility. It is suggested that the coronary vein is of resistance to coronary flow under conditions of arteriolar dilatation [22]. Although the coronary venous system possesses 7% of total coronary vascular resistance in the resting condition, its contribution greatly increases to 31% under the vasodilated condition and acts as the potential modulation of coronary blood flow.

We have recently demonstrated that both NO and EDHF are involved in the regulation of the coronary vascular tone in the coronary venous system [23]. In contrast, the role of PGI2 in the venous system has not been well-established [23] and this is mainly related to the common agreement that NO and EDHF are the two major determinants in the coronary resistance [18–20]. When the coronary venous system contributes a greater fraction of total coronary resistance under pathological conditions, for example when the heart is subjected to ischemia-reperfusion injury, the endothelial response of cardiac veins may become more important. Further, during cardiac surgery, when the retrograde cardioplegia is delivered from the coronary sinus through the cardiac venous system, the protection of the endothelial function in the venous system may prove to be an important clinical issue. The present study was therefore designed to examine the effects of H-R with or without cardioplegia under normothermia or hypothermia on EDHF-mediated relaxation in porcine cardiac veins.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
All experiments were in accordance with institutional guidelines. This investigation conformed to the "Guide for the Care and Use of Laboratory Animals," published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Preparation and Mounting of Microvein Rings
From a local slaughterhouse, when the hog was slaughtered, fresh porcine hearts were immediately placed in a container filled with cold (4°C) Krebs solution and transferred to the laboratory. Upon receipt of the heart, intramyocardial coronary microveins (usually adjacent to the tertiary branches of the left anterior descending artery) in diameters of 200 to 450 µm were carefully dissected and removed under a microscope, with care taken to protect the endothelium. The vessels were cleaned of fat and connective tissue and cut into cylindrical rings of 2-mm length under a microscope. The rings were guided a suitable length through the lumen by a pair of stainless steel wires (40 µm in diameter). One wire was fixed tightly on a jaw in a 4-channel myograph (model 610M; JP Trading, Aarhus, Denmark) [24, 25], and the other wire was passed lightly through the vascular lumen and anchored onto another jaw. These two wires were attached to a force transducer or to a micrometer, respectively. An adjustable micrometer can pull the jaws apart and stretch the vein between the two parallel wires. A calibrated force transducer was used to measure the force, with the output shown on a computer screen and printed in a printer. Data were digitized and stored in the computer. Two organ chamber arrangements in the same myograph were run concurrently.

The Krebs solution had the following composition (in mM): Na+, 144; K+, 5.9; Ca2+, 2.5; Mg2+, 1.2; Cl–, 128.7; HCO3 –, 25; SO4 2–, 1.2; H2PO4 –, 1.2; and glucose, 11.

Normalization
All rings were equilibrated for 45 minutes before and after normalization in the myograph. The normalization had the following procedures: the microvenous rings were progressively stretched until the passive transmural pressure reached 30 mm Hg, and then the pressure was immediately released. The computerized program determines the length-tension exponential curve for each ring and gives the internal circumference and diameter at a pressure of 30 mm Hg [24, 25]. The internal circumference was then set to the value, estimated to be equivalent to 90% of the circumference at a passive transmural pressure of 30 mm Hg [26, 27]. This pressure was maintained throughout the experiments.

Hypoxia
During normalization or relaxation studies, the solution was aerated with a gas mixture of 95% O2 and 5% CO2 (normoxia, the partial pressure of oxygen [PO 2] was more than 200 mm Hg). Hypoxic condition was induced by switching bubbling gas from 95% O2 and 5% CO2 to 95% N2 and 5% CO2 (hypoxia, PO 2< 5 mm Hg) in the specially designed myograph chamber for hypoxia experiments by our workshop. The myograph chamber was closely covered and well-sealed by a plastic cover with two holes. One hole was used to insert a N2 delivery tube and the other, for inserting a PO 2 probe that could continuously measure the PO 2 in the chamber solution. The PO 2 was measured by an oxygen meter (model 781; Strathkelvin Instrument, Glasgow, Scotland).

Experimental Protocol: Bradykinin (BK)-Induced, EDHF-Mediated Relaxation
In each relaxation group, NG-nitro-L-arginine (L-NNA, 300 µM, a nitric oxide synthase inhibitor), oxyhemoglobin (HbO, 20 µM), a NO scavenger, and indomethacin (Indo, 7 µM), a cyclooxygenase inhibitor, were added into the chamber for 30 minutes. The U46619 (100 nM), a thromboxane mimetic, was then added to contract the rings. When the contraction reached a stable plateau (usually 15 minutes), cumulative concentration-relaxation curves to BK (–10 to –6 log M) were established. From each group of rings a mean concentration-relaxation curve was constructed. To test whether the EDHF-mediated relaxation to BK is repeatable after the normal incubation period (90 minutes) in our experiments, in 8 rings under normalization in the same vein, before and after wash procedure with Krebs solution and equilibrium for 90 minutes, the BK-induced EDHF-mediated relaxation in U46619-precontraction was induced.

Effect of H-R on EDHF-Mediated Relaxation
Group IA: Thirty-Minute Hypoxia, Followed by 30-Minute Reoxygenation in Krebs Solution at 37°C. The rings of microvein were incubated in Krebs solution at 37°C and subjected to hypoxia (PO 2 < 5 mm Hg) for 30 minutes, followed by 30-minute-reoxygenation (n = 8). In this group of experiments, before (as the control) and after H-R, the EDHF-mediated relaxation was induced by BK.

Group IB: Sixty-Minute Hypoxia, Followed by 30-Minute Reoxygenation in Krebs Solution at 37°C. The protocol was similar to group IA, except that the hypoxia was prolonged to 60 minutes (n = 8).

Combined Effect of H-R and ST Solution on EDHF-Mediated Relaxation
Group IIA: Sixty-Minute Hypoxia in ST at 37°C, Followed by 30-Minute Reoxygenation at 37°C. The rings were incubated in Krebs solution (as the control) at 37°C and the EDHF-mediated relaxation was induced by BK. After this procedure, the rings were washed with Krebs solution and then incubated in ST at 37°C, subjected to hypoxia for 60-minutes and followed by 30-minute reoxygenation (n = 8). The EDHF-mediated relaxation was then induced by BK again.

Group IIB: Sixty-Minute Hypoxia in ST at 4°C, Followed by 30-Minute Reoxygenation at 37°C. Two rings from the same microvein were allocated into two groups (n = 8 in each group). One was incubated in ST and the other in Krebs (as the control) at 4°C for 60-minute hypoxia, followed by 30-minute reoxygenation. The EDHF-mediated relaxation to BK was established.

The ST had the following composition (in mM): Na+, 138; K+, 20; Ca2+, 2.7; Mg2+, 16; Cl–, 157; HCO3 –, 8; lactate, 28; and procaine, 1. The osmolarity is 370.

Data Analysis
Relaxation is expressed as the percentage decrease in isometric force induced by U46619. Mean maximal relaxation for each group was calculated from the maximal relaxation of different rings induced by BK. The effective concentration (EC) 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 = MAP/ (AP + KP), where E is response, M is maximal relaxation, A is concentration, K is EC50 concentration, and P is the slope parameter. A computerized program was used for the curve fitting. From this fitted equation, the mean EC50 ± standard error of the mean (SEM) was calculated for each group.

Statistical Analysis
All statistical analysis was performed with SPSS9.0 software (SPSS Inc, Chicago, IL). 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. For comparisons between curves that have multiple points related to multiple concentrations of a substance, repeated measurement of 2-way ANOVA was used to determine the significance. Values of p less than 0.05 were considered significant.

Drugs
Drugs used and their sources are as follows: BK, L-NNA, Indo, HbO (Sigma, St Louis, MO); and U46619 (Cayman Chemical, Ann Arbor, MI). The L-NNA (dissolved in distilled water) and Indo (dissolved in ethanol) were stored at 4°C. The solutions of U46619, HbO, and BK were held frozen until needed. The ST was purchased from David Bull Laboratories (Mulgrave, Victoria, Australia).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Resting Force
With regard to the resting force, there were no significant differences between the veins incubated with Krebs and ST solution either at 37°C or at 4°C for H-R (p > 0.05). Table 1 gives the details of the resting force in these microveins.


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  		Table 1. Resting Force and U46619-Induced Contraction Before and After Hypoxia-Reoxygenation (H-R) in Each Group in Porcine Cardiac Veins
 
U46619-Induced Precontraction
The concentration of U46619 varied from –7 log M to –6.5 log M to maintain a similar stable contraction force between the control and H-R condition in each group (p > 0.05). Table 1 gives the details of the U46619-induced precontraction before and after H-R in each group.

Partial Pressure of Oxygen in Hypoxia
The normal oxygen pressure (> 200 mm Hg) was quickly lowered to 30 mm Hg in 5 minutes and further lowered to less than 5 mm Hg in another 10 minutes, and maintained a low level throughout the experiment. In the 30-minute-hypoxia group, the partial pressure of oxygen was 4.5 ± 0.6 mm Hg and in the 60-minute-hypoxia group it was 2.2 ± 0.3 mm Hg (Fig 1).


Figure 1
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  		Fig 1. Partial pressure of oxygen (Po2) in hypoxia. Data are shown as mean ± standard error of the mean.

 
BK-Induced, EDHF-Mediated Relaxation in Microveins
The BK-induced relaxation was 91.7% ± 2.3%. The EDHF-mediated relaxation in the presence of Indo, L-NNA, and HbO was significantly reduced to 59.0% ± 2.3% (p < 0.001, 95% confidence interval [CI]: 25.2% to 40.2%). However, the EC50 value was not significantly affected (–7.76 ± 0.09 vs –8.05 ± 0.14 log M in the control group, p = 0.094, 95% CI: –0.06 to 0.65 log M).

After wash procedure with Krebs solution and equilibrium for 90 minutes, the BK-induced EDHF-mediated relaxation in U46619-precontraction remained unchanged (before: 62.7% ± 5.5%; after: 59.7 ± 5.1%, p = 0.5, 95% CI: 11.9% to 21.3%; 2-way ANOVA: p = 0.4, n = 8; Fig 2). The EC50 value was also unchanged (before: –7.96 ± 0.09 log M; after: –7.76 ± 0.12, p = 0.991, 95% CI: –0.33 to 0.33 log M).


Figure 2
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  		Fig 2. Concentration-relaxation curves for bradykinin in the U46619 (100 nM) precontracted cardiac veins before (open circles) and after (control; solid circles) 90-minute incubation in Krebs solution in the presence of indomethacin (7 µM), NG-nitro-L-arginine (300 µM), and oxyhemoglobin (20 µM). Data are shown as mean ± standard error of the mean (n = 8; p = 0.4; 2-way analysis of variance).

 
EDHF-Mediated Relaxation After H-R in Krebs Solution at 37°C
Group IA: Thirty-Minute Hypoxia, Followed by 30-Minute Reoxygenation in Krebs Solution at 37°C. In control rings incubated in Krebs solution in the presence of Indo, L-NNA, and HbO, BK induced a maximal relaxation of 61.1% ± 2.3% with EC50 of –8.08 ± 0.18 log M. After H-R, the BK-induced maximal relaxation was significantly reduced to 38.7% ± 2.0% (p < 0.001, 95% CI: 17.3% to 27.6%; 2-way ANOVA: p = 0.006; n = 8; Fig 3A) although there was no change on the EC50 (–7.78 ± 0.16 log M; p = 0.23, 95% CI: –0.84 to 0.24 log M).


Figure 3
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  		Fig 3. Concentration-relaxation curves for bradykinin in the U46619 (100 nM) precontracted cardiac veins (see Material and Methods) with the presence of indomethacin (7 µM), NG-nitro-L-arginine (300 µM), and oxyhemoglobin (20 µM); (A) (group IA) before (control; solid circles) and after (H-R; open circles) 30-minute hypoxia followed by 30-minute reoxygenation in Krebs solution at 37°C; or (B) (group IB) before (control; solid circles) and after (H/R; open circles) 60-minute hypoxia followed by 30-minute reoxygenation in Krebs solution at 37°C. Data are shown as mean ± standard error of the mean. **p less than 0.01 compared with the control (n = 8, 2-way analysis of variance); p less than 0.01 compared with H/R (30/30) in (A). (H/R = hypoxia reoxygenation.)

 
Group IB: Sixty-Minute Hypoxia, Followed by 30-Minute Reoxygenation in Krebs Solution at 37°C. Exposure to hypoxia for 60 minutes in Krebs solution at 37°C followed by 30-minute reoxygenation significantly decreased BK-induced, EDHF-mediated relaxation from 56.6% ± 2.5% to 27.8% ± 1.2% (p < 0.001, 95% CI: 20.6% to 36.9%). The EC50 of BK was right shift from –8.36 ± 0.13 to –8.07 ± 0.14 log M (p = 0.043, 95% CI: –0.56 to –0.01 log M; 2-way ANOVA: p = 0.001, n = 8; Fig 3B). Further, the reduction of the relaxation was more severe during prolonged H-R (27.8% ± 1.2% vs. 38.7% ± 2.0%, p < 0.001, 95% CI: –15.8% to –5.9%).

EDHF-Mediated Relaxation After H-R in ST at Different Temperatures
Group IIA: Sixty-Minute Hypoxia in ST, Followed by 30-Minute Reoxygenation at 37°C. Exposure to ST solution at 37°C for 60-minute hypoxia significantly decreased EDHF-mediated relaxation from 57.1% ± 1.5% to 23.8% ± 4.1% (p < 0.001, 95% CI: 24.1% to 42.5%; 2-way ANOVA: p = 0.001, n = 8; Fig 4A) with unchanged EC50 (–8.02 ± 0.14 vs –8.13 ± 0.10 log M in the control group, p = 0.4, 95% CI: –0.19 to 0.41 log M). Comparing ST with Krebs solution, there were no significant differences regarding the relaxation (23.8% ± 4.1% vs 27.8% ± 1.2%, p = 0.37, 95% CI: –13.2% to 5.2%).


Figure 4
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  		Fig 4. Concentration-relaxation curves for bradykinin in the U46619 (100 nM) precontracted microveins (see Material and Methods) with the presence of indomethacin (7 µM), NG-nitro-L-arginine (300 µM), and oxyhemoglobin (20 µM); (A) (group IIA) before (control; solid circles) and after (H/R; open circles) 60-minute hypoxia followed by 30-minute reoxygenation in ST at 37°C; or (B) (group IIB) 60-minute hypoxia followed by 30-minute reoxygenation in Krebs (H/R; solid circles) or in ST (H/R; open circles) at 4°C. Data are shown as mean ± standard error of the mean. **p less than 0.01 compared with the control (n = 8, 2-way analysis of variance). (H/R = hypoxia reoxygenation; ST = St. Thomas solution.)

 
GROUP IIB: Sixty-minute hypoxia in ST, Followed by 30-Minute Reoxygenation at 4°C. Incubation of the vein in Krebs or ST at 4°C for 60-minute hypoxia also reduced the EDHF-mediated relaxation to BK (Krebs: 25.3% ± 3.3%, p < 0.001; ST: 29.1% ± 4.4%, p < 0.001). However, there were no significant differences between Krebs and ST regarding the relaxation (p = 0.5, 95% CI: –16.08% to 8.42%; 2-way ANOVA: p = 0.6, n = 8; Fig 4B). With regard to the EC50, there were also no differences (–8.31 ± 0.22 vs –8.19 ± 0.21 log M in the control group, p = 0.70, 95% CI: –0.55 to 0.80 log M). Further, there were no significant differences regarding the relaxation after exposure to ST at either 37°C or 4°C (p = 0.4, 95% CI: –7.99% to 18.65%).


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The present study demonstrates, in porcine cardiac veins, that (1) H-R impairs EDHF-mediated relaxation and the injury is more severe during prolonged H-R and (2) under either profound hypothermia (4°C) or normothermia (37°C), the alteration of the EDHF-mediated relaxation by H-R is not eliminated by ST.

Methodology
The present study used cardiac veins (diameter ranging from 200 to 450 µm) to examine the effect of H-R with or without cardioplegic solutions on the endothelial function. 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 physiological pressure.

As mentioned above, although blood cardioplegia is standard practice in most cardiac surgery units, the basis of blood cardioplegia is ST. In addition, in a few units ST alone is still used as a crystalloid cardioplegia and as a preservation solution for the donor heart in cardiac transplantation. As a cardioplegic solution, ST is used under either hypothermia or normothermia and as a preservation solution it is used at 4°C. During this period, the myocardial temperature is moderately or profoundly hypothermic. The present study mimics the clinical settings on this aspect and we explored the combined effect of ST and H-R on the coronary venous system, particularly the effect on EDHF-mediated endothelial function.

BK-Induced, EDHF-Mediated Relaxation
In this study, BK was used to evoke the endothelium-dependent relaxation. Bradykinin, through a receptor mechanism, increases the intracellular Ca2+ concentration and stimulates the release of endothelium-derived relaxing factors (NO, PGI2, and EDHF) in the endothelial cell. Because the other two main components (NO and cyclooxygenase pathways) were abolished by L-NNA, HbO, and Indo [27], the residual relaxation is through the non-NO and non-cyclooxygenase mechanism (ie, related to EDHF). In the present study, in the rings pretreated with L-NNA, Indo, and HbO, the relaxation by BK was about 59.0% suggesting that, in the presence of L-NNA, HbO, and Indo, EDHF plays an essential role in endothelium-dependent relaxation in the porcine cardiac veins. This is in accordance with the results from our previous reports [23].

Effects of H-R on EDHF-Mediated Relaxation
The cardiovascular diseases that are initiated by local or systemic tissue ischemia remain the main cause of death [28]. It has become apparent that reperfusion, the restoration of blood flow after a period of ischemia, can place ischemia organs at risk of further cellular necrosis and thereby limit the recovery of function. The microvasculature, particularly the endothelial cells lining blood vessels, is vulnerable to the deleterious consequences of ischemia and reperfusion. Ku [29] showed that a 90-minute period of ischemia followed by 1-hour reperfusion was associated with a decreased endothelium-dependent relaxation, in response to thrombin in canine coronary arteries. Later studies found that NO-mediated relaxation was impaired by H-R. On the other hand, despite of the fact that the effect of H-R on membrane hyperpolarization was studied [30], the effect on EDHF-mediated relaxation in the coronary venous system has not been reported.

In the present study, after 30-minute hypoxia followed by 30-minute reoxygenation, EDHF-mediated (L-NNA, HbO, and Indo-resistant) relaxation was significantly reduced and this effect was more significant after 60-minute hypoxia followed by 30-minute reoxygenation. These results showed that the EDHF-mediated relaxation is impaired by the H-R in the cardiac veins and the injury is more severe during prolonged H-R.

In the present study, the U46619-induced contraction was slightly affected by H-R. To fairly compare the relaxation, a similar precontraction force was essential and this was achieved in the present study by adding a higher concentration of U46619 (between 100 and 1,000 nM) after H-R to reach a similar precontraction (Table 1).

EDHF-Mediated Relaxation After H-R in ST at Different Temperatures
The extracellular type of preservation solution ST was initially designed to eliminate the H-R injury of myocardium and has been demonstrated to be effective in cardiac protection. However, recent studies have provided evidence of the impairment of this solution on the endothelial function [26, 31–32]. The combined effect of H-R and ST on the EDHF-related endothelial function under clinically relevant temperatures remains unknown and therefore this became one of the aims of the present study. The results from this study indicate that exposure to ST solution at 4°C or at 37°C for 60-minute hypoxia reduced EDHF-mediated relaxation, suggesting that ST does not provide protection to the EDHF-mediated relaxation impaired by H-R. Interestingly, the detrimental effect of ST on EDHF-mediated relaxation discovered in coronary arteries without ischemia-reperfusion injury [12–14] was not seen in the cardiac microvein under H-R (compare Figs 3 and 4, open circles). However, the detrimental effect of H-R on the microartery [33] is also seen in the microvein in the present study.

Clinical Implications
The coronary venous system collects one-third blood of the coronary circulation and is considered to be an important site for the blood-tissue exchange of water and nutrients. Apart from the physiological role of the coronary venous system, the understanding of the coronary venous system has direct pathological and clinical implications. During cardiac surgery, the heart is inevitably subjected to ischemia and subsequent reperfusion injury. Hyperkalemic solutions are commonly used in cardiac surgery to protect the heart in order to produce better postoperative recovery of myocardial function. The solution is usually delivered from the coronary artery (antegrade) and drains to the right atrium from the coronary venous system. However, retrograde perfusion of cardioplegic solutions from the coronary venous system through the coronary sinus in the right atrium to the cardiac muscle is indicated under a number of situations; for example, in coronary artery diseases or in aortic or aortic valve surgery. The present study indicates that under the normothermia and hypothermic condition the EDHF-mediated endothelial function of coronary microveins is damaged by H-R and this damage cannot be eliminated by ST.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This study was supported in full by grants from the Research Grant Council of the Hong Kong Special Administrative Region (Projects CUHK4127/01M & CUHK4383/03M), China, and the Providence St. Vincent Medical Foundation, Portland, Oregon.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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