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Ann Thorac Surg 2005;80:757-767
© 2005 The Society of Thoracic Surgeons

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Review

Effect of Cardioplegic and Organ Preservation Solutions and Their Components on Coronary Endothelium-Derived Relaxing Factors

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

^_* Address reprint requests 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 Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
Cardioplegic (and organ preservation) solutions were initially designed to protect the myocardium (cardiac myocytes) during cardiac operation (and heart transplantation). Because of differences between cardiac myocytes and vascular (endothelial and smooth muscle) cells in structure and function, the solutions may have an adverse effect on coronary vascular cells. However, such effect is often complicated by many other factors such as ischemia-reperfusion injury, temperature, and perfusion pressure or duration. To evaluate the effect of a solution on the coronary endothelial function, a number of points should be taken into consideration. First, the overall effect on endothelium should be identified. Second, the effect of the solution on the individual endothelium-derived relaxing factors (nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor) must be distinguished. Third, the effect of each major component of the solution should be investigated. Lastly, the effect of a variety of new additives in the solution may be studied. Based on available literature these issues are reviewed to provide information for further development of cardioplegic or organ preservation solutions.

	Introduction

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
Ischemia-reperfusion injury is the major problem in open-heart operations, including heart transplantation. The goal of cardioplegia was to eliminate this injury. Cardioplegic (and organ preservation) solutions were initially designed to protect the myocardium (cardiac myocytes) from ischemia-reperfusion injury. Subsequently, researchers found that, despite their protective effect on cardiac myocytes, these solutions may impair coronary vascular endothelial cells. These opposite effects are due to the differences in structure and function between cardiac myocytes and coronary vascular (endothelial and smooth muscle) cells.

A number of issues should be taken into account in the investigations regarding the effect of cardioplegic/organ preservation solutions on the coronary endothelial function. First, in the whole heart, the overall effect of these solutions is a result of the combination of the effect on the cardiac myocytes and the effect on the coronary endothelium. Second, even in studies focused on endothelial function, the effect of the solutions has often been mixed with ischemia-reperfusion injury and other factors. The goal of this review is to discuss the effect of the solutions and their components on the endothelium to provide information for further development of protective solutions. In addition, this review emphasizes the effect of cardioplegic/organ preservation solutions on individual endothelial factors.

	Literature Selection Methods

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
A comprehensive English literature search through PubMed was performed with closing date of June 28, 2004. Multiple keywords were used for searching as follows: [(endothelium OR endothelial function OR EDRF)] AND [(cardiac operation OR heart transplantation)] OR [(cardioplegia OR cardioplegic solution OR organ preservation solution OR hyperkalemia)] AND [(cardioplegic additives OR supplementation OR adjunct)]. The focus of the literature selection was original studies. Book chapters or references before 1970 were occasionally cited for historic or technical aspects.

	Cardioplegic and Organ Preservation Solutions

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
Crystalloid Cardioplegic Solutions
The introduction of cardioplegia for myocardial protection was a breakthrough in cardiac operations. Melrose and colleagues [1] in 1955 proposed the concept of chemical heart arrest with the mixture of blood and 2.5% solution of potassium citrate with the concentrations of potassium varying from 9 to 245 mEq/L in experimental studies, and the method was soon used clinically by Effler in 1957 [2]. However, the extremely high potassium concentrations in the Melrose solution were later demonstrated to induce myocardial necrosis and then abandoned. Bretschneider [3] reported to use low-sodium, mildly hyperkalemic (7 mEq/L), calcium-free procaine solution in Germany 1975, and Gay and Ebert [4] found that potassium crystalloid cardioplegia at a potassium concentration of 24 mEq/L increased recovery after ischemia. The effectiveness of moderately hyperkalemic potassium cardioplegia was then supported and was widely demonstrated by excellent clinical results in the 1970s. One of the widely used cardioplegic solutions was St. Thomas Hospital (ST) solution [5]. During the 1980s and early 1990s, hundred of studies of cardioplegia were published.

Due to its superb operating conditions and its effectiveness in myocardial preservation, cold cardioplegic arrest became the most commonly used method until blood cardioplegia became popular. During this period, a number of crystalloid cardioplegic solutions were developed, including histidine-tryptophan-ketoglutarate (HTK) solution [6]. Although the composition varies in different solutions, the pharmacologic principles involved in myocardial protection are similar [7, 8]. First, immediate arrest induced by K⁺, Mg²⁺, procaine, profound hypocalcemia, or a combination of these modalities lowers energy demand to avoid depletion of the high-energy phosphate pool and conserves the myocardial energy reserves that can be used during the period of ischemia to maintain ionic and metabolic homeostasis and, consequently, leads to the better tolerance to ischemia. Second, reduction of myocardial temperature during arrest or storage lowers metabolic rate during ischemia. Third, supplementation of glucose and amino acid (ie, glutamate or aspartate) enhances anaerobic or aerobic energy production (or both) with a buffer such as tris (hydroxymethyl) aminoethane, bicarbonate, phosphate, and histidine and optimizes the small energy output of anaerobic glycolysis during ischemia. Fourth, membrane stabilization provided by exogenous additives (ie, steroids, procaine, oxygen radical scavengers, Ca²⁺ antagonists), hypocalcemia, or enrichment of Mg²⁺ may counteract the myocardial injury during ischemia and reperfusion. Lastly, addition of an osmotic agent such as mannitol and adjustment of infusion pressure limit myocardial edema and prevent the occurrence of "no-flow phenomenon" [7, 8].

Blood Cardioplegia
Cold oxygenated blood with potassium as the arresting agent was described in the 1970s [9–11]. The presence of red cells and plasma proteins as well as other blood components provides a strong capacity for oxygen carrying, buffering, and antioxidation [11]. The most commonly used method is mixing the crystalloid cardioplegia with blood in a ratio of 1:4 [8, 11]. The final delivery concentrations of potassium remain at 20 to 25 mEq/L for the initial prompt arrest of the heart and 8 to 10 mEq/L for the subsequent delivery of the solution [8]. The final concentration of Mg²⁺ and procaine is less than that in crystalloid cardioplegic solutions such as ST. For example, the concentration of Mg²⁺ is 5 to 6 mEq/L in cold blood cardioplegia and 10 to 12 mEq/L in warm blood cardioplegia in the Buckberg blood cardioplegic solution [8]. Since the 1980s, blood cardioplegia has become the choice of myocardial protection in most cardiac units, although crystalloid cardioplegia is still used today because of either the preference of the surgeon or economic concerns.

Organ Preservation Solutions Including Crystalloid Cardioplegic Solutions Used for Heart Preservation
Preservation of the heart with organ preservation solutions has been demonstrated as a useful strategy in heart transplantation. These solutions protect the heart by controlling the risk factors involved in ischemic-reperfusion injury with similar principles of cardioplegic solutions, including reduction of oxygen demand, cellular swelling, extracellular edema, and Ca²⁺ overload [12]. In fact, commonly in cardiac operations one solution serves as both a cardioplegic and heart preservation solution. The composition of various crystalloid cardioplegic or heart preservation solutions are listed in Table 1.

St. Thomas Hospital solution
This crystalloid cardioplegic solution is an extracellular type of solution [5]. The subsequent discovery of the efficacy of St. Thomas Hospital (ST) solution in heart storage promoted its application in the field of heart preservation for transplantation [17, 18]. St. Thomas Hospital solution was widely used for cardioplegia in the 1970s and the 1980s until blood cardioplegia became popular. This solution is still used as crystalloid cardioplegia in some hospitals and also used as a cardioplegic additive for blood cardioplegia. Composition of ST solution varies between its two forms—the original ST solution and ST solution No. 2 (Plegisol). The composition of the ST solution used in our clinical and experimental studies is listed in Table 1.

Histidine-Tryptophan-Ketoglutarate solution
Similar to ST solution, HTK solution was initially developed for cardioplegia. Use of HTK solution was expanded to preservation of the liver, kidney, and pancreas as well as the heart and lung [19–23]. The protective effect of HTK solution is based on the high buffering capacity provided by histidine and its low electrolyte content, thus restricting tissue acidosis induced by ischemia [24].

Celsior solution
Celsior solution was developed for heart preservation not only as a storage medium but also as a perfusion fluid during initial donor-heart arrest, poststorage graft reimplantation, and early reperfusion [25]. Celsior formulation prevents cell swelling (by mannitol and lactobionate), oxygen-derived free radical injury (by reduced glutathione, histidine, and mannitol), and contracture by enhancement of energy production (glutamate) and limitation of calcium overload (high magnesium content, slight degree of acidosis) [25].

	Overall Effect of Cardioplegic and Organ Preservation Solutions on Coronary Endothelial Function

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
Coronary circulation plays the key role in myocardial perfusion. Injury to the coronary circulation may change the coronary resistance and therefore affect the coronary flow. The reduction of coronary flow may damage myocardial perfusion. During cardiac arrest or heart preservation, the coronary vascular endothelium is immersed in the solution. Although the preservation effect of crystalloid cardioplegic or organ preservation solutions on the endothelial function has been reported [26–28], numerous studies provided evidence for endothelial damage after exposure to these solutions. Histologic examination and cell culture approach also revealed that crystalloid hyperkalemic cardioplegic solutions impair the vascular endothelium and reduce the ability of coronary endothelial cells to replicate [29, 30].

Factors Involved in the Effect of Cardioplegic and Organ Preservation Solutions
As mentioned before, the so-called effect of solutions on coronary endothelium is often mixed with the effects due to other factors combined with the cardioplegia procedure. That is, damage (or protection) to the endothelium may result from not only the solution per se, but also from other components of the procedure. Theoretically, these factors may include the following aspects:

1 Direct action of the solutions due to their intrinsic characteristics (the components of the solution)

2 Adjuncts to the cardioplegic procedure such as hypothermia or the infusion pressure or duration acting both as independent factors and through their interaction with cardioplegic solutions

3 The effect of ischemia-reperfusion injury

4 Other factors involved in isolated working heart models or in vivo models when these models are used to study endothelial function.

Due to the influence of these factors, studies suggested that crystalloid-perfused isolated heart is an inappropriate model to interpret the interaction between coronary flow reserve and ischemic injury [33]; use of vascular bed preparation was recommended in the assessment of vascular function [34].

Taken together, the "true" effect of the solutions on the endothelium should be carefully distinguished from other factors in order to identify the possible damaging effect due to the intrinsic characteristics of the solution.

Hyperkalemia on Endothelium-Regulated Coronary Flow or Endothelium-Dependent Relaxation
In Langendorff preparation of the rat heart [35] and an in vivo porcine model of cardiopulmonary bypass [36–38], cold hyperkalemic (25 or 40 mmol/L K⁺) cardioplegia reduced vasodilatory response to 5-hydroxytryptamine [35], adenosine phosphate, calcium ionophore A23187 [36–38], bradykinin, and the -agonist BHT-920 [38]. However, ischemia-reperfusion injury was another factor in these studies. The effect of the time of cold ischemic storage with cardioplegic solution was demonstrated [39, 40].

Comparison Between Crystalloid and Blood Cardioplegia
Compared with crystalloid cardioplegia, blood cardioplegia preserves the coronary endothelium [38, 40, 41] and prevents endothelial cells from deformity [40]. The impact of the ischemic period was also confirmed [40, 41]. However, with prolonged ischemic intervals, the preservative effect of intermittent antegrade warm blood cardioplegia was lost [40].

Effect of University of Wisconsin, Histidine-Tryptophan-Ketoglutarate, and Celsior Solutions
Numerous studies regarding the effect of heart preservation solutions on the endothelial function have been published. These experimental studies were performed in either hearts or coronary arteries of rat or piglet, aorta of rabbits, endothelial cells of bovine aorta or human saphenous or umbilical veins, but rarely in the coronary arteries of large mammals or humans. The results are largely conflicting because of the variety of experimental methods including the differences of the models, preservation duration and solution temperature, and the index for endothelial function. The results associated with UW and HTK solutions were well summarized in a recent review by Parolari and colleagues [42] and will not be repeated in this review. It has been reported that loss of endothelium-dependent vasodilatation and nitric oxide (NO) release on bradykinin stimulation after myocardial protection with UW solution in neonatal piglet heart [43].

Fewer studies have been conducted on the effect of Celsior solution. Kevelaitis and associates [44] reported protective effect of reduced glutathione on endothelial function of coronary arteries subjected to prolonged cold storage. Use of Celsior solution for induction of cardioplegia and storage better preserved endothelium-dependent G-protein-mediated relaxation compared with the other arrest and preservation strategies using blood and crystalloid solutions in heart transplantation [45]. Further, a comparison of UW and Celsior solutions revealed that total ischemic time correlated with impaired endothelial function in the Celsior but not in the UW group [46]. Again, in most of these studies, the effect of the solution was combined with other factors.

	Possible Mechanisms Underlying the Damage of Cardioplegia and Organ Preservation on Endothelial Function

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
Possible mechanisms underlying the damage of cardioplegia and organ preservation on endothelial function include hyperkalemic components of the solutions (to be discussed in the next section), hypothermia and mechanical damage (perfusion pressure and duration; see details in a previous review [42]), and ischemia-reperfusion injury.

Oxidative stress-induced endothelial cell activation is the key component of the detrimental effect of ischemia-reperfusion. With expression of a set of proinflammatory, procoagulant, and vasoactive genes, a series of protein production steps in endothelial cells is promoted that causes intravascular microthrombosis, reduced blood flow, and activation of inflammatory cells. Among those proteins, the production of E-selectin, P-selectin, and intercellular adhesion molecules (ICAMs) lead to recruitment of neutrophils, which have been recognized as the principal effector cells of ischemia-reperfusion injury [47]. Leukocyte-endothelium adherence has indeed been observed after cardioplegic arrest and reperfusion [37].

Recently, the cellular mechanism underlying endothelial activation has been revealed with the study of nuclear factor -B (NF-B). Oxidative stress activates the tyrosine phosphorylation of IB, an inhibitor of NF-B that binds to NF-B in the cytoplasm; such phosphorylation dissociates IB from NF-B. The translocation of functional NF-B to the nucleus with binding to the target genes results in transcriptional activation of those genes [48]. In patients undergoing cardiopulmonary bypass with cardioplegic arrest, NF-B increased dramatically after reperfusion compared with before cardioplegia [49]. Therefore, targeting on the signaling pathway of endothelial cell activation may ameliorate cardioplegic arrest and reperfusion-induced endothelium impairment. Better recovery of coronary vascular response to serotonin and bradykinin in porcine coronary vessels and to acetylcholine in the neonatal lamb heart [51] was obtained by adding deferoxamine or manganese superoxide dismutase to the cardioplegic solution to reduce the oxygen-derived free radicals [50] or with leukocyte molecule CD18 (ligand for ICAM-1) antibody before cardioplegic arrest [51]. Moreover, transfection of NF-B decoy oligonucleotides into isolated heart blocked ICAM-1 upregulation and inhibited increase in neutrophil adhesion [52].

	Endothelial-Derived Relaxing Factors

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
Endothelium has multiple functions, including regulation of vascular tone, prevention of platelet aggregation, antiproliferation, and so on [53]. This article reviews the effect of cardioplegic and organ preservation solutions on the endothelial function related to regulation of vascular tone—the endothelium-dependent relaxation. Endothelium-dependent relaxation is known to be mediated by three different endothelium-derived relaxing factors (EDRFs): nitric oxide (NO), prostacyclin (PGI₂), and an unidentified endothelium-derived hyperpolarizing factor (EDHF).

Prostacyclin
Prostacyclin is the first defined relaxing factor derived from endothelium. Earlier in 1970s, Moncada, Vane, and others proposed that PGI₂, a product of arachidonic acid metabolism of endothelium, has relaxant effect on vascular smooth muscle [54, 55]. When activated by stimuli, the enzyme phospholipase A₂ in endothelial cells converts membrane phospholipids to arachidonic acid that is subsequently metabolized to PGI₂, thromboxane A₂, and several other prostaglandins (PGE₂, PGF₂, PGD₂) through the action of cyclooxygenase (COX). When activated by sheer stress, hypoxia, or receptor-operated mechanisms, endothelial cells produce 10 to 20 times more PGI₂ than the smooth muscle cells [55, 56]. Prostacyclin causes vasorelaxation in most arteries including the coronary bed [56–58], mediated by the rise of cyclic 3', 5'-adenosine monophosphate (cAMP) [59] that leads to the extrusion of Ca²⁺ from the cytosol [60] and the decreased sensitivity of the contractile apparatus to Ca²⁺ [61]. The relaxation may involve a Ca²⁺-independent mechanism [62] and membrane hyperpolarization of smooth muscle cell through potassium (K⁺) channels including ATP-sensitive K⁺ (K_ATP) channels [63, 64], calcium-activated K⁺ (K_Ca) channels [65], and voltage-gated K⁺ (Kv) channels [66, 67].

Nitric Oxide
In 1980, Furchgott and Zawadzki postulated that in rabbit aortic arteries, on stimulation of acetylcholine, endothelial cells release another vasodilator substance distinguished from PGI₂ [68]. The existence of the EDRF was demonstrated by bioassay techniques and layered preparations [69, 70] and was subsequently identified as NO [71, 72]. Nitric oxide is synthesized from amino acid L-arginine by nitric oxide synthase (NOS). At least two major NOS isoforms exist. One isoform is expressed constitutively in neurons and vasculature that is involved in cell communication and is activated by an increase in intracellular calcium. The other isoenzyme exists in macrophages to participate in host defense and is not normally found in endothelial cells or vascular smooth muscle unless induced by cytokines [73]. As a mixed-function monooxygenase using NADPH, NOS oxidizes L-arginine to form NO and citrulline [74]. In the vascular system, the endothelium-derived NO diffuses from endothelial cell and acts on the underlying smooth muscle cell layer. The subsequent upregulation of cyclic 3', 5'-guanosine monophosphate (cGMP) in smooth muscle results in the activation of cGMP-dependent protein kinase that leads to vasorelaxation [75, 76]. Several types of K⁺ channels are involved in NO-mediated hyperpolarization—K_ATP and K_Ca channels through cGMP-dependent manner [77–79], K_Ca channels by direct activation [80], and the cGMP-independent stimulation of Na⁺-K⁺-ATPase [81].

Endothelium-Derived Hyperpolarizing Factor
Since Bolton and associates demonstrated that a muscarinic agonist elicited endothelium-dependent hyperpolarization of vascular smooth muscle cells [82], the hyperpolarizing phenomenon has been confirmed in various blood vessels, including coronary arteries [83, 84]. Further studies revealed the existence of a novel vasorelaxant agent termed endothelium-derived hyperpolarizing factor (EDHF) [83–86].

Certain investigators suggested that the so-called EDHF is merely an electrical signal conducted from the endothelial cell to the underlying smooth muscle cell through myoendothelial gap junctions, the intercellular connection between the layer of endothelium and smooth muscle [87–93]. Failure in detecting bioassayable hyperpolarizing substances may support this hypothesis [94]. Contrary to this viewpoint, others [95–97] and we [98] have found evidence that this factor is bioassayable and therefore is a transferable chemical(s). Several substances have been suggested to be EDHF, including epoxyeicosatrienoic acid (EET) [96, 99], anandamide [100], K⁺ [101], H₂O₂ [102], citrulline, NH₃, and ATP [103]. The EDHF evokes hyperpolarization/relaxation of the smooth muscle by opening KCa channels, resulting in the closure of voltage-dependent Ca²⁺ channels and the reduction of intracellular free Ca²⁺ [84, 95–98, 104, 105]. The mechanisms of these three factors are shown in Figure 1.

View larger version (24K): [in this window] [in a new window]   Fig 1. Diagram describing the pathways of endothelium-dependent vasorelaxation. Under sheer stress or the stimulation of agonists, [Ca2+]i rise in endothelial cells that leads to the release of prostacyclin (PGI2 ), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). These three endothelium-derived relaxing factors decrease the [Ca2+]i in smooth muscle cells through different mechanisms and ultimately relax the smooth muscle. (BKCa = large conductance calcium-activated potassium channel; cAMP = cyclic 3', 5'-adenosine monophosphate; cGMP = cyclic 3', 5'-guanosine monophosphate; EET = epoxyeicosatrienoic acid; IKCa = intermediate conductance calcium-activated potassium channel; KATP = ATP-sensitive potassium channel; Kir = inward rectifier potassium channel; R = receptor; SKCa = small conductance calcium-activated potassium channel.)

	Influence of Cardioplegia and Organ Preservation Solutions on Individual Endothelium-Derived Relaxing Factors

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

Nitric Oxide
Studies have suggested impaired NO-related endothelial function during cardiopulmonary operations. By measuring the end products of NO—nitrite and nitrate—Gohra and coworkers demonstrated that NO release decreased significantly at approximately 70 minutes of crystalloid cardioplegic arrest in human coronary vasculature and was further reduced after reperfusion [109]. Similarly, the inability of the endothelium to release NO associated with reduced endothelium-dependent vasodilatation after infusion with UW solution [43] or loss of NO production after cardioplegia-reperfusion associated with decreased protein level of constitutive NO synthase [108] was demonstrated. The NO loss after cold (4°C) ischemic storage with crystalloid cardioplegia was recovered by chronic oral administration of L-arginine, the physiologic substrate of NO [110]. However, the combined ischemia-reperfusion injury was probably the main cause of the NO-related endothelial dysfunction in these and other studies [111, 112].

We have demonstrated that when the effect of ischemia-reperfusion is excluded, the NO-related, endothelium-dependent vasorelaxation after exposure to oxygenated crystalloid hyperkalemic cardioplegia to acetylcholine or substance P is well preserved in either porcine epicardial coronary arteries [113] or neonatal rabbit aorta [114]. Although in these studies, the indomethacin-resistant relaxation was actually mediated by both NO and EDHF, the unchanged endothelium-dependent response and the susceptibility of EDHF to cardioplegic solution [115–117] provided convincing evidence for the minimal impact of hyperkalemic cardioplegic solution on the NO-related function after exposure for a certain period (1 or 2 hours).

Endothelium-Derived Hyperpolarizing Factor
We conducted a series of experiments to investigate the effect of cardioplegic solution and organ preservation solutions on EDHF-mediated function. With exclusion of the effect of ischemia-reperfusion and elimination of the effect of PGI₂ and NO, we have demonstrated that hyperkalemia [115, 116] as well as ST [117] and UW solutions [118] impair EDHF-related function either in porcine or human coronary arteries [115, 116]. The mechanism is due to the opposite effect of EDHF and hyperkalemia. Hyperkalemia depolarizes whereas EDHF hyperpolarizes the smooth muscle membrane. The persistent depolarizing effect of hyperkalemia even after washout of cardioplegic solutions restricts the hyperpolarizing effect of EDHF [105, 116, 118]. Use of potassium-channel openers as hyperpolarizing cardioplegia may overcome this shortage of hyperkalemic cardioplegia [119].

	Influence of Different Components in Cardioplegic and Organ Preservation Solutions on Endothelial Function

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
Potassium
Potassium is the key component in cardioplegic or organ preservation solutions, as mentioned above. The concentration of K⁺ varies in different solutions. In UW solution, [K⁺] is as high as 125 mmol/L compared with only 20 mmol/L in ST and 10 mmol/L in HTK solution, respectively. The importance of K⁺ concentration with regard to coronary endothelial impairment has been revealed: K⁺ at 30 mmol/L but not at 20 mmol/L abolished endothelial-dependent, 5-hydroxytryptamine-induced vasodilatation [120].

In contrast, studies from others [26] and us have demonstrated that hyperkalemia per se does not significantly alter the endothelium-dependent relaxation as a whole to acetylcholine or substance P in porcine coronary arteries (to K⁺ 50 mmol/L) [113] and neonatal rabbit aorta (to K⁺ 100 mmol/L) [114]. These contradictory results should stimulate further investigation into the effect of hyperkalemia on individual relaxing factors derived from endothelial cells. As of now, little direct evidence exists to show that the reduction of the production of NO is due to hyperkalemia. Rather, reduced NO production is most likely due to the combined ischemia-reperfusion injury. When the capability of the endothelium to release NO is preserved or specific NO inhibitors are not present, the endothelium is tolerant to hyperkalemia as far as the endothelium-dependent relaxation is concerned, as shown above [26, 113, 114].

However, in contrast to NO, susceptibility of EDHF to high concentration of K⁺ has been demonstrated in accumulating studies. When the effect of PGI₂ and NO is inhibited by indomethacin and N ^G-nitro-L-arginine (L-NNA), the endothelium-dependent relaxation or hyperpolarization (mediated by EDHF) to a number of EDRF stimuli is impaired by incubation with K⁺ ranging from 20 to 125 mmol/L in porcine and human coronary arteries [105, 115, 116]. Realizing that L-NNA cannot abolish the production of NO, we further added oxyhemoglobin, a scavenger of NO, to abolish the effect of residual NO and demonstrated again the detrimental effect of hyperkalemia on EDHF-mediated relaxation and hyperpolarization in porcine coronary microarteries [121].

The mechanism of the reduced EDHF-mediated relaxation in hyperkalemic solutions is twofold. First, hyperkalemia depolarizes the coronary smooth muscle membrane and the prolonged depolarization increases the difficulty for subsequent hyperpolarization. Second, EDHF hyperpolarizes the vascular smooth muscle cell by opening K⁺ channels, the function of which may be blocked by K⁺ that inhibits K⁺ channels [105, 116].

Magnesium
The introduction of Mg²⁺ into cardioplegia helps to achieve immediate heart arrest during cardiac operations, and the enrichment of Mg²⁺ may counteract the unfavorable effect of hypocalcemia on sarcolemmal membrane by preventing calcium influx, thus obtaining better membrane stabilization [7]. In addition to the protective effect on myocardium [122, 123], Mg²⁺ has been proven to be a potent vasodilator through both endothelium-dependent and -independent mechanisms [124, 125]. The fact that Mg²⁺ infusion improves methacholine-induced vasorelaxation demonstrated the importance of endothelium in the effect of Mg²⁺ in human forearm vessels [126]. Pretreatment with the NOS inhibitor L-NAME (Nw-Nitro-L-arginine methyl ester) reduces Mg²⁺-mediated vasodilatation, and NO donor sodium nitroprusside or a cGMP analog, 3-guanosine monophosphate, restores the response to Mg²⁺, indicating the role of the NO-cGMP pathway in the action of Mg²⁺ [124, 125]. Moreover, the involvement of the COX system marked with the production of PGI₂ has also been suggested in Mg²⁺-induced relaxation [125, 127]. Tofukuji and associates reported that hyper-Mg²⁺ cardioplegia (25 mmol/L Mg²⁺) is superior to hyper-K⁺ cardioplegia in terms of preserving ß-adrenoceptor-mediated and endothelium-dependent regulation of the coronary microcirculation in pigs undergoing cardiopulmonary bypass [128]. Further, Pearson and colleagues observed the inhibitory effect of hypomagnesemia on endothelium-dependent vasodilatation induced by acetylcholine or adenosine diphosphate; they proposed that such impairment of endothelial function is due to the decreased release of NO [129]. In addition, a recent study from our laboratory demonstrated that in porcine coronary arteries, Mg²⁺ preserves the EDHF-mediated relaxation and hyperpolarization and restores the EDHF function impaired by hyperkalemia [121].

Procaine
Similar to Mg²⁺, the local anesthetic procaine is added to cardioplegia to induce asystole and obtain membrane stabilization [7]. Procaine is recognized as a vasodilator [130, 131]. At a concentration of 1 mmol/L, procaine relaxes vascular smooth muscle not only in an endothelium-independent manner but also in an endothelium-dependent manner that is closely related to NO but not PGI₂ [131]. We further showed that procaine does not affect EDHF function in coronary circulation [132] despite the fact that it depolarizes the membrane of vascular smooth muscle cells by reducing K⁺ conductance [133].

	Effect of Different Additives to Cardioplegic or Organ Preservation Solutions on Endothelial Function

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
The additives that are intended to protect endothelial function may be categorized as follows:

1 Nitric oxide substrates or donors. The benefit of supplementation of NO precursor L-arginine or NO donors such as nitroglycerin in cardioplegia on postischemic ventricular performance and endothelial function has been well established; thus such supplementation is considered an effective replacement therapy in cardiac operation [134–136].

2 Prostacyclin analogs. Prostacyclin analogs such as iloprost and OP-41483, added to crystalloid cardioplegia, provide better preservation of cardiac function after ischemic arrest [137, 138].

3 Endothelium-derived hyperpolarizing factor analogs. Addition of epoxyeicosatrienoic acid_11,12 (EET_11,12), the possible analog of EDHF, to hyperkalemic solution may partially restore the bradykinin-induced, EDHF-mediated endothelial function in porcine coronary arteries [139]. Further, the benefit of EET_11,12-supplementation in ST solution was shown under moderate hypothermia [140].

4 Potassium channel openers (KCOs). When added to hyperkalemic cardioplegia, aprikalim (a known adenosine triphosphate-sensitive KCO) reduces the Na⁺-Ca²⁺ exchange outward current elevated by hyperkalemia and thus may attenuate the [Ca²⁺]_i elevation, leading to improved contractile function after cardioplegia in the ventricular myocyte [141]. In addition, supplementation with aprikalim in traditional hyperkalemic solutions preserves EDHF-mediated coronary relaxation, indicating its protective effect on endothelial function [142]. Other KCOs such as KRN4884 have been demonstrated to have a preconditioning effect on the EDHF-mediated relaxation and may be beneficial if added to cardioplegic solutions [143].

5 Scavengers of oxygen-derived free radicals. Supplementation of crystalloid cardioplegic solutions with free radical scavengers, such as ascorbate and deferoxamine, reduces postreperfusion myocardial injury [144] and preserves the endothelium-dependent vasodilatation in coronary microvessels [50].

6 Sodium-hydrogen ion exchange (NHE) inhibitors. The cardioprotective effect of NHE inhibitors is not only observed with perioperative administration to patients undergoing bypass operation [145], but also demonstrated when added into blood cardioplegia [146]. The decreased accumulation of intracellular Na⁺ and subsequently decreased Ca²⁺ overload contribute to the reduced postischemic infarct size and tissue edema due to the supplementation of the selective NHE type 1 isoform inhibitor, cariporide. In isolated postischemic left anterior descending coronary artery rings, maximum relaxation in response to acetylcholine was significantly greater in the cariporide group than in the vehicle group, indicating the preservation effect of cariporide on endothelium [146].

7 Other substances. Adenosine has been shown to partially attenuate the microcirculatory injury by cardioplegia [147] but the effect is controversial [148]. Adding phosphodiesterase III-inhibitor (E-1020) to Bretschneider’s HTK cardioplegic solution has been shown to improve myocardial functional recovery [149]. The finding that 17ß-estradiol prevented intracellular Ca²⁺ loading or hypercontracture of ventricular cardiomyocytes in high K⁺ exposure [150], together with its direct favorable effects on vascular endothelial function [151], raises the possibility of 17ß-estradiol acting as a cardioprotective adjunct in hyperkalemic cardioplegia. The effect of prevention of Ca²⁺ overloading by Ca²⁺ antagonists highlights the cardiovascular protective effect of Ca²⁺ antagonist-supplemented cardioplegic solutions [152, 153]. In addition to these strategies, solutions enriched with metabolic substrates (ie, glutamate, aspartate, fumarate, etc) have been used clinically for a long time for better myocardial protection [154]. However, little is known regarding the consequence of such enrichment on endothelial performance.

	Conclusion

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
In summary, due to the differences between the cardiac myocytes and vascular (endothelial and smooth muscle) cells in structure and function, cardioplegic or organ preservation solutions primarily designed to protect the myocardium may have detrimental effects on coronary vascular endothelial cells, as demonstrated over the last few decades. One must be aware that under either experimental or clinical settings, the reported effects of the solutions on coronary endothelium are often mixed, with many other factors contributing to the outcome, such as ischemia-reperfusion injury, temperature, and perfusion pressure and duration.

In evaluating a clinically used solution and in developing a new solution, these factors should be carefully distinguished from the effect of the solution. The key component of the cardioplegic and heart preservation solutions that causes cardiac arrest—high concentrations of potassium ion (hyperkalemia)—is the major component that has been studied regarding endothelial function. Despite numerous studies that reported "impaired endothelial function or endothelium-dependent relaxation by hyperkalemia," most of these studies had combined effects due to ischemia-reperfusion injury or other factors. Little evidence exists to show that hyperkalemia changes NO-related endothelial function. The impairment of the NO pathway is mainly due to ischemia-reperfusion injury. In fact, without ischemia-reperfusion, the coronary-endothelial NO pathway is resistant to the moderately increased potassium concentrations used for cardioplegia (about 20 mEq/L). This fact explains the excellent clinical results by using either crystalloid or blood cardioplegia. However, the second endothelium-dependent relaxation pathway that is usually a "back-up" of the NO pathway—the EDHF pathway—is significantly altered (damaged) by hyperkalemia even at the moderately high concentration of potassium. This is because hyperkalemia inhibits K⁺ channels in the endothelium and smooth muscle that are related to either the release of EDHF from the endothelium or the target of the action of EDHF. Magnesium has a protective effect on this pathway because it hyperpolarizes the coronary smooth muscle membrane and therefore has "synergetic" effect with EDHF. When combined with ischemia-reperfusion and other factors that significantly impair the NO pathway, the effect of hyperkalemia on the EDHF pathway becomes an important issue in the protection of coronary endothelium.

A variety of new additives designed to protect these two major endothelium-dependent pathways may further improve the protection of the coronary endothelium from other factors such as ischemia-reperfusion injury. All these issues should be taken into account in the development of new cardioplegic and heart preservation solutions in the future in order to provide "perfect" cardiac protection.

	Acknowledgments

Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 
This review was fully supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region (Project No. CUHK4127/01 M & CUHK4383/03 M), China and the Providence St. Vincent Medical Foundation, Portland, OR.

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Top Abstract Introduction Literature Selection Methods Cardioplegic and Organ... Overall Effect of Cardioplegic... Possible Mechanisms Underlying... Endothelial-Derived Relaxing... Influence of Cardioplegia and... Influence of Different... Effect of Different Additives... Conclusion Acknowledgments References

 

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