Ann Thorac Surg 2003;76:385-390
© 2003 The Society of Thoracic Surgeons
Original article: general thoracic
Warm ischemia lung protection with pinacidil: an ATP regulated potassium channel opener
Daniel G. Tang, MDa,
Dawn R. Pavot, MDb,
Michelle M. Mouria, BAa,
Dana M. Holwitt, MDa,
Neri M. Cohen, MD, PhDa*
a Division of Cardiothoracic Surgery, Department of Surgery,, Richmond, VA, USA
b Department of Pathology, Medical College of Virginia Hospitals & Physicians of Virginia Commonwealth University Health System, Richmond, Virginia, USA
* Address reprint requests to Dr Cohen, Division of Cardiothoracic Surgery, Department of Surgery, Box 0068, Medical College of Virginia Hospitals & Physicians of Virginia Commonwealth University Health System, Richmond, VA 23298-0068, USA.
e-mail: nmcohen{at}hsc.vcu.edu
Presented at the Forty-ninth Annual Meeting of the Southern Thoracic Surgical Association, Miami Beach, FL, Nov 7–9, 2002.
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Abstract
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BACKGROUND: Ischemia/reperfusion injury remains a limiting factor in lung transplantation. Traditional hyperkalemic preservation solutions are associated with a host of metabolic derangements. ATP-regulated potassium channel openers (PCOs) may provide an attractive alternative to traditional solutions by utilizing inherent mechanisms of ischemic preconditioning. The purpose of this study was to assess warm ischemia graft protection with pinacidil, a nonspecific PCO.
METHODS: An isolated recirculating blood perfused ventilated rabbit lung model was used (n = 15). No ischemia control lungs underwent immediate reperfusion (n = 5). Warm ischemia control lungs were flushed with lactated Ringers (LR), stored at 37°C for 2.5 hours and then reperfused for 2 hours (n = 5). PCO protected lungs were flushed with LR + 100 µmol/L pinacidil, stored, and then reperfused (n = 5). Intermittent blood gases were taken from the pulmonary artery and left atria. Every 30 minutes, graft function was assessed with a 10-minute 100% fractional inspired oxygen concentration challenge to measure maximal gas exchange. Lung samples were graded for histologic injury and assayed for myeloperoxidase activity.
RESULTS: A mixed-models repeated measures ANOVA demonstrated a significant difference between groups. Tukey’s honestly significant difference multiple comparison test demonstrated significantly improved graft function and reduced histologic injury with pinacidil protection compared with the warm ischemia controls. There was no significant difference in graft function or pathology grade between the pinacidil protected lungs and the no ischemia controls. A similar trend, although not significant, was seen in myeloperoxdiase activity.
CONCLUSIONS: Potassium channel openers with pinacidil can provide pulmonary protection against warm ischemia reperfusion injury.
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Introduction
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Lung transplantation as a treatment modality for end-stage pulmonary disease has been limited by a shortage in donor organs. Recent data from the United Network for Organ Sharing listed approximately 3800 patients awaiting lung transplants with an expected median waiting time close to 3 years. Unfortunately, both of these figures continue to grow [1].
With increased demand, emphasis has been placed on better utilization of available organs; however, ischemia/reperfusion injury remains a significant obstacle to the long-term success of lung transplantation. Chronic rejection, manifested by bronchiolitis obliterans, remains the most common cause for mortality after 1 year. Annual multivariate analysis of the Transplant Registry for the International Society of Heart Lung Transplantation has continued to identify organ ischemia time as a significant predictor for both bronchiolitis obliterans and perioperative death [2].
Despite a large volume of research into strategies to enhance allograft protection, hypothermic/hyperkalemic solutions continue to remain the mainstay of thoracic organ preservation. A recent American College of Chest Physician survey of lung transplant programs demonstrated that 
60% still use such solutions (data reported at the Transplant/Immunology NetWork meeting during CHEST 2002 [San Diego, CA]; Chest Soundings 2003;17:10). Extracellular hyperkalemic solutions create a depolarized environment that, in myocardial preservation, produces diastolic arrest. However, this is accompanied by significant derangements in transmembrane ionic gradients, which result in abnormal ion fluxes, eg, Na+, K+, Cl-, and Ca2+. This is further associated with ongoing metabolic costs, including ATP depletion, cell swelling, and calcium overload [3].
The ATP regulated potassium channel openers (PCOs) may provide an alternative to traditional hypothermic/hyperkalemic solutions. First described by Noma in 1983 [4], ATP regulated potassium (KATP) channels have been demonstrated to play an essential role in coupling membrane excitability, molecular bioenergetics, and cellular protection in a host of organs and tissues. Molecular studies of KATP channels have reported the basic underlying structure to be a heteromultimer consisting of pore forming potassium inward rectifying (Kir 6.x family) 
subunits, and sulfonyl urea receptor (SUR) ß subunits [5]. Pharmacologic opening of KATP channels activates a large outward potassium current that causes a marked shortening of the action potential duration and hyperpolarization of the cell membrane near the normal resting potential. This conductance cannot be overcome electrically, "clamping" the membrane potential at the potassium equilibrium potential (EK) and short-circuiting membrane excitability.
In the myocardium this has been demonstrated to decrease calcium overload and minimize other abnormal large ion fluxes. In addition to these electrophysiologic effects, pharmacologic opening of KATP channels has been associated with stimulation of mitochondrial respiration and matrix swelling enhancing ATP synthesis [6]. In vitro models of ischemic injury have further demonstrated PCO associated suppression of neutrophil-free radical production [7, 8].
A large volume of research in myocardial protection implicate KATP channels as the active site for the phenomenon of ischemic preconditioning [9]. This has been demonstrated both in the immediate early protective effects of preconditioning and during the "second window of preconditioning" [10]. Within the myocardium, PCOs have been revealed to induce a chemical preconditioning while blockade of KATP channels blocks induction of ischemic preconditioning. Multiple studies in myocardium have since confirmed that PCOs can limit infarct size and protect against ischemia/reperfusion injury [6, 11].
Our laboratory has extended these studies to pulmonary allograft protection as well. The purpose of this study was to assess warm ischemia graft protection using PCO protection alone with functional, histologic, and biochemical assays.
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Material and methods
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All animals received humane care in accordance with guidelines set in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication 85 to 23, revised 1985). Protocols were reviewed and approved by Virginia Commonwealth University’s Institutional Animal Care and Use Committee.
Rabbit lung harvest and injury
New Zealand white rabbits (4 to 5 kg) of either sex were sedated with acepromazine (10 mg/kg intramuscularly) and xylazine (100 mg/kg intramuscularly), and then anesthetized with ketamine (50 mg/kg intramuscularly). Tracheostomy was performed and pressure controlled mechanical ventilation established with an infant ventilator (BP2001; Bear Medical Systems, Inc, Riverside, CA) at 30% FiO2. Median sternotomy and thymectomy was performed and snares were placed about the superior and inferior vena cavae. The pericardium was opened and snares were placed about the aorta and pulmonary artery (PA). Purse-string sutures for cannulation were placed in the walls of the right and left ventricles. Heparin (1000 U/kg) was given as an injection into the right ventricle. The PA was then cannulated through a right ventriculotomy, and the snare about the PA was tightened isolating the lung inflow. The left atrium (LA) was similarly cannulated through a left ventriculotomy and the aorta was ligated isolating the lung outflow. A venotomy was made in the inferior vena cava, blood was collected, and stored at 4°C. Lung preservation solution was infused (50 mL/kg) through the PA cannula at a fixed pressure (column height 30 cm). The inflow (PA) and outflow (LA) cannulas were clamped; and the heart-lung block was excised with the tracheostomy tube clamped at end-inspiration. The lungs were then suspended in a warm (37°C) humidified tissue chamber.
Study protocol
Fifteen rabbits underwent lung protection in three groups (five in each group). Group 1 (no ischemia control) underwent harvest and immediate reperfusion. Groups 2 and 3 underwent harvest followed by 2.5 hours of warm (37°C) ischemic storage before reperfusion. Groups 1 and 3 received lactated Ringer’s solution as the pulmonary flush at induction of ischemia. Group 2 received lactated Ringer’s plus pinacidil (100 µmol/L).
Isolated lung model
Following ischemic storage, the heart-lung block was then connected by the PA inflow and the LA outflow cannulas to a blood reperfusion circuit as illustrated in Figure 1;
and mechanical ventilation was reestablished at 30% fractional inspired oxygen concentration (FiO2). The perfusion circuit recirculates warmed autologous blood with a roller pump at a rate of 60 mL/min. After passage through the lungs, oxygenated blood collected from the LA cannula is deoxygenated by passing a CO2/N2 gas mixture over a thin layer of blood in a recirculating exchange column. The blood is then passed through a 40-µm filter and returned to the lung through the PA cannula. Continuous recordings of PA pressure, LA pressure, and airway pressure were made using a standard analog physiologic data acquisition system (Gould 13-4615-50, Valley View, OH). Blood samples were collected for blood gas analysis (System 1306; Instrument Lab, Lexington, MA) at 40-minute intervals after the start of reperfusion. At each sampling time point, paired blood samples were obtained (deoxygenated blood before infusion through the PA and oxygenated blood from the LA after passing through the lungs) to obtain accurate measurements of pulmonary venous blood oxygen content step up. Graft function was measured by oxygen challenge technique. Fractional inspired oxygen concentration (FiO2) was transiently increased from 30% to 100% for 10 minutes every 30 minutes, and paired blood samples were taken to measure gas exchange. Reperfusion was continued for 2 hours.
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Fig 1. Reperfusion setup. Warm autologous blood is circulated through the pulmonary artery and collected from the left atria. Blood is then deoxygenated in a gas exchange column with nitrogen and carbon dioxide, filtered, and then recirculated.
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Myeloperoxidase activity assay
Two grams of lung tissue from the right upper and middle lobes were homogenized in 8 mL of 0.5% hexadecyltrimethylammonium bromide, 50 mmol/L potassium phosphate solution (pH 6.0), and then stored at -70°C (Sigma Chemical Company, St. Louis, MO). At assay, samples were disrupted with three freeze/thaw cycles. Samples were then centrifuged at 10,400g for 20 minutes at 4°C, and had the supernatant aliquoted. Duplicate samples of 1:400 to 1:800 dilutions in 50 mmol/L potassium phosphate were then mixed with 50x 0-dianisidine and 0.15% H2O2 (Sigma). The reaction in the microplate well was allowed to sit for 5 minutes and its absorbance at 460 nm was measured. Human leukocyte derived myeloperoxidase (MPO) was used to create a standard curve (0.35 to 35.4 mU; CalBiochem, La Jolla, CA). Total protein in the sample was assayed by the Micro-BCA protein assay protocol (Pierce, Rockford, IL). The MPO activity was expressed as milliUnits of MPO per micrograms of protein.
Histopathologic assessment
A section of lung from the dependent portion of the right lower lobe was excised, fixed in 10% buffered formalin, and then stained with hematoxylin and eosin. A semiquantitative score (range 0 to 4), as described by Tassiopoulos and coworkers [12], for histologic grading of lung ischemia reperfusion injury was used. A blinded pathologist scored the specimens based on the amount of vascular congestion, interstitial edema, and intraalveolar hemorrhage.
The left lower lobe was excised, blotted to drain intravascular fluid, and dried in an oven at 40°C for 48 hours to measure wet to dry tissue weight ratios.
Statistical analysis
Summary results are expressed as the mean ± standard error of the mean (SEM). Error bars in the figures likewise represent SEMs. Differences in gas exchange between study groups were assessed with a repeated measures multiple analysis of variance using JMP 4.0 statistical software (SAS Institute, Pacific Grove, CA). Analysis of variance was used to assess differences in mean MPO production and histopathologic injury score. When a significant F value for comparison across all three groups was found, Tukey’s honestly significant difference (HSD) test for intergroup comparisons was performed. A p value less than 0.05 was considered significant.
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Results
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Lung graft function was assessed by gas exchange, as expressed by the arterial-alveolar (A-a) gradient is illustrated in Figure 2. Then, 2.5 hours of warm ischemia produced a profound graft injury (control injury) relative to the no ischemia controls. There was a significant difference in graft function between groups (F[2,22] = 22.6, p < 0.0001). Tukey’s HSD demonstrated significantly improved graft function with pinacidil protection compared with the warm ischemia controls. In contrast, there was not a significant difference in graft function between the pinacidil protected lungs and the no ischemia controls. Warm ischemia control lungs had a least squares mean A-a gradient of 499 ± 40.0 mm Hg; pinacidil protected lungs had a mean gradient of 256 ± 40.0 mm Hg; and no ischemia control lungs had a mean gradient of 132 ± 40.0 mm Hg.
Histopathologic grading of lung injury correlated with graft function. Warm ischemia control specimens demonstrated marked amounts of vascular congestion, interstitial edema, hypercellularity, and intraalveolar hemorrhage. As with graft function, there was a significant difference in pathologic scoring between groups (F[2,13] = 11.4, p = 0.002) as illustrated in Figure 3.
Tukey’s HSD again demonstrated significantly better graft protection with pinacidil protection versus warm ischemia controls, and no significant difference with no ischemia controls. No ischemia control lungs had a mean score of 0.8 ± 0.35; pinacidil protected lungs had a mean score of 1.2 ± 0.7; and warm ischemia control lungs had a mean score of 3.8 ± 0.2. Representative micrographs are illustrated in Figure 4.
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Fig 3. Histopathologic grading. There is significantly decreased histologic injury in the pinacidil group compared with warm ischemia controls; no difference compared with no ischemia controls.
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Fig 4. Representative micrographs of (A) no ischemia control; (B) pinacidil protection, revealing marked preservation; and (C) warm ischemia control, demonstrating marked intraalveolar hemorrhage, capillary congestion, interstitial edema, and hypercellularity. (Hematoxylin and eosin stain, original magnification x200.)
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Myeloperoxidase activity was used as a measure of neutrophilic migration and activation. There was a trend towards decreased MPO production in the pinacidil protected group although this did not reach statistical significance (F[2,15] = 1.5, p = 0.259), which is depicted in Figure 5.
The mean MPO production in the warm ischemia control lungs was 7.25 ± 1.83 mU/µg, in pinacidil protected lungs was 4.60 ± 0.90 mU/µg, and in the no ischemia control lungs was 4.04 ± 1.15 mU/µg.
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Fig 5. Myeloperoxidase activitiy. A trend similar to the histopathologic grading towards decreased myeloperoxidase activity was seen in the pinacidil protected group. (MPO = myeloperoxidase.)
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There was a slight trend towards decreased edema with pinacidil protection, although this was also not significant (F[2,13] = 0.4, p = 0.712). The mean wet to dry tissue weight ratio in the warm ischemia control was 7.19 ± 1.65, in the pinacidil protected group was 6.91 ± 0.73, and in the no ischemia control was 5.84 ± 0.53.
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Comment
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The phenomenon of ischemic preconditioning and protection against ischemia/reperfusion injury was first demonstrated in the myocardium, but has since been demonstrated in almost all other organ systems including the lungs [13]. The ATP-regulated potassium channel openers are postulated to provide a more physiologic form of protection against ischemia/reperfusion injury by utilizing this inherent protective mechanism.
In summary, our results demonstrate that the PCO pinacidil can provide pulmonary graft protection against ischemia/reperfusion injury. Lung function, as assessed by gas exchange, was well preserved in the pinacidil protected group. This protection was also seen in the marked preservation of cell structure at the histologic level. In conjunction with previously described electrophysiologic and intracellular effects, a trend towards decreased MPO activity further suggests that PCO protection may in part be due to a decrease in neutrophil activation. Further studies are warranted to explore the clinical utility and underlying mechanisms of PCO pulmonary protection.
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Discussion
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DR STEPHEN D. CASSIVI (Rochester, MN): Thank you for that very interesting presentation. It is always good to see that people are still working on making improvements in the field of lung transplantation. We have still got a ways to go, obviously.
I have a few questions after reading your abstract and listening to your talk. You used lactated Ringers as your preservation solution and you talked about its benefits as opposed to the hyperkalemic solutions used for cardiac arrest. Currently we are using low potassium dextran as the de facto standard across North America. Do you think that it would make any difference in your findings if you flushed with what we are actually using in the clinical setting along with your pinacidil?
And then the next question I have is, whether you are considering doing these experiments in an in vivo lung transplant animal model such as the rat lung transplant model that is being used currently in St. Louis and in Toronto?
DR TANG: Doctor Cassivi, thanks for your comments. A recent survey of lung transplant programs regarding preservation solutions demonstrated that 60% still used traditional hyperkalemic solutions as opposed to low potassium dextran so both types of solutions are certainly relevant [1].
At this years Surgical Forum for the American College of Surgeons, we presented other data on the effect of an extracellular potassium gradient on pulmonary protection by potassium channel openers (PCOs). In theory, membrane depolarization with a high potassium gradient could interfere with membrane hyperpolarization associated with KATP channel opening.
There have been mixed results regarding the effect of hyperkalemic solutions on PCOs in the myocardium. One study demonstrated that increasing hyperkalemia decreased PCO mediated coronary vasodilatation [2]. In contrast, another study demonstrated that hyperkalemia improved myocardial protection by potassium channel openers [3].
We found that the pulmonary protective effect of PCOs is independent of the extracellular potassium gradient. We hypothesize that activation of the mitochondrial population of KATP channels may be more relevant to pulmonary protection as opposed to plasmolemmal KATP channels, and our current studies are testing this.
With regards to dextran, other studies have shown that this may provide additional endothelial protection and it would be interesting to see if this enhances PCO pulmonary protection further. However, for this study we wanted to focus solely on the effects of PCOs.
As for an in vivo model, our current studies are focused on defining the mechanisms of PCO pulmonary protection and defining an ideal protection solution. Once defined, an animal lung transplant model would certainly be appropriate as the next step towards clinical implementation of PCOs.
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Acknowledgments
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This work was funded in part by the Association for Academic Surgery Joel J. Roslyn Faculty Research Award (Dr Cohen).
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References
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Abstract
Introduction
= no ischemia control; 
= warm ischemia control; 
= pinacidil protection.)