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Ann Thorac Surg 2004;78:620-627
© 2004 The Society of Thoracic Surgeons
a Department of Surgery, Division of Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, Missouri, USA
Accepted for publication February 23, 2004.
* Address reprint requests to Dr Damiano, Division of Cardiothoracic Surgery, Washington University School of Medicine, 660 S Euclid Ave, Box 8234, St. Louis, MO, USA 63110
e-mail: damianor{at}msnotes.wustl.edu
Presented at the Thirty-ninth Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 31-Feb 2, 2003.
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Abstract |
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METHODS: Thirty-two rabbit hearts received one of four preservation solutions in a Langendorff apparatus: (1) UW; (2) a solution containing 0.5 mmol/L pinacidil; (3) pinacidil with Hoechst-Marion-Roussel 1098 (HMR-1098), a sarcolemmal channel blocker; and (4) pinacidil with 5-hydroxydecanote, a mitochondrial channel blocker. Left ventricular pressure-volume curves were generated by an intraventricular balloon. All hearts were placed in cold storage for 8 hours, followed by 60 minutes of reperfusion.
RESULTS: Postischemic developed pressure was better preserved by pinacidil than by UW. This cardioprotective effect was eliminated by 5-hydroxydecanote and diminished by HMR-1098. Diastolic compliance was better preserved by pinacidil when compared with UW. This protection was abolished by the addition of 5-hydroxydecanote and moderately decreased by HMR-1098.
CONCLUSIONS: Our results support the superiority of pinacidil over UW after 8 hours of storage. The cardioprotective role of pinacidil is mediated primarily by the mitochondrial KATP channel.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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Over the last decade, our laboratory has shown the benefits of using the nonspecific potassium-adenosine triphosphate (KATP) channel agonist, pinacidil, in the preservation of cardiac function and physiology [4–6] after global ischemia. It also has been shown that hyperpolarized cardiac arrest using a KATP channel opener is beneficial in the maintenance of cardiac energy stores after prolonged ischemia and cold storage [7]. Two separate KATP channels are abundantly present in the heart, one on the sarcolemma and the other on the mitochondrial membrane. Both have been shown to play a role in cardiac protection mediated by KATP agonists. The present study was designed to investigate pinacidil as a preservation solution and to determine which KATP channel is responsible for the cardioprotection it offers.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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Animal preparation
Anesthesia was accomplished through an intramuscular injection of acepromazine (1 mg/kg) and xylazine (17.5 mg/kg), followed by ketamine (62.5 mg/kg). A tracheotomy was performed and mechanical ventilation (Model 683; Harvard Apparatus, South Natick, MA) was implemented through an endotracheal tube. Intravenous heparin was administered (3,000 U) through an ear vein. Sternotomy and rapid cardiectomy were performed, and the aorta cannulated and rapidly attached to a crystalloid perfusion column. A polyethylene vent (inside diameter, 0.86 mm) was inserted through the apex of the left ventricle.
A fluid-filled latex balloon was inserted into the left ventricle and secured to the mitral valve annulus with a pursestring suture of 5-0 Prolene (Ethicon, Somerville, NJ). The intraventricular balloon was connected through polyethylene tubing to a pressure transducer (model P23ID; Gould Instrument Systems, Valley View, OH) and amplifier (model 20-4615-50; Gould). Two needle electrodes were placed on the LV epicardium to monitor the bipolar ventricular electrogram. The signal was filtered between 0.05 and 1,000 Hz. Two other electrodes were secured to the right atrial appendage and connected to a pacemaker (model 5320; Medtronic, Minneapolis, MN). Heart rate was maintained at a constant rate of 190 to 205 beats per minute with the pacemaker. The pressure and electrogram tracings were displayed continuously and digitized in real time on a personal computer (Compaq Computer, Houston, TX) using a waveform recording system (WinDAQ/400; DATAQ Instruments, Akron, OH) at a sampling rate of 1000 Hz. A needle temperature probe (model BAT 8; Bailey Instruments, Saddle Brook, NJ) was placed in the right ventricle for myocardial temperature monitoring (model TM147T; Electromedics, Parker, CO). The heart was enclosed in a plastic jacket and was maintained at 37°C by a water-jacketed beaker connected to a warm-water bath (model D1/L; Haake, Berlin, Germany).
Experimental preparation
The isolated rabbit heart was suspended through an aortic cannula at a column height of 70 cm and perfused with Krebs-Henseleit buffered solution (37°C) using a Masterflex Model 7520-10 roller pump (Cole Parmer, Chicago, IL). The Krebs-Henseleit solution consisted of the following (in mmol/L distilled water): NaCl, 118.5; NaHCO3, 25.0; KCl, 3.2; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; and glucose, 5.5. The Krebs-Henseleit solution was maintained at a strict pH range between 7.40 and 7.48 by bubbling with a gas mixture of 95% O2 and 5% CO2. An inline ultrasonic flow probe (model T101; Transonic Systems, Ithaca, NY) measured flow of the Krebs-Henseleit solution down the perfusion column. A separate 70-cm cardioplegia arrest column was maintained at 4°C by water-cooling jackets.
Experimental protocol
The isolated rabbit hearts underwent a 30-minute equilibration period after mounting onto the Langendorff circuit. Baseline LV pressure–volume curves were generated by inflating the left ventricle balloon. Hearts were excluded from the study if they were unable to generate pressures of at least 80/10 mm Hg during baseline data acquisition. Intracavitary LV pressures and bipolar LV electrograms were recorded by more than 7 balloon volumes, each corresponding to a fixed, intracavitary LV end-diastolic pressure (EDP), namely, 0, 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mm Hg). Coronary flow was measured at each of these LV pressures. After baseline data acquisition, the left ventricle balloon was deflated to generate an EDP of 2.5 mm Hg.
After baseline data acquisition, hearts were arrested with 50 mL of hypothermic (4°C) solution followed by 8 hours of hypothermic (7°C to 8°C) storage in 50 mL of the same solution. Hearts were randomly allocated to receive one of four solutions: (1) UW (n = 8); (2) Washington University Solution (WashU) containing 0.5 mmol/L pinacidil (n = 8); (3) WashU with 100 µmol 5-hydroxydecanoate (5-HD) solution (n = 8); or (4) WashU with 100 µmol HMR-1098 (n = 8) solution. After arrest, all remaining fluid was evacuated from the left ventricle balloon.
Each heart was returned to the Langendorff circuit and reperfused for 60 minutes (37°C) after storage. After reperfusion, LV pressure–volume measurements were generated using the same balloon volumes employed for baseline data acquisition. The percent recovery of developed pressure, diastolic pressure–volume relationships and perfusate flow were compared between treatment groups. After reperfusion data acquisition, a portion of the left ventricle apex was excised, blotted, and weighed with a balance (Model TM 400; Mettler Instruments, Hightstown, NJ) and then dried in an oven (Isotemp Model 625G; Fisher Scientific, Houston, TX) until a constant dry weight was reached. Myocardial edema was expressed as percent tissue water according to the equation: percent tissue water = (wet weight – dry weight) / wet weight x 100.
Solution preparation
The UW solution was purchased in the form of ViaSpan (Du Pont Merck Pharmaceuticals, Wilmington, DE). The UW solution was modified with the standard 0.016 g/L of dexamethasone, 200,000 U/L penicillin, and 40 U/L insulin. Each component of the WashU solution was added directly to deionized water (Table 1). Pinacidil was purchased from Sigma-Aldrich Corporation (St. Louis, MO). The pinacidil solution was titrated to pH 7.65 with KOH and filtered. This solution was further modified to produce the final two experimental solutions by adding 100 µmol of the specific mitochondrial channel blocker, 5-HD, or the specific sarcolemmal channel blocker, HMR-1098. The 5-HD was purchased from Sigma-Aldrich Corporation (St. Louis, MO). The HMR-1098 was a generous gift from Aventis Pharmaceuticals (Frankfurt, Germany).
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Systolic function
Systolic function was evaluated using both recovery of developed pressure (DP) and maximal +dP/dT.
Recovery of developed pressure
The recovery of DP was calculated as the ratio of the postischemic DP to the baseline DP at the corresponding balloon volume. The average percent recovery of DP (% DP) was calculated by a program developed in our laboratory by means of the trapezoidal rule [9].
Maximal systolic +dP/dT
The systolic maximal +dP/dT (ddmax) of a beat was calculated as the maximum derivative of left ventricular pressure during systolic contraction. The mean ddmax was calculated by averaging 20 seconds of data. Average ddmax values were obtained for each balloon volume at baseline and after reperfusion (postischemic). The ddmax versus balloon volume data for baseline and postischemic data were fitted to second-order polynomial regressions. Each regression was integrated to calculate area under the curve (AUC) ranging from minimum to maximum matched balloon volume. To express the systolic contractile function over multiple LV volumes, the percent recovery of maximal systolic +dP/dT was calculated as a ratio of the postischemic AUC divided by the baseline AUC multiplied by 100.
Diastolic properties
The diastolic properties of the left ventricle were evaluated using minimal diastolic –dP/dT and percent change of baseline EDP at a fixed volume.
Minimal diastolic –dP/dT
The minimal –dP/dT (ddmin) of a beat was calculated as the minimal derivative of LV pressure during diastolic relaxation. The mean ddmin was calculated as the mean of 20 seconds of data. Average ddmin values were obtained for each balloon volume at baseline and after reperfusion (postischemic). The ddmin versus balloon volume data for baseline and postischemic data were fitted to second-order polynomial regressions. Each regression was integrated to calculate AUC ranging from minimum to maximum matched balloon volume. The percent recovery of minimal diastolic –dP/dT was calculated as a ratio of the postischemic AUC divided by the baseline AUC multiplied by 100.
Percent change in EDP at a fixed volume
The percent change in EDP at a fixed volume was calculated by taking the mean of 20 seconds of data and recording the EDP at that volume both at baseline and reperfusion. The values were compared by dividing the reperfusion diastolic pressure with the baseline diastolic pressure and multiplying by 100.
Recovery of coronary flow
Coronary flow was recorded continuously through out the data acquisition period. It was measured through an in-line flow probe and subsequently averaged. Reperfusion coronary flow was divided by preischemic coronary flow and the quotient multiplied by 100.
Statistical analysis
Statistical analysis was performed using the computer software SigmaStat (version 2.03; Jandel, San Rafael, CA) and Excel 97 (Microsoft, Redmond, WA). A paired t test was used to compare preischemic to postischemic endpoints within groups. A Kruskal-Wallis one-way analysis of variance on ranks was used to compare means between groups. Results were expressed as mean ± SEM. Statistical significance was at the level of p less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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In agreement with our prior work, pinacidil provided superior systolic and diastolic myocardial protection to an ischemic heart during prolonged cold storage when compared with the standard, hyperkalemic, depolarizing UW solution. The primary mediator of this cardiac protection appears to be the mitochondrial KATP channel. When 5-HD was added to the WashU solution, blocking the mitochondria KATP channel, the superior myocardial protection seen with pinacidil was eliminated. In contrast, when the sarcolemmal KATP channel was blocked by the addition of HMR-1098, the benefits of pinacidil were decreased but remained better than the UW solution, in most instances. Although not to the extent of the mitochondrial KATP channel, however, our data revealed that the sarcolemmal KATP channel played a significant role in the cardioprotection provided by pinacidil including compliance, contractility and developed pressure. These findings have been supported by other groups [15, 16]. Further studies will be required to determine the time course and precise mechanisms involved in the myocardial protection afforded by potassium channel openers and mediated by both the mitochondrial and sarcolemmal KATP channels.
When systolic function was investigated, the WashU solution was significantly superior to UW solution. Developed pressure was better protected by the hyperpolarized arrest of the WashU solution when compared with that of the UW solution. Once again, the presence of 5-HD abolished the beneficial effects of pinacidil, and the presence of HMR-1098 lessened but did not eliminate pinacidil's myocardial protection. Furthermore, 5-HD lowered myocardial protection to levels below those observed in the UW solution. These data were repeated when contractility was examined using the +dP/dT. Our laboratory has shown the beneficial effects of pinacidil on systolic function in the past [4, 8]. These results were recently confirmed by Ikizler and associates [20] as it was shown that pinacidil improved systolic indicators when added to custadiol after 60 minutes of cold storage. Additionally, it has been shown that 5-HD abolishes the systolic benefits of diazoxide, a selective mitochondrial KATP channel opener, and ischemic preconditioning [21, 22]. In this study, we observed repeatedly that the addition of a mitochondrial KATP channel blocker, 5-HD, not only reversed the effects of hyperpolarized arrest but also actually lowered recovery of systolic function to levels less than that seen after depolarized arrest.
The diastolic properties of active relaxation and passive compliance were studied by using –dP/dT and percent change of EDP at a fixed volume, respectively. The WashU solution was superior to the UW solution when these properties were compared. The addition of 5-HD completely abrogated the diastolic protection provided by the pinacidil as well as the protection afforded by UW solution. The addition of HMR-1098 significantly lowered the diastolic protection seen with hyperpolarized arrest but remained equivalent or superior to that observed with UW solution. Our laboratory has previously shown that the active compliance of the heart was better maintained by a pinacidil containing solution after cold storage and nonsurgical ischemia [8]. Recently, other groups have also shown the diastolic benefits of hyperpolarized arrest with potassium channel openers [23]. Additionally, Lin and colleagues [24] showed that the –dP/dT was improved with pinacidil but reversed with glibenclamide, a sulfonurea compound known to block KATP channels. Our study is unique in showing that the opening of the KATP channels beneficially effects the passive compliance of the heart as well.
The WashU solution maintained coronary flow better than all of the solutions, particularly UW solution. When the mitochondrial KATP channel blocker, 5-HD, was added to the solution, the protective effects of the pinacidil were eliminated. In contrast, when the sarcolemmal KATP channel blocker, HMR-1098 was added to the solution, a decrease in coronary flow was observed but the solution still possessed superior protection to UW. It has been shown that potassium channel openers when used in cardioplegia hyperpolarize endothelial cells and cause smooth muscle relaxation and vasodilatation [17]. In a previous study, hypoxia and reoxygenation reduced the relaxation mediated by endothelium-derived hyperpolarizing factor in the coronary microartery. This function was restored by either hypoxic preconditioning or by a KATP channel opener [18]. Matsuda and associates [19] showed that high potassium cardioplegia augmented the coronary calcium-mysosin light chain pathway and resulted in vasoconstriction. Pinacidil effectively blocked the activation of this pathway and maintained adequate vasorelaxation during cardioplegia [19]. The WashU solution, with both reduced potassium concentration as well as pinacidil, is ideal for the maintenance and restoration of the cardiac epithelium after cold storage.
The WashU solution containing pinacidil offers promise in extending the cold storage time for heart transplantation. That could have substantial clinical benefits by expanding the number of hearts available for transplant, thereby lessening waiting list time and the number of deaths while waiting. Human leukocyte antigen typing could also be safely performed allowing for better matches and less immunosuppression therapy. Continued research in larger animal transplant models will need to be conducted before clinical implementation. The search for the exact mechanism of the cardioprotection pinacidil confers will also need further attention. However, the WashU solution offers much hope for the future of producing a solution designed specifically for the successful cold storage and preservation of human hearts.
Study limitations
The Krebs-Henseleit–perfused Langendorff preparation was used in this study because it allowed for analysis of intervention of the isolated heart without humoral, neural, adrenergic, or anesthetic influences. This preparation is less complicated than a blood-perfused parabiotic preparation because of the independence from physiologic variations produced by the support animal. Moreover, for long-term preservation of hearts, the maintenance of the support animal becomes logistically difficult. The findings of this study should be interpreted with caution, however, as the current preparation lacked blood perfusion and the important dynamics of the intact organism. As with any experimental results, these findings should be extrapolated with caution to the clinical setting.
Moreover, the precise contribution of each component of the pinacidil vehicle is not fully known. Isolated comparison of the individual components, which differed between the hyperpolarizing pinacidil and UW solutions, was not performed. The differences between solutions in ionic concentrations of potassium, calcium, and magnesium available to the cell should not, however, affect the protective mechanism observed with the pinacidil. Specifically, the potassium was kept low in the WashU solution to prevent the depolarization of the myocardial cells. A small amount of calcium was added to prevent calcium paradox, a phenomenon observed in mammalian cells upon reperfusion. The function of the magnesium in both solutions is to displace calcium from the sarcolemma. The UW solution did provide a clinical frame of reference for comparison in these studies, as many investigators consider it the "gold standard" for donor organ preservation. Furthermore, we were unable to directly relate the improved cardioprotective efficacy of pinacidil compared with UW to prevention of intracellular calcium accumulation, cell swelling, or promotion of glycolytic flux, because these endpoints were not measured. These limitations will serve as the focus for future studies.
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Acknowledgments |
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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DR DIODATO: In order to investigate the role of Pinacidil in our solution we actually performed a second set of experiments, which were presented at the ISHLT in April. In these experiments, we removed Pinacidil from the Washington University solution and we then placed it in the University of Wisconsin solution. We compared the 4 groups in the following way—the University of Wisconsin, Washington University, normal; Washington University without Pinacidil; and University of Wisconsin with Pinacidil. We saw the recovered function in the Washington University solution without the Pinacidil decreased to the University of Wisconsin levels. With the addition of Pinacidil to the University of Wisconsin solution, we did see a slight but not significant increase in preservation of left ventricular systolic and diastolic functions. We did not, however, test any of the other components of the solution individually.
DR JACOBS: The second question really was why does Wisconsin solution work poorly in St. Louis and well in Wisconsin?
DR DIODATO: I cannot answer that question. The initial studies from 10 years ago were done at shorter time periods, 4 hours. I know in previous work from our laboratory, Dr Hoenicke showed that after 12 hours of cold storage, Washington University solution had a much greater recovery of function than the University of Wisconsin, which had almost no recovery of function.
DR PEDRO J. DEL NIDO (Boston, MA): If I could just follow up on that question, having done some experiments with UW solution, most people use UW with low potassium as compared to what you used, which as 120 mM KCl. There is at least theoretical evidence that you get endothelial injury in myocardium after prolonged exposure as opposed to liver. My question relates more to mechanism; it has been proposed that KATP channels play a protective role in preventing apoptosis. You have a relatively short reperfusion period, so you haven’t addressed that question really. Is there a possibility that you’re going to see much more injury if you reperfuse these animals for longer and that the differences may not be so great if you had a longer period of reperfusion because you are not going to see apoptosis in a relatively short period of reperfusion.
DR DIODATO: Those are both great points. We are actually looking at longer periods of reperfusion, and we are also planning to look at these solutions on a single cellular level to look for mechanisms of apoptosis.
DR DEL NIDO:The one difference that I noticed is you have a markedly increased buffering capacity in your solution. There is histidine in the solution and that has been shown to be a good cardioprotective mechanism. UW solution has no buffering capacity. Do you have an idea whether that contributed anything in your model?
DR DIODATO: Well, we believe it did. Again, in order to find out exactly how much that did contribute to the cardioprotection we observed, we would have to remove all of the other confounding variables and test each one at a time. We did not do that for this experiment.
DR RICHARD N. GATES (Orange, CA): I have just one question. In this model, which is very well controlled, one potential oversight might be that you are actually testing the ability of these two solutions to protect a heart that has been perfused with crystalloid for 30 minutes. Ideally, it would have been nice to initially arresting the heart directly in the chest cavity with either solution. Have you done any experiments where you have compared these two solutions with a direct in-chest blood perfused heart?
DR DIODATO:We have not done that at this time, but we are looking to move the experiment to a larger animal model and perform heart transplants at that time. That is part of our next series of experiments.
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