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J Thorac Cardiovasc Surg 2005;129:407-415
© 2005 The American Association for Thoracic Surgery
Cardiothoracic Transplantation |
Toronto General Hospital, University Health Network, Division of Cardiac Surgery, University of Toronto, Toronto, Ontario, Canada
Read at the Eighty-fourth Annual Meeting of The American Association for Thoracic Surgery, Toronto, Ontario, Canada, April 25-28, 2004.
Received for publication May 4, 2004; accepted for publication September 2, 2004. * Address for reprints: Vivek Rao, MD, PhD, FRCS, EN14-222, Toronto General Hospital, 200 Elizabeth St, Toronto, Ontario, M5G 2C4, Canada (E-mail: vivek.rao{at}uhn.on.ca).
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Abstract |
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METHODS: Porcine orthotopic transplantations (n = 16) were performed after 6 hours of ischemic storage. Intermittent donor blood perfusion (control, n = 8) was compared with donor blood perfusion enhanced with 100 µmol/L of an endothelin receptor blocker (n = 8). Left ventricular performance was assessed after caval occlusion with a Millar micromanometer and conductance catheter. Coronary endothelial function was assessed in vitro with a macrovascular tissue bath apparatus. Myocardial endothelin, tumor necrosis factor 
, and transforming growth factor ß protein expression were determined. Oxidative stress was inferred on the basis of 8-isoprostane levels, and myocardial metabolism was inferred on the basis of the extraction or production of oxygen, acid, and lactate by the heart.
RESULTS: Endothelial function was diminished 48 hours after transplantation but not earlier. Endothelin receptor blocker treatment during preservation limited coronary endothelial dysfunction 48 hours after reperfusion (P = .001). Weaning from cardiopulmonary bypass and left ventricular performance after transplantation was improved in endothelin receptor blocker–treated hearts (P = .02). Myocardial endothelin expression was equivalent in both groups and increased during reperfusion after transplantation (P = .001). Tumor necrosis factor levels decreased with endothelin receptor blocker treatment (P = .02), whereas transforming growth factor ß levels did not change (P = .86). 8-Isoprostane, oxygen, acid, and lactate levels were similar, suggesting that oxidative stress and metabolism were not important mechanisms of benefit.
CONCLUSIONS: Endothelin accumulates during allograft storage and contributes to endothelial and myocardial dysfunction after transplantation. Endothelin blockade during allograft preservation limits endothelial injury and enhances ventricular recovery after transplantation.
Endothelin (ET-1) is a potent inflammatory peptide with profound biologic effects on both endothelium and myocardium. ET-1 accumulates in the heart during the ischemia and reperfusion associated with cardiac surgery.4 In coronary artery bypass graft surgery, increased perioperative ET-1 signaling is associated with poor postoperative clinical outcomes.5 In patients undergoing cardiac transplantation, heightened ET-1 bioactivity is associated with early myocardial dysfunction, late coronary vasculopathy, and reduced overall survival.3,4,6-8
We developed a preservation method by using limited coronary perfusion provided by donor blood harvested at the time of organ procurement that can improve both metabolic and functional recovery after transplantation compared with conventional storage techniques.9 Our perfusion technique is simple, straightforward, and clinically applicable. In addition, our perfusion approach provides an opportunity to add pharmacologic supplements to the perfusate and by this means identify novel agents to enhance recovery. We hypothesized that adding an ET-1 antagonist in the blood perfusate during cardiac allograft storage would provide dual endothelial and myocardial protection and, in so doing, limit early endothelial dysfunction and improve myocardial performance after transplantation.
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Methods |
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Donor procedure
Female Yorkshire pigs (approximately 70 kg) were used for both donor and recipient, and the donor heart was harvested as previously described.9 After median sternotomy, umbilical tapes were placed around the superior and inferior venae cavae to permit adjustment of cardiac preload by occlusion with snares. In animals randomized to ET-1 receptor blockade (ERB group), the standard cardioplegic solution was supplemented with 100 µmol/L of the mixed endothelin A (ETA) and endothelin B (ETB) receptor antagonist bosentan (Actelion Ltd).
Harvesting donor blood for subsequent perfusion during organ preservation
The method of delivery and final composition of donor shed blood perfusate has been described previously.9 In brief, the perfusate was delivered antegradely through the aortic root intermittently (200 mL every 20 minutes) at a pressure of 60 mm Hg by means of gravity throughout the 6-hour storage period. The temperature was allowed to drift to room temperature (approximately 21°C ± 1°C). In the ERB group 100 µmol/L bosentan was added to the perfusate.
Recipient procedure
Orthotopic implantation of the donor heart to the recipient proceeded as described previously.9 Bosentan was not added to the cardioplegic solution once cardiopulmonary bypass (CPB) was established and was only administered in the ERB group during harvest and ex vivo perfusion. After completion of all anastomoses, the aortic crossclamp was removed, and all hearts were reperfused for a period of 60 minutes. Weaning from CPB was deemed successful if the animal maintained a mean systemic pressure of 65 mm Hg for 30 minutes. The animal was then killed during anesthesia by intravenous potassium chloride injection and exsanguination.
Measurement of myocardial metabolic recovery
Myocardial metabolic recovery was assessed by obtaining simultaneous arterial and coronary sinus blood samples, as well as full-thickness left ventricular (LV) myocardial biopsy specimens. Blood samples were analyzed for lactate, oxygen, acid, and hemoglobin content, as described previously.9 Samples were obtained at baseline, before donor organ arrest, and at the end of organ storage. In addition, samples were obtained during each cardioplegic dose at the recipient operation and at 15-minute intervals during the reperfusion period.
ET-1 and inflammatory cytokine expression
LV tissue was homogenized on ice with Tris-buffered saline (TBS; 10 mmol/L Tris and 100 mmol/L NaCl, pH 7.5) containing 1% Triton X-100 and protease inhibitors (0.5 µg/mL each of aprotinin, pepstatin, and leupeptin and 0.05 mmol/L phenylmethylsulfonyl fluoride). The extract was diluted with TBS and assayed in duplicate with an ET-1 ELISA kit (Biomedica). The protein concentration was determined by using the Bio-Rad DC Dye.
Tumor necrosis factor (TNF-) and transforming growth factor ß (TGF-ß) levels were determined by Western immunoblotting. The LV tissue was homogenized on ice with TBS containing 1% Triton X-100 and protease inhibitors (as above). The protein concentration was determined by using the Bio-Rad DC Dye. Extracts were subjected to sodium dodecylsulfate–polyacrylamide gel electrophoresis. Nonreducing conditions were used for TGF-ß. The proteins were transferred onto polyvinylidene difluoride membrane and probed with polyclonal primary antibodies (Sigma). The bands were detected by means of chemiluminescence (Amersham Biosciences), scanned, and quantified by means of densitometry (Quantity One, Bio-Rad).
Measures of oxidant stress
Total 8-isoprostane (free and esterified) levels were assessed as a marker of oxidant injury10 by using an EIA kit (Cayman Chemical Co). The sample preparation protocol outlined in the kit was used with some modification. In brief, LV tissue was homogenized on ice in 10 mmol/L phosphate buffer, pH 7.4, containing 0.005% butylated hydroxy toluene at a ratio of 10 µL of buffer per milligram of tissue. An equal volume of 15% KOH was added to the whole homogenate and incubated at 40°C for 60 minutes. The sample was centrifuged, and the supernatant was neutralized with 1 mol/L KH2PO4. One milliliter of eicosanoid affinity column buffer (0.1 mol/L potassium phosphate, pH 7.4, containing 0.5 mol/L NaCl) was added to the sample, which was then purified by using an 8-isoprostane affinity column, according to the manufacturer's instructions. The samples were assayed at 2 dilutions, in duplicate, with the EIA kit and read at an absorbance of 405 nm.
Assessment of ventricular function
A Millar (Millar Instruments, Inc) high-fidelity micromanometer catheter (for pressure measurements) and a 5F NUMED conductance catheter (for volume measurements) were introduced sequentially into the left ventricle through an apical ventriculotomy. The outputs from the micromanometer and conductance catheter were linked to an analog digital converter and then transferred to a laptop computer for construction of pressure-volume loops. Measurements were obtained by using the methods of Baan and colleagues,11 and parallel conductance was corrected by using the method of Szwarc and coworkers.12 Once baseline pressure-volume loops were constructed, the venae cavae were snared for a minimum of 6 cardiac cycles to allow for the recording of pressure-volume loops at varying preloads. End-systolic elastance was calculated as the slope and position of the end-systolic point of the pressure-volume loops. We have found this to be a more sensitive measure of contractility compared with maximal systolic elastance.13 Preload recruitable stroke work (PRSW) was evaluated by calculating the relationship between stroke work (area within the pressure-volume loop) and the end-diastolic volume determined by our conductance catheter. The linear relationship between stroke work and end-diastolic volume provides a load-insensitive index of cardiac function (PRSW).14,15
Assessment of endothelial function
Endothelium-dependent and endothelium-independent vascular relaxation was assessed in vitro by constructing concentration-response curves with a small-vessel myograft for isometric tension recording. Our preliminary investigations determined that endothelial function was similar immediately after cardiac transplantation compared with that of normal freshly isolated control arterial segments (without storage and cardiac transplantation). Jeanmart and colleagues16 previously determined that 48 hours of cold storage does not itself induce endothelial dysfunction in normal porcine epicardial coronary segments but can be used to study the effects of late reperfusion. Accordingly, to expose the progressive endothelial dysfunction that occurs after transplantation, we stored our vascular segments for 48 hours at 4°C after transplantation. Normal freshly isolated control arterial segments were performed on coronary segments obtained from the recipient's native heart, which was harvested after cardioplegic arrest and stored similarly for 48 hours.
In brief, the left anterior descending coronary artery was cleaned of fat and connective tissue and placed into Krebs-Henseleit solution (118.0 mmol/L NaCl, 4.6 mmol/L KCl, 1.2 mmol/L MgSO4, 2.5 mmol/L CaCl2, 1.2 mmol/L KH2PO4, and 25.0 mmol/L NaHCO3). The tissue was stored at 4°C for 48 hours. Vessels were cut into 5.0-mm segments and placed into a 25-mL organ chamber. The rings were suspended between 2 wires, one of which was connected to an isometric force transducer. Data were collected with AcqKnowledge software (Biopac Systems Inc). The rings were bathed at all times with Krebs solution containing 11.0 mmol/L glucose at 37°C and oxygenated with a 95% oxygen/5% carbon dioxide mixture. The tissue was stretched to 3 g and allowed to stabilize for 90 minutes. The rings were contracted with 100 mmol/L KCl and, after washout, stabilized once again for 30 minutes.
The tissue rings were precontracted with a thromboxane A2 analog, U46619 compound (30 nmol/L). Endothelium-dependent vasorelaxation was assessed on the basis of a dose response to bradykinin (0.25 nmol/L to 1.0 µmol/L), and similarly, endothelium-independent vasodilation was assessed on the basis of a dose response to sodium nitroprusside (10 nmol/L to 5 µmol/L). The percentage of maximum relaxation from preconstriction was compared between groups. Two segments of left anterior descending coronary artery were obtained from each heart, and the data were averaged, provided the variability between segments was less than 10%; otherwise, the data was excluded from analysis.
Statistical analysis
Statistical analysis was performed by the SAS program (Version 8.2 for Windows, SAS Institute). Two-way analysis of variance was used to compare continuous variables by simultaneously evaluating the main effects of time and group. Categorical data were analyzed by 
2 or Fisher exact tests where appropriate.
Analysis of covariance was used to compare the slope of the stroke work/end-diastolic volume relationship to simultaneously evaluate the main effects of treatment group and end-diastolic volume. Exact P values are provided to enable the reader to determine clinical and statistical significance.
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Results |
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LV function
Despite the intermittent perfusion of shed donor blood during the period of ischemic storage, control hearts had reduced LV function after orthotopic heart transplantation compared with baseline measures (Figure 1). Notably, LV functional recovery was improved in ERB-treated hearts after transplantation (Figure 1), as assessed on the basis of PRSW and end-systolic elastance. The PRSW relationship showed the characteristic downward (reduced slope) and rightward (increased volume-axis intercept) displacement after transplantation compared with baseline curves, indicating allograft LV contractile dysfunction.15 However, this displacement of the PRSW relationship was attenuated in ERB-treated hearts (Figure 1). In addition, successful weaning from CPB was greater in the ERB-treated hearts (7/8 vs 5/8), although the difference did not reach statistical significance (P = .23).
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Endothelial dysfunction was apparent in hearts subjected to prolonged storage and transplantation and then assessed after an extended period of storage (Figure 2). Notably, ERB treatment during storage limited the endothelial dysfunction associated with transplantation and surgical reperfusion (Figure 2). ERB treatment had no effect on segments harvested immediately after storage before transplantation and reperfusion (Figure 2). Endothelium-independent coronary vasoreactivity was not different between groups at any time, indicating that the improved coronary vasomotor function was the result of improved endothelial function after ERB treatment (Figure 2).
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did not differ with time but was less in ERB-treated hearts during the storage period (Figure 3). Expression of TGF-ß was similar over time (P > .20) and between groups (ERB-treated subjects vs control subjects: 120 ± 24 vs 121 ± 28 arbitrary units; P > .20).
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Myocardial metabolism
Aerobic metabolism was determined by measuring the extraction or production of oxygen, acid, and lactate from the heart. ERB treatment did not influence the recovery of aerobic metabolism at any time point during the study (Figure 4).
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Discussion |
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Enhanced endothelial protection
The results of our study indicate that ET-1 signaling might directly contribute to endothelial dysfunction after allograft preservation and subsequent reperfusion after transplantation. Allograft coronary endothelial dysfunction was a late consequence of reperfusion after prolonged storage. However, it should be noted that animals were killed immediately after functional assessments made after the surgical reperfusion period. Thus, we did not assess endothelial function after prolonged reperfusion in vivo. We stored our vascular rings ex vivo in a solution for 48 hours to enhance our ability to detect endothelial dysfunction after transplantation, recognizing that this might not necessarily reflect changes consistent with in vivo reperfusion.
Importantly, ET-1 receptor blockage during the period of donor cardioplegic arrest and storage was sufficient to prevent the endothelial dysfunction observed after reperfusion. In our preliminary studies addition of an ERB during CPB at the time of donor implantation resulted in profound systemic vasodilatation and instability. Thus, in this study we assessed the role of targeted ET-1 blockade during harvest and allograft storage alone. Our results indicate an extended benefit with targeted ERB administration during storage while avoiding adverse consequences on systemic hemodynamics during CPB. We used a high dose of bosentan in this study, and these adverse effects on hemodynamics during CPB could perhaps be attenuated by reducing the dose of bosentan.
Because early endothelial injury from ET-1 bioactivity in the heart is believed to mediate the subsequent development of coronary allograft vasculopathy,3,7 the enhanced endothelial protection we observed might have considerable late benefits. These will be assessed in future studies.
Enhanced allograft functional recovery
Allograft contractile performance was evaluated by using load-insensitive measures of LV systolic function from pressure-volume loop analysis after caval occlusion. ET-1 blockade enhanced the recovery of LV function after transplantation, as assessed on the basis of both PRSW and end-systolic elastance. Importantly, the PRSW relationship is a sensitive measure of porcine allograft LV functional recovery after orthotopic transplantation.15 Transplanted hearts revealed a characteristic downward and rightward shift after transplantation, indicating LV contractile dysfunction.15 However, decrease of PRSW was attenuated in the ERB-treated allografts, indicating beneficial effects on LV recovery.
Myocardial interstitial ET-1 expression is associated with ischemic injury in human transplant recipients.7 Because allograft performance is negatively influenced by ischemia-reperfusion injury, it follows that this mode of injury was likely minimized by ET-1 blockade. Interestingly, myocardial metabolism was unchanged by ET-1 blockage, and consequently, the improved recovery of LV function might not necessarily be related to an altered metabolic state. Similarly, oxidative stress, as inferred on the basis of 8-isoprostane levels, was comparable between groups, suggesting that ET-1–mediated attenuation of free radical production might not be a primary mechanism of benefit. However, the techniques used in this study might not be able to discriminate subtle changes in myocardial metabolism and free radical injury, and accordingly, we cannot completely rule out these critical pathways as potential mediators in the beneficial effects of ERB on allograft recovery.
It is conceivable that improved endothelial function could result in enhanced contractile function by improving myocardial perfusion and a washout of toxic metabolites that accumulate during storage. We were not able to demonstrate endothelial dysfunction early after transplantation and reperfusion by using a macrovascular tissue bath apparatus. It is possible that microvascular endothelial dysfunction was present at the time of LV performance assessment but could not be measured by our technique. The demonstration of significant endothelial dysfunction at a later time suggests that injury was indeed ongoing and induced by the period of storage.
Although ET-1 accumulation was not suppressed by ERB, receptor blockage likely resulted in attenuated ET-1 bioactivity, despite its enhanced expression. However, we did not specifically measure ET-1 bioactivity. Because ET-1 levels accumulated during storage and were increased at the time of LV performance assessment, it is possible that reduced ET-1 bioactivity by the nonselective ERB with bosentan directly improved myocardial contractility. Whereas the vasoconstrictor effects of ET-1 are widely recognized, activation of the ETA receptor has direct effects on myocyte biology, including contractile protein interactions, inotropic state, protein expression, and electrophysiology.4 Numerous studies indicate that ET-1 has a direct effect on myocardial contractility.4,5,18,19 Accordingly, the benefits of ERB on allograft LV functional recovery might be a direct result of this relationship between the ETA receptor and cardiomyocyte contractile function. Furthermore, expression of TNF- was reduced in hearts treated with ET-1 blockade, which might also have provided enhanced functional recovery, given that the proinflammatory cytokine TNF- is known to directly impair myocardial performance after ischemia-reperfusion.20
Limitations
These data are limited to the acute setting after transplantation, and the vascular rings were not exposed to 48 hours of in vivo reperfusion. Although we have reason to believe that combined myocardial and endothelial protection will provide long-term benefits, these issues remain to be evaluated. In addition, our technique for the assessment of endothelial function is an ex vivo method and is limited to the epicardial coronary vasculature. Myocardial perfusion was not measured, and it is conceivable that regional perfusion differences could result from enhanced endothelial function. We have inferred a cause-and-effect relationship between the use of ERB and the observed benefits on myocardial and endothelial function, particularly because we have attempted to eliminate other confounding mechanisms that have been implicated in ET-1 signaling, such as metabolic effects, inflammatory cytokine pathways, and oxidant stress. However, our techniques for determining myocardial metabolism and oxidative stress might be insensitive to detect subtle changes in these indices. Further confirmation with other techniques of assessment might be appropriate before it can be concluded that metabolism and oxidative stress do not contribute to the benefits afforded by ERB. In addition, our study design does not allow us to directly conclude that attenuated ET-1 bioactivity was the one and only mechanism leading to the significant benefits observed in this study. We also cannot exclude a sex-related effect because only female swine were used in this study.
Discussion
Dr Robert C. Robbins (Stanford, Calif). Were the donor heart ischemic times similar, the same?
Dr Fedak. The ischemic times between groups were identical. We maintained a protocol that was carefully controlled to provide a 6-hour ischemic period. Any transplantation that went longer than the 6-hour limit was eliminated from the study. We measured ischemic times during the experiment, but there was really no difference between the groups because we removed the aortic crossclamp at 6 hours in every animal.
Dr Robbins. You mentioned nitric oxide in your video cartoon. I did not see anything about it in your article, or did you not mention anything about nitric oxide production?
Dr Fedak. We did not study nitric oxide production. We do not believe that ET-1 blockade with a receptor antagonist will directly affect nitric oxide production. However, it will restore the balance of nitric oxide with respect to ET-1 bioactivity, which is the critical balance influencing endothelial function. Accordingly, although nitric oxide levels might not be influenced by ET-1 blockade, we are restoring the balance of nitric oxide relative to ET-1 by blocking ET-1 receptors. Importantly, this is also the reason that we suggest a combination of L-arginine to increase nitric oxide in addition to an ET-1 antagonist to decrease ET-1 activity. It might be best to maximize the balance of these mediators in both directions. Therefore, that is why we did not study nitric oxide levels in this study.
Dr Robbins. I was a little disappointed in your article and in your presentation. You really did not show us any of the pressure-volume loops and did not really dwell too much on the functional data, which in my mind would be kind of the whole point of this. Can you make some comments on specifics of hemodynamics other than to say that it was better in the treated group?
Dr Fedak. We performed two measures of LV function, both PRSW and systolic elastance, which are load-insensitive measures and, as far as I am aware, probably the best measures we have at the moment to measure ventricular function. On the basis of these two measures, we saw very similar data indicating improved cardiac function in the ET-1 antagonist group. We also observed more of the hearts weaned successfully from CPB. Some of the more subjective benefits included less myocardial edema and the appearance of more vigorous contractions after reperfusion. Therefore, as far as I am aware, we used the most objective and single best measures of in vivo myocardial function in this study.
Dr David T. Cooke (Palo Alto, Calif). Very interesting study. I have a question on your cytokine data. You show a significant difference in TNF- production, yet there were no statistical differences in your level of oxidative stress. What do you think is the cause of your differences in TNF- levels? And to follow up on that, did you check for evidence of apoptosis or differences in apoptosis that could possibly cause that? Did you look at other cytokines, such as MCP-1 or IL-1ß?
Dr Fedak. Thank you for your question. We did not look at the other cytokines that you mentioned, but we did look at TGF-ß. We did not see a difference in TGF-ß at any other time points or between groups. Our measure of TNF- was by means of immunoblotting, which might not be as sensitive as other methods. We did only see it at one significant time point. In terms of the mechanism as to why TNF- levels were being reduced, I cannot say from the data that we have. I can only say that perhaps with the blockade of the ET-1 receptor, it is also an inflammatory cytokine—and there is a lot of crosstalk between these cytokines—I would take it as a surrogate marker of less inflammation and perhaps less injury. We did not measure cell death, as you mentioned, but that would probably be a good thing to do in the future.
Dr Carmelo A. Milano (Durham, NC). I would like to congratulate you on an excellent article. You underplayed perhaps one of the most important potential contributions of this, and that would relate to graft vasculopathy. Preservation of the endothelium early on might have very important late effects, and that will be another important area of study.
Clinically, the time of graft storage is a fairly inert time, and you did not mention the temperature at which you stored the grafts, but I was wondering if you were considering administration of this ET-1 antagonist, perhaps in an experiment in which you treat the donor during the time of brain death, which is one time when potential endothelial damage might occur, or treat the graft and the recipient after reperfusion?
Dr Fedak. Absolutely. Thank you for your points. I did not stress the allograft vasculopathy because I really have no data to support it, but that is part of this process, and we do believe that by protecting the endothelium early, it will have late effects, particularly on allograft vasculopathy. We have studies ongoing in the rat heterotopic transplant model to look specifically at that.
In terms of your other point of when to deliver this agent, that is very important. There is an intravenous form of bosentan, tezosentan, and it could be given to the brain-dead donor systemically before cardioplegic arrest, which I think might have benefits. In addition, we could give it just as we did in this study quite easily by adding it into the cardioplegia during the arrest period at the time of donor organ harvest. Perhaps that alone might have significant benefits in the long run without using donor shed blood perfusion, as we suggest here, which is not done clinically. Therefore I think there are direct clinical consequences that we could do quite easily. We would have to study those further, obviously.
As to your second question regarding temperature, in our patients undergoing cardiac surgery, we try to compromise between warm and cold heart surgery, and we allow patients to drift in the operating room, which is what we did for these studies. In previous studies we found that providing limited coronary perfusion at room temperature led to improved preservation as opposed to static cold storage.
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Footnotes |
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