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Eur J Cardiothorac Surg 2005;27:226-234
© 2005 Elsevier Science NL
a Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany
b Department of Cardiovascular Surgery, Semmelweis University, Budapest, Hungary
c Institute of Pathology, University of Heidelberg, Heidelberg, Germany
d Department of Cardiology, University of Heidelberg, Heidelberg, Germany
e Inotek Pharmaceuticals Corporation, Beverly, MA, USA
f Department of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary
Received 31 July 2004; received in revised form 27 September 2004; accepted 13 October 2004.
* Corresponding author. Address: Department of Cardiac Surgery, University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg 69120, Germany. Tel.: +49 6221 566 111; fax: +49 6221 565 585. (E-mail: dzsi{at}hotmail.com).
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Abstract |
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 Top Abstract 1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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Key Words: PARP • Heart transplantation • Reperfusion
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1. Introduction |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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The mechanisms leading to tissue injury and organ dysfunction after ischemia/reperfusion or hypoxia/reoxygenation are multiple. However, there is good evidence that reactive oxygen species such as superoxide anion, hydroxyl radical and hydrogen peroxide, as well as the reactive nitrogen species peroxynitrite contribute to reperfusion injury in the previously ischemic myocardium [2–4] which in turn leads to PARP activation, with subsequent myocardial and vascular injury. Activated PARP catalyses an energy consuming cycle by transferring ADP ribose units to nuclear proteins. The results of this process are rapid depletion of the intracellular NAD+ and ATP pools which slows the rate of glycolysis and mitochondrial respiration leading to cell necrosis [1,5–7].
Acute graft dysfunction caused by ischemia-reperfusion injury is recognized as a major source of early morbidity and mortality following transplantation. Recently, it has been shown in rodent [8] and large animal models [9] that the blockade of PARP improves postischemic myocardial and endothelial function after short periods of cardioplegic arrest. Therefore, the primary aim of the study was to test the hypothesis that PARP inhibition reduces reperfusion injury after cardiac preservation in a setting of canine orthotopic heart transplantation.
Orthotopic heart transplantation in patients with congestive heart failure and severe pulmonary arterial hypertension can result in acute perioperative right ventricular failure of the allograft and subsequent death. The normal donor right ventricle, when abruptly presented with the high-resistance pulmonary vasculature of the recipient's lungs, initially dilates in an attempt to maintain pulmonary blood flow and left atrial filling [10]. If the pulmonary hypertension is severe, the right ventricle may become hypocontractile, resulting in a low cardiac output state. However, recent studies suggest that not pulmonary hypertension per se but pre-existent myocardial damage [11–15] as a result of brain death and/or ischemia-reperfusion injury in association with pulmonary hypertension may cause graft dysfunction. Therefore, we specially focused on postischemic right ventricular function and examined the adaptation potential of the right ventricle to the increase in right ventricular afterload.
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2. Material and methods |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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2.2. Orthotopic heart transplantation
The donor and recipient dogs were premedicated with propionylpromazine and anesthetized with pentobarbital (15mg/kg initial bolus and then 0.5mg/kg/h i.v.), paralyzed with pancuronium bromide (0.1mg/kg as a bolus and then 0.2mg/kg/h i.v.) and endotracheally intubated. The dogs were ventilated with a mixture of room air and O2 (FiO2=60%) at a frequency of 12–15/min and a tidal volume starting at 15ml/kg per minute. The settings were adjusted to maintain arterial partial carbondioxide pressure levels between 35 and 40mmHg. The femoral artery and vein were cannulated to record aortic pressure (AoP) and to take blood samples for biochemical analysis. Basic intravenous volume substitution was carried out with Ringer's solution. According to the values of potassium, bicarbonate and base excess, substitution included administration of potassium chloride and sodium bicarbonate (8.4%). Neither catecholamines nor other hormonal or pressor substances were administered.
Left anterolateral thoracotomy was performed in the fourth intercostal space. After pericardiotomy, the great vessels were dissected.
After systemic anticoagulation with sodium heparin (300U/kg) the heart was arrested with 25ml/kg HTK solution (in mmol: 15 NaCl, 9 KCl, 4 MgCl2 6 H2O, 18 histidine hydrochloride monohydrate, 180 histidine, 2 tryptophan, 30 mannitol, 0.015 CaCl2, 1 potassium-hydrogen-2-oxopentandioat, H2O) in the donor. The heart was excised and stored at 4°C in the same solution for 4h.
In the recipient animal, the left subclavian artery was cannulated for arterial perfusion after systemic anticoagulation with sodium heparin (300U/kg). The venous cannulas were placed in the vena cava superior and inferior. After initiation of cardiopulmonary bypass, the body temperature was cooled to 28°C. After crossclamping of the aorta on total bypass, the recipient heart was excised. The donor heart was implanted according to the bicaval technique. Twenty minutes prior to anticipated cross-clamp removal, rewarming was initiated. Implantation time was standardized at 60min. After deairing, the aorta was declamped. If necessary, ventricular fibrillation was counteracted with DC cardioversion of 40J. Ventilation was restarted with 100% oxygen. All animals were weaned from CPB without inotropic support 40min after the release of the aortic cross clamp.
2.3. Cardiac function
Left and right ventricular systolic and diastolic pressures and volumes were measured by combined 6F Millar pressure-conductance catheters with 6mm spacing which were inserted via the apex and the pulmonary artery, respectively. Stroke volume (SV) was calculated from the integrated flow signal measured by an aortic ultrasonic flow probe and was used to calibrate the volume signal from the conductance catheter. Parallel conductance was estimated by rapid injection of one ml of hypertonic saline into the pulmonary artery or superior vena cava, respectively. Vena cava occlusions were performed to obtain a series of pressure–volume loops. The slope (Ees) and intercept (V0) of the left and right ventricular end-systolic pressure–volume relationships and preload recruitable stroke work (PRSW) were calculated as load-independent indices of myocardial contractility. Diastolic function was assessed by the end-diastolic pressure–volume relationships.
To examine the adaptation potential of the right ventricle to an afterload increase, the pulmonary artery was constricted by tightening a snare around the pulmonary artery 3–4cm distal to the right ventricular outflow tract. An increase in RVSP to 
35 and 55mmHg, respectively, was achieved by progressive constriction of the pulmonary artery. Measurements were taken 10min after pulmonary banding at both levels in steady state. After the second pulmonary banding level the snare was loosened and the dogs were then allowed to return to baseline steady state. If necessary, volume substitution was applied to keep cardiac output at a constant level during this protocol.
Coronary blood flow was measured on the left anterior descendent artery with a perivascular ultrasonic flow probe. Coronary endothelium-dependent vasodilatation was assessed after intracoronary administration of a single bolus of acetylcholine (ACH, 10–7M) and endothelium-independent vasodilatation after sodium-nitroprusside (SNP, 10–4M). The vasoresponse was expressed as percent change of baseline coronary vascular resistance.
Functional assessments were performed before explantation of the donor heart and 2h after declamping the aorta within the recipient.
2.4. Determination of high energy phosphates
Before explantation and after 2h of reperfusion left ventricular biopsies were taken and immediately immersed in fluid nitrogen (–196°C) and stored frozen at –80°C until the biochemical measurements. The heart tissue was homogenized in 3.5% HClO4 and than centrifuged with 20,000U/min at 5°C. The supernatant was neutralized with Triethanolamin-HCL/K2CO3 solution. Creatine phosphate (CP) and adenosine triphosphate (ATP) degradation was assessed with standard photometry using the enzyme kinetic assay containing glycerinaldehyd-3-phosphate dehydrogenase, 3-phosphoglycerat-kinase, glycerin-3-phosphat dehydrogenase and triosephosphate-isomerase. CP, ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) contents were expressed as [µmol/g dry weight]. The values obtained were used to calculate energy charge potential as [ATP+0.5ADP]/ [ATP+ADP+AMP].
2.5. Poly(ADP-Ribose) immunohistochemistry
Poly(ADP-Ribose) (PAR), the product of PARP, was detected in order to assess the activation of PARP [8,9]. Heart biopsy specimens were taken at baseline and after 2h of reperfusion and fixed in formalin and embedded in paraffin. After section of the probes, slides were deparaffinized, antigen was retrieved by incubation in boiling 0.1M sodium citrate (pH 6), then slides were rinsed in water. Slides were incubated in 10% (W/v) trichloro-acetic acid (TCA) for 10min to prevent catabolism of the polymer by poly-ADP-ribose glycohydrolase. Slides were rinsed in PBS, then endogenuos peroxidase activity was quenched with 1.5% (vol/vol) hydrogen peroxide in methanol for 15min. Non-specific binding sites were blocked using 2% (vol/vol) normal goat serum in PBS for 1.5h at 37°C. Preliminary experiments determined optimal antibody concentrations. Chicken antibody against PAR was a generous gift from Dr John R. Simon (Tulip BioLabs, Inc. West Point, PA) and it was used 1:250 or 1:500 dilutions, slides were incubated overnight at 4°C, then washed in PBS and as a secondary antibody, biotinylated goat anti-chicken IgG (Vector Laboratories, Burlingame CA) was used for 30min at 30°C. After PBS washes, slides were incubated with VECTASTAIN Elite ABC (peroxidase) standard kit (Vector Lab.) for 30min at 30°C, and developed using diaminobenzidine substrate. Slides were counterstained with nuclear fast red.
2.6. Data analysis
All values were expressed as mean±standard error (SEM). Paired t-test was used to compare two means within groups. Individual means between the groups were compared by one-way analysis of variance. A probability value less than 0.05 was considered statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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35mmHg) resulted in an isolated increase (P<0.05) in RV Ees. A severe increase of right ventricular afterload (RVSP55mmHg) led to a further increase (P<0.05) of RV Ees with concomitant slight increase of end-diastolic pressure and volume which, however, did not reach the level of significance.
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3.3. Coronary blood flow and vascular function
Coronary blood flow was similar in both groups before explantation. After transplantation, coronary blood flow decreased significantly (P<0.05) in the control group, while it remained unchanged in the INO-1001 group (Table 1). Endothelium-dependent vasodilatation after ACH was significantly (P<0.05) reduced in the control group after transplantation in comparison to values before explantation (Fig. 4) while it remained unchanged in the INO-1001 group. Endothelium-independent vasodilatation after SNP showed no significant differences over the time and between the groups (Fig. 4).
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4. Discussion |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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Previous studies have shown PARP-activation in the reperfused heart [6–9,16] and reduced reperfusion injury in PARP-knock out animals [6,17] or after PARP inhibition [6,8,9,16,17]. This is the first study which shows the effectiveness of PARP-inhibition in a clinically relevant large animal model of orthotopic transplantation. The mechanisms of the protective action by PARP inhibition are multiple [overviewed in 17]. Ischemia/reperfusion injury initiates a pathophysiological cascade including an inflammatory response with liberation of cytokines and free radicals. Triggered by peroxynitrite-induced DNA single strand breaks, PARP catalyzes an energy-consuming polymerization of ADP-ribose, resulting in NAD+ depletion, inhibition of glycolysis and mitochondrial respiration, and the ultimate reduction of intracellular high energy phosphates in the reperfused heart [1,8,18]. Indeed, immunhistologic studies demonstrated that PARP rapidly activated in the reperfused myocardium [8,18,19]. Most of poly(ADP-ribose) staining was seen in cardiac myocytes indicating that the heart tissue itself, rather than the infiltrating mononuclear cells, is the main site of PARP activation. In addition, we showed previously [8] that PARP activation occurs in cardiac endothelial cells after cardioplegic arrest and reperfusion. Accordingly, PARP inhibition improves not only myocardial but endothelial function during reperfusion [9]. These findings are in accordance with the immunhistologic and functional findings of the present study. In various types of ischemia/reperfusion, the prevention of PARP-activation results in a better preservation of the high-energy phosphate content resulting in an improved energy status [1,6–8,16]. Beside its direct effects on myocardial metabolism, PARP-activation contributes to the expression of P-selectin and ICAM-1 during cardiac ischemia/reperfusion [6,8] and consequently to the recruitment of neutrophils into the jeopardized tissue. It is likely that both an inhibition of the energetic component of PARP-mediated cell dysfunction and the suppression of pro-inflammatory pathways contribute to cardioprotective effects [1].
As right ventricular afterload elevation is a common condition after orthotopic heart transplantation, special emphasis was focused on the adaptation potential of the right ventricle to an increased afterload. In previous studies [20,21], it was demonstrated that an isolated increase of right ventricular afterload leads to an increase of myocardial contractility (homeometric autoregulation) under physiological conditions. In damaged hearts, the heterometric autoregulation (i.e. increase of preload) was the primary compensatory mechanism to maintain physiological cardiac output values [20].
The present data confirm previous observations that the elevation of afterload resulted in a compensatory increase of contractility (RV Ees). Even a severe increase of right ventricular afterload led only to a tendency towards higher right ventricular volumes without reaching the level of significance indicating that inotropic adaptation is the primary compensatory mechanism of the right ventricle to an increased afterload. In contrast, after hypothermic cardiac arrest and reperfusion, an elevation of right ventricular afterload resulted in a progressive increase of RVEDV and RVEDP while RV Ees showed only a moderate increase. This indicates the utilization of the Frank–Starling mechanism (heterometric autoregulation) as a primary form of adaptation to increased afterload while inotropic adaptation remains limited.
However, the question arises whether the decreased ability of the right ventricle to an increased afterload by increasing contractility is a result of only ischemia reperfusion/injury, or other factors after transplantation such as denervation. Even if sympathetic innervation is important in the regulation of contractility there is an evidence that myocardial contractility may increase in response to an increased afterload in the absence of neural control [22,23]. The cellular mechanisms include increased affinity of contractile proteins to Ca2+, increased intracellular Ca2+-release and increased activation of mechano-sensitive Ca2+-channels [22–25]. The fact that PARP inhibition completely abolished the detrimental effects of cardioplegic arrest and reperfusion on the right ventricular inotropic adaptation potential indicates that rather ischemia-reperfusion injury and reduced energy reserves than denervation are responsible for the attenuated homeometric autoregulation in the control group.
Whether the decreased right coronary perfusion pressure in the control group (as a consequence of decreased blood pressure) influences right ventricular contractility remains unclear. Only very limited information is available even under physiological conditions which describe the effect of coronary perfusion pressure on right ventricular function, and the results are controversial. The current study was not designed to address this point.
The present protocol investigated the adaptation mechanisms of the right ventricle to an acute gradual increase of afterload in recipients with normal pulmonary resistance at baseline. How the right ventricle behaves in the setting of chronic pulmonary hypertension in the recipient was investigated by Chen et al. [15] in a model of monocrotaline induced pulmonary hypertension. They described increased right ventricular power and decreased transpulmonary efficiency. This suggests that even if contractility showed some increase in response to the chronically elevated pulmonary afterload, it does not fulfill the requirements to keep cardiac output constant only by means of homeometric autoregulation. Unfortunately, the authors did not provide data of end-diastolic volumes which would be necessary to elucidate the role of the heterometric autoregulation (Frank–Starling mechanism). Nevertheless, these data support our findings, which postulate limited inotropic adaptation potential of the right ventricle after heart transplantation.
The present study has some limitations. Similarly to the clinical situation, in the present in vivo model, different types of pathophysiological stimuli are counteracting: cardiac preservation/reperfusion and systemic inflammatory response to extracorporal circulation. The systemic inflammatory reaction may worsen reperfusion injury and in turn reduced myocardial function may worsen the hemodynamic consequences of the systemic inflammatory reaction. As we applied INO-1001 systemically during reperfusion, the beneficial effects of PARP inhibition may not be restricted to the improvement of cardiac function. Indeed, we could see for example [9] that cardiopulmonary bypass per se induces PARP activation in the lungs. Systemic application of PARP inhibitors could completely prevent PARP activation in lung tissue and thereby improve lung function after cardiopulmonary bypass. Under these aspects, the systemic effects of PARP inhibition may also contribute to improved hemodynamic performance after transplantation.
In summary, in the present study INO-1001, a novel PARP inhibitor, was able to markedly attenuate reperfusion injury resulting in a better recovery of biventricular and endothelial function as well as energy reserves.
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Appendix A. Conference discussion |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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Dr Szabo: In fact, we have started now a clinical trial this year in normal coronary artery bypass patients, 40 patients at first as a Phase II trial. We also use the conductance catheter measurements in patients. This first study should be a study for proof of principle. And we intend to start a multicenter study, and here I would like to invite you to participate in this multicenter study in heart transplantation patients.
Dr Laufer: I think one of the differences to the real world setting in your experiments is that we take out the donor organs from brain dead individuals, and we know that brain death itself causes this adrenergic storm, especially if it is an explosive brain death, and that causes structural changes.
The question is if the maintenance of the protective effect of your drug is still there if you start out with an organ that is not really normal. In other words, these fox hearts have been normal. You didn't induce brain death and then you took out the organ. So you took out a vital organ which is perfectly normal. It's different. The product with which you start out is very different in the clinical transplantation setting and, as well, there is, I think, all the time the problem to evaluate the outcome in the clinical setting as opposed to the experimental one. Can you comment on that?
Dr Szabo: I absolutely agree. Our group has also a large experience with brain death experiments and published a couple of papers and, indeed, there is a lot of data which suggests that there is a decreased ischemic tolerance of these hearts. We made a histologic examination from brain dead dogs, and we could also see that there is also PARP activation as a result of brain death. So it might be a concept for donor pretreatment and optimizing donor function within donor organisms. So I think this question is of good relevance.
Dr Laufer: Did you infuse this protective drug into the blood, or did you infuse it with the cardioplegia solution?
Dr Szabo: We infuse this drug during the reperfusion.
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Footnotes |
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Presented at the joint 18th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 12th Annual Meeting of the European Society of Thoracic Surgeons, Leipzig, Germany, September 12–15, 2004. |
References |
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TopAbstract1. Introduction2. Material and methods3. Results4. DiscussionAppendix A. Conference...References |
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